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Octopus arms, notable for their complex anatomy and remarkable flexibility, have sparked significant interest within the neuroscience community. However, there remains a dearth of knowledge about the neurochemical organization of various cell types in the arm's nervous system. To address this gap, we used hybridization chain reaction (HCR) to identify distinct neuronal types in the axial nerve cords of the pygmy octopus, Octopus bocki , including putative dopaminergic, octopaminergic, serotonergic, GABAergic, glutamatergic, cholinergic, and peptidergic cells. We obtained high-resolution multiplexed fluorescent images at 0.28 × 0.28 × 1.0 μm voxel size from 10 arm base and arm tip cross sections (each 50 μm thick) and created three-dimensional reconstructions of the axial ganglia, illustrating the spatial distribution of multiple neuronal populations. Our analysis unveiled anatomically distinct and molecularly diverse scattered neurons, while also highlighting multiple populations of dense small neurons that appear uniformly distributed throughout the cortical layer and potential glial cells in the neuropil. Our data provide new insights into how different types of neurons may contribute to an octopus's ability to interact with its environment and execute complex tasks. In addition, our findings establish a benchmark for future studies, allowing pioneering exploration of octopus arm molecular neuroanatomy and offering exciting new avenues in invertebrate neuroscience research. [Display omitted] • 3D octopus arm cord maps reveal stratified monoaminergic and peptidergic neurons • Significant neurochemical differences between the arm tip and base ganglia • Cholinergic and glutamatergic neurons are abundant; GABAergic neurons are scarce • Novel identification of neuronal cotransmission and possible protoplasmic glia Winters-Bostwick et al. reveal complex neurochemical organization within the arm nerve cord of Octopus bocki , using HCR to identify diverse neuronal types. High-resolution 3D imaging and reconstruction demonstrate spatial distribution of cell types, offering new insights into octopus neuroanatomy and enhancing understanding of limb function. [ABSTRACT FROM AUTHOR]

The choice of fixation method significantly impacts tissue morphology and visualization of gene expression and proteins after in situ hybridization chain reaction (HCR) or immunohistochemistry (IHC), respectively. In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation techniques prior to HCR and IHC on chicken embryos. Our findings underscore the importance of optimizing fixation methods for accurate visualization and subsequent interpretation of HCR and IHC results, with implications for probe and antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei and neural tubes compared to PFA fixation. Additionally, TCA fixation altered the subcellular fluorescence signal intensity of various proteins, including transcription factors, cytoskeletal proteins, and cadherins. Notably, TCA fixation revealed protein signals in tissues that may be inaccessible with PFA fixation. In contrast, TCA fixation proved ineffective for mRNA visualization. These results highlight the need for optimization of fixation protocols depending on the target and model system, emphasizing the importance of methodological considerations in biological analyses. [Display omitted] • TCA fixation alters cell and tissue morphology compared to PFA fixation. • PFA fixation is superior to TCA fixation prior to fluorescence in situ hybridization chain reaction (HCR). • Signals are detectable after immunohistochemistry (IHC) by fluorescence microscopy using both PFA and TCA fixation methods. • Tissue-specific fluorescence intensity of cadherin and microtubule protein signals differ between PFA and TCA-fixed embryos. [ABSTRACT FROM AUTHOR]

Abstract Venom is a remarkable innovation found across the animal kingdom, yet the evolutionary origins of venom systems in various groups, including spiders, remain enigmatic. Here, we investigated the organogenesis of the venom apparatus in the common house spider, Parasteatoda tepidariorum. The venom apparatus consists of a pair of secretory glands, each connected to an opening at the fang tip by a duct that runs through the chelicerae. We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series. These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip, progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion. The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells. Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium, forkhead and sum-1 in the surrounding muscle layer, while Distal-less was predominantly expressed at the gland extremities. Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders, offering key insights into their evolution and development.

An electrochemical aptasensor was used for the fast and sensitive detection of zearalenone (ZEN) based on the combination of Co3O4/MoS2/Au nanocomposites and the hybrid chain reaction (HCR). The glassy carbon electrode was coated with Co3O4/MoS2/Au nanomaterials to immobilize the ZEN-cDNA that had been bound with ZEN-Apt by the principle of base complementary pairing. In the absence of ZEN, the HCR could not be triggered because the ZEN-cDNA could not be exposed. After ZEN was added to the surface of the electrode, a complex structure was produced on the modified electrode by the combination of ZEN and ZEN-Apt. Therefore, the ZEN-cDNA can raise the HCR to produce the long-strand dsDNA structure. Due to the formation of dsDNA, the methylene blue (MB) could be inserted into the superstructure of branched DNA and the peak currents of the MB redox signal dramatically increased. So the concentration of ZEN could be detected by the change of signal intensity. Under optimized conditions, the developed electrochemical biosensing strategy showed an outstanding linear detection range of 1.0×10−10 mol/L to 1.0×10−6 mol/L, a low detection limit (LOD) of 8.5×10−11 mol/L with desirable selectivity and stability. Therefore, the fabricated platform possessed a great application potential in fields of food safety, medical detection, and drug analysis. [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.

This study presents a hybrid recognition system for multi-class compound Marathi characters, which addresses the problem of handwritten Marathi character recognition. The methodology efficiently bridges the gap between feature extraction and classification by integrating a Convolutional Neural Network (CNN) and Support Vector Machine (SVM). The first step is gathering and preprocessing a wide range of handwritten Marathi compound characters that are written in different styles. Using conventional supervised learning methods, the CNN is trained on this dataset, paying special attention to data augmentation and validation in order to reduce overfitting. High-level features taken from the final fully connected layer of the CNN are fed into an SVM classifier in the next step. By using these features in its training, the SVM improves prediction accuracy. For multi-class classification, the one-vs-all method is used. The hybrid CNN-SVM algorithm demonstrates its effectiveness in the crucial phases of feature extraction and classification by identifying handwritten compound Marathi characters with remarkable accuracy. Evaluation metrics, such as accuracy, precision, recall, F1-score, and confusion matrix analysis, are employed in the process of evaluating the effectiveness of the model. This assessment is carried out on a different testing dataset, offering a thorough examination of the model's functionality. The proposed algorithm demonstrates its superior performance and potential for improved character recognition by achieving training accuracy of 98.60% and validation accuracy of 97.69%. The development of handwriting recognition systems has benefited greatly from this research, especially when it comes to intricate scripts like Marathi. The suggested hybrid algorithm shows encouraging outcomes and has a great deal of potential for use in document processing, natural language comprehension, and character recognition in languages that use the Marathi script. Subsequent efforts will centre on refining the model and investigating ensemble methods to increase the robustness and accuracy of recognition. [ABSTRACT FROM AUTHOR]

Recognition of handwritten characters has been a challenging task so far. There exist thousands of official languages across the globe which are used to communicate through documentation. Optical character recognition (OCR) being the challenging domain in such context where images of such documents are to be recognized either offline or online. Online and offline recognition of documents refer to the approaches whereby the basic operation of character recognition is performed during documentation itself and recognizing characters from stored documents, respectively. Numerous applications from a range of fields like medical transcription, digitization of ancient manuscripts, language translations, etc. are solely dependent on the task of OCR. In this work, an efficient framework is presented for the purpose of handwritten character recognition that can be well utilized for both offline and online processes. The proposed work takes the handwritten character images as input. It applies set of pre-processing such that the samples become suitable for the feature extraction task. The novelty lies in the process of feature extraction whereby three distinct types of feature are considered based on the shape primitives of the images. These features are global to the sample. Subsequently, local shape features are further extracted out of this shape features after suitable quantization process. These local features are the evidences which can be generically used to recognize test samples. These local feature vectors are dubbed as evidences and are preserved into a glossary dubbed as evidence glossary. The efficiency of the proposed scheme is well justified as it utilizes only a fraction of the feature vector and still it can recognize the characters. Other advantages of the proposed work are scale invariance and rotations invariance. Suitable datasets from two distinct languages, namely Odia and English are utilized for evaluating the efficiency of the framework. Comparison of the performance of the framework with six distinct state-of-the-art machine learning models is conducted whereby it outclass the competent in terms of several performance metrics. [ABSTRACT FROM AUTHOR]

A novel three-dimensional (3D) porous nitrogen-sulfur co-doped carbon (N–S–C) mesh was synthesized and used for the first time as the quenching material to construct a fluorescent aptasensor for ochratoxin A (OTA) detection. The fluorescent aptasensor with enzyme-free signal amplification strategy was developed by using cDNA as a promoter to trigger hybridization chain reaction (HCR), which effectively improved the sensitivity of this aptasensor. In the absence of OTA, 3D porous N–S–C mesh can adsorb carboxyfluorescein FAM-labeled hairpin DNA1 (H1-FAM) and hairpin DNA2 (H2) and quench the fluorescence of FAM. In the presence of the OTA, the OTA specifically binds to the aptamer strand and the DNA duplex undergoes dissociation. The released cDNA in turn serves as a promoter for HCR, and the strand assembly of H1-FAM and H2 is triggered by the promoter to generate long-strand DNA polymers via HCR, resulting in an increasing fluorescent signal. Under optimal conditions, there was a good linear relationship between lgCOTA and fluorescence intensity difference in the range 0.01–500 ng/mL (R2 = 0.993), and the detection limit was 2.7 pg/mL. The designed sensor platform was applied to determine spiked OTA in peanut, wheat flour, corn flour, black tea, and wine with recoveries in the range of 94.4–119.6%. [ABSTRACT FROM AUTHOR]

Ergonomic reliability plays a significant role in the safe operation of devices.With the spread of infectious diseases around the world, in work environments with high loads and high infection rates, medical staff work in a state of high self-protection. The use of visual display terminal (VDT) for medical equipment has undergone fundamental changes, and the traditional medical equipment human-machine interface design needs to be improved. After the completion of design and development, a VDT design enters the experimental testing stage, which has significant limitations for simulating the work of medical staff in the high-load and high-infection environments. The testing cost is high, and subjects face harsh conditions; thus, an ergonomic reliability model that can predict the use of VDT in such special high-infection and high-load circumstances must be established. An ergonomic reliability model based on an improved backpropagation neural network (BPNN) and human cognition reliability (HCR) is proposed for predicting and evaluating operation flows according tomedical equipment VDTs. Firstly, a small data sample can be used to train BPNN to generate a network that can ensure suitable accuracy. To prevent the model from falling into local optimal solutions, the bat algorithm is introduced to improve the BPNN. Compared to a traditional BPNN, the superiority of the improved BPNN is clearly demonstrated. Secondly, the HCR method is used to analyze and highlight changes in the human factor reliability of VDTs for medical equipment in different time processes and operating processes according to BPNN prediction results, to provide a reference for selecting the optimalmethod. Finally, the validity and availability of the proposedmethod are verified through an eye tracker experiment and statistical analysis results. [ABSTRACT FROM AUTHOR]

Background: Long non-coding RNAs (lncRNAs), which are generally less functionally characterized or less annotated, evolve more rapidly than mRNAs and substantially possess fewer sequence conservation patterns than proteincoding genes across divergent species. People assume that the functional inference could be conducted on the evolutionarily conserved long noncoding RNAs as they are most likely to be functional. In the past decades, substantial progress has been made in discussions on the evolutionary conservation of non-coding genomic regions from multiple perspectives. However, understanding their conservation and the functions associated with sequence conservation in relation to further corresponding phenotypic variability or disorders still remains incomplete. Results: Accordingly, we determined a highly conserved region (HCR) to verify the sequence conservation among long non-coding RNAs and systematically profiled homologous long non-coding RNA clusters in humans and mice based on the detection of highly conserved regions. Moreover, according to homolog clustering, we explored the potential function inference via highly conserved regions on representative long non-coding RNAs. On lncRNA XACT, we investigated the potential functional competence between XACT and lncRNA XIST by recruiting miRNA-29a, regulating the downstream target genes. In addition, on lncRNA LINC00461, we examined the interaction relationship between LINC00461 and SND1. This interaction or association may be perturbed during the progression of glioma. In addition, we have constructed a website with user-friendly web interfaces for searching, analyzing, and downloading to present the homologous clusters of humans and mice. Conclusion: Collectively, homolog clustering via the highly conserved region definition and detection on long non-coding RNAs, as well as the functional explorations on representative sequences in our research, would provide new evidence for the potential function of long non-coding RNAs. Our results on the remarkable roles of long non-coding RNAs would presumably provide a new theoretical basis and candidate diagnostic indicators for tumors. [ABSTRACT FROM AUTHOR]

POU genes are a family of evolutionarily conserved transcription factors with key functions in cell type specification and neurogenesis. In vitro experiments have indicated that the expression of some POU genes is controlled by the intercellular signaling molecule retinoic acid (RA). In this work, we aimed to characterize the roles of RA signaling in the regulation of POU genes in vivo. To do so, we studied POU genes during the development of the cephalochordate amphioxus, an animal model crucial for understanding the evolutionary origins of vertebrates. The expression patterns of amphioxus POU genes were assessed at different developmental stages by chromogenic in situ hybridization and hybridization chain reaction. Expression was further assessed in embryos subjected to pharmacological manipulation of endogenous RA signaling activity. In addition to a detailed description of the effects of these treatments on amphioxus POU gene expression, our survey included the first description of Pou2 and Pou6 expression in amphioxus embryos. We found that Pit-1, Pou2, Pou3l, and Pou6 expression are not affected by alterations of endogenous RA signaling levels. In contrast, our experiments indicated that Brn1/2/4 and Pou4 expression are regulated by RA signaling in the endoderm and the nerve cord, respectively. The effects of the treatments on Pou4 expression in the nerve cord revealed that, in developing amphioxus, RA signaling plays a dual role by (1) providing anteroposterior patterning information to neural cells and (2) specifying neural cell types. This finding is coherent with a terminal selector function of Pou4 for GABAergic neurons in amphioxus and represents the first description of RA-induced changes in POU gene expression in vivo. [ABSTRACT FROM AUTHOR]

Based on a dual signal amplification strategy of novel accordion-like Bi2O3-decorated Ti3C2 (Ti3C2@Bi2O3) nanocomposites and hybridization chain reaction (HCR), an ultra-sensitive electrochemical biosensor was constructed for miRNA-21 detection. By etching Ti3AlC2 with HF, Ti3C2 with an accordion-like structure was first obtained and subsequently covered by Bi2O3 nanoparticles (NPs), forming Ti3C2@Bi2O3. A layer of Au NPs was electrodeposited on the glassy carbon electrode coated with Ti3C2@Bi2O3, which not only significantly improved the electron transport capacity of the electrode but also greatly increased its surface active area. Upon the immobilization of the thiolated capture probe (SH-CP) on the electrode, the target miRNA-21 specifically hybridized with SH-CP and thus opened its hairpin structure, triggering HCR to form a long double strand with the primers H1 and H2. A large number of the electrochemical indicator molecules were thus embedded inside the long double strands to produce the desirable electrochemical signal at a potential of − 0.19 V (vs. Ag/AgCl). Such dual signal amplification strategy successfully endowed the biosensor with ultra-high sensitivity for miRNA-21 detection in a wide linear range from 1 fM to 100 pM with a detection limit as low as 0.16 fM. The excellent detection of miRNA-21 in human blood plasma displayed a broad prospect in clinical diagnosis. An ultra-sensitive electrochemical biosensor was successfully constructed for miRNA-21 detection in human blood plasma based on the dual signal amplification strategy of novel accordion-like Bi2O3 decorated Ti3C2 (Ti3C2@Bi2O3) nanocomposites and hybridization chain reaction. [ABSTRACT FROM AUTHOR]

Abstract Background Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are widely used in the treatment of coronary heart disease, but the best revascularization method for multivessel coronary artery disease (MVD) patients is still controversial. Hybrid coronary revascularization (HCR), together with CABG and PCI, have been proved to be feasible methods, but the long-term effect of HCR is not as clear as CABG. Method By October 2020, we retrieved articles from PubMed, Web of science, EMBASE and Cochrane library databases. The main results are based on major adverse cardiovascular and cerebral events (MACCE). Result A total of 18 articles (3 randomized controlled trials (RCTs) and 15 observational studies) were included in this meta-analysis. The outcomes of MACCE in the HCR group at perioperative, short-term (30 days to 1 year), medium-term (1 year to 5 years) and long-term (5 years and above) follow-up period were similar to those in the CABG group. The mortality rates of patients in perioperative, short-term and medium-term follow-up were similar to those in the CABG group, but lower than that in the CABG group at long-term follow-up (OR = 0.35, 95% CI 0.18–0.69, p = 0.002). The revascularization rate was higher in the HCR group during the perioperative period (OR = 3.50, 95% CI 2.07–5.94, p

Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (Apt EpCAM , Apt HER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 103−107 particles/μL, with a limit of detection (LOD) of 3.3 × 102 particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine. [Display omitted] • Dual-aptamers provide high specificity and accurate analysis of TEV. • CRISPR increases the sensitivity and reduces the LOD compared to HCR. • TEV are able to obtain a high accuracy of cancer diagnosis (100 %). [ABSTRACT FROM AUTHOR]

Accurate detection of cancer biomarkers like mucin 1 (MUC-1) is significantly important for diagnosis and prognosis of tumor. Herein, a self-correction electrochemical immunoassay for MUC-1 detection was developed based on bioinspired nanochannels and electrochemistry of molybdophosphate. MUC-1 can be captured in the bioinspired polymer nanochannels via the formation of immunocomplex. Then, diffusion of MoO 4 2− in the nanochannels would be sterically hindered and the in-situ generation of phosphomolybdic acid onto the electrode surface which was modified with phosphate chitosan hydrogel, would be influenced. Therefore, the two well-defined redox peaks of phosphomolybdic acid can be applied to the detection of the antigen. To improve detection sensitivity, the phosphate chitosan was incorporated with carbon nanotubes. Moreover, the occurrence in nanochannels was amplified by hybridization chain reaction, which enhanced the hindrance for transport of MoO 4 2− by increasing steric effect, electrostatic repulsion as well as anchoring sites. Under optimized conditions, currents of the both anodic peaks on square wave voltametric voltammograms decreased linearly with the logarithm of the concentration of MUC-1 ranging from 10 fg∙mL−1 to 100 ng∙mL−1 with a detection limit of 6.0 fg∙mL−1. And thus, the detection results can be self-corrected by the results acquired from the two peaks, which had valuable implications for the reliable MUC-1 detection in disease diagnosis and therapy application. [Display omitted] • A self-correction electrochemical immunoassay was constructed. • Phosphate chitosan modified electrode was applied to monitor MoO 4 2−. • Target-triggered HCR was performed in nanochannels. • This immunoassay exhibited low detection limit for MUC-1. [ABSTRACT FROM AUTHOR]

Coronary artery bypass surgery has been for decades the gold standard for the treatment of coronary artery disease, especially in patients with 3-vessel disease. Despite the excellent results achieved through the application of this technique, problems related to median sternotomy and the use of cardiopulmonary bypass such as wound infections and cerebral stroke still play an important role in the postoperative course of these patients. Minimally invasive coronary revascularization techniques aim at reducing the rate of such complications while offering the same quality of revascularization. Minimally invasive coronary surgery includes a set of coronary revascularization techniques that are completed without the use of cardiopulmonary bypass or through a left mini-thoracotomy at the level of the 4th or 5th intercostal space and aim at reducing the level of the aforementioned complications compared to the standard technique. Hybrid coronary revascularization is an alternative approach that combines surgical revascularization with percutaneous coronary interventions to extend the treatment spectrum even further. Through the application of these techniques, surgeons aim at minimizing the risk of infection of the surgical wound and significantly reduce the number of post-operative cerebral strokes. Also, numerous studies have shown that minimally invasive coronary surgery can offer better results in terms of duration of the mechanical ventilation, length of stay in intensive care, transfusions of blood products and consequently in reducing treatment costs. Minimally invasive revascularization techniques are not only an important alternative to standard coronary surgery, but in the near future will constitute the standard treatment for this group of patients. This article aims to describe the techniques used to achieve minimally invasive coronary revascularization and to highlight the outcomes of the different approaches.CABG,

In situ hybridization techniques are powerful methods for exploring gene expression in a wide range of biological contexts, providing spatial information that is most often lost in traditional biochemical techniques. However, many in situ hybridization methods are costly and time-inefficient, particularly for screening-based projects that follow on from single-cell RNA sequencing data, which rely on of tens of custom-synthetized probes against each specific RNA of interest. Here we provide an optimized pipeline for Hybridization Chain Reaction (HCR)-based RNA visualization, including an open-source code for optimized probe design. Our method achieves high specificity and sensitivity with the option of multiplexing using only five pairs of probes, which greatly lowers the cost and time of the experiment. These features of our HCR protocol are particularly useful and convenient for projects involving screening several genes at medium throughput, especially as the method include an amplification step, which makes the signal readily visible at low magnification imaging.

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 P1 a 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 P1 a 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 P1 a 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 ., Competing Interests: KW, HC, DA No competing interests declared, (© 2023, Watanabe et al.)

Minimal-access cardiac surgery appears to be the future. It is increasingly desired by cardiologists and demanded by patients who perceive superiority. Minimal-access coronary artery revascularisation has been increasingly adopted throughout the world. Here, we review the history of minimal-access coronary revascularization and see that it is almost as old as the history of cardiac surgery. Modern minimal-access coronary revascularization takes a variety of forms—namely minimal-access direct coronary artery bypass grafting (MIDCAB), hybrid coronary revascularisation (HCR), and totally endoscopic coronary artery bypass grafting (TECAB). It is noteworthy that there is significant variation in the nomenclature and approaches for minimal-access coronary surgery, and this truly presents a challenge for comparing the different methods. However, these approaches are increasing in frequency, and proponents demonstrate clear advantages for their patients. The challenge that remains, as for all areas of surgery, is demonstrating the superiority of these techniques over tried and tested open techniques, which is very difficult. There is a paucity of randomised controlled trials to help answer this question, and the future of minimal-access coronary revascularisation, to some extent, is dependent on such trials. Thankfully, some are underway, and the results are eagerly anticipated.

In this work, a dual-signal ratiometric electrochemical aptasensor based on the hybrid chain reaction (HCR) and streptavidin-modified magnetic beads (SA-MBs) was developed to rapidly detect zearalenone (ZEN). The HCR, as a powerful signal amplification technique to imporve the signal of sonser. When the target is present, they specifically bind with ZEN-Apt and release ZEN-cDNA to trigger HCR. Simultaneously, more double-stranded DNA causes the signal of Thi to be blocked. As a result, the two signals tend to change in the opposite direction as the ZEN concentration changes. Additionally, the peak current ratio of I Thi /I Fc showed a positive correlation with the ZEN concentration. Under optimal conditions, the constructed biosensor showed an excellent linear detection range (1.0 × 10−10 mol/L to 1.0 × 10−6 mol/L), a low detection limit (4.4 × 10−11 mol/L) and high specificity for ZEN. In addition, the detection method retains the characteristics of low cost and rapid detection of electrochemical detection, while improving the detection limit and detection accuracy via SA-MBs and internal reference signal. This provides a new idea for the practical detection of ZEN. • The dual-signal ratiometric biosensor owns higher detection accuracy. • MoS 2 -Thi is used as the second signal to provide internal parameter comparison. • Synergistic effect of magnetic bead and HCR achieve the signal amplification. • This approach was desirable regarding to sensitivity, detection limit and range. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) has been identified as a vital bioindicator of food contamination, closely associated with human health. This study innovatively integrates hybridization chain reaction (HCR) with G-quadruplex DNAzyme, forging a highly efficient S. aureus surface-enhanced Raman spectroscopy (SERS) biosensor that markedly elevates its detection abilities. Specifically, the triggering of HCR relies on the unique single-stranded DNA (ssDNA) sequence released by the target bacteria (S. aureus), which then pairs with the H1 and H2 probes, initiating an alternate hybridization process, forming long-chain DNA nanostructures. The G-quadruplex DNAzyme emerges through the intricate folding of G bases within its structure, simulating the catalytic activity of peroxidase and facilitating the oxidation of cysteine to cystine. This reaction operation further modulates the discrete state of Au NPs, thereby influencing the conduction of SERS signals. Under optimized conditions, the S. aureus sensor exhibits excellent linear response characteristics within the concentration range of 8.0 to 8.0×106 cfu/mL, featuring an impressively limit of detection (LOD) down to 1.5 cfu/mL. Consequently, the HCR-mediated G-quadruplex DNAzyme assembly strategy not only establishes a highly sensitive S. aureus detection platform but also opens new paths for precise identification, significantly impacting food safety and public health. • Novel SERS biosensor integrating HCR & G-quadruplex DNAzyme for S. aureus detection. • HCR triggered by S. aureus -specific ssDNA boosts SERS signals via long-chain DNA formation. • G-quadruplex DNAzyme peroxidase-like activity modulates Au NPs state. • Detection of S. aureus in real samples with high recoveries and low variations. • HCR-mediated G-quadruplex DNAzyme assembly paves the way for precise S. aureus detection. [ABSTRACT FROM AUTHOR]

Accurate and reliable detection of Staphylococcus aureus (S. aureus) is essential for preventing infections, particularly in healthcare and food safety contexts. This work presents a novel dual-mode biosensor that integrates the CRISPR/Cas12a system with an enzyme-free isothermal amplification method for detecting S. aureus. Hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) amplify the aptamer-triggered response, significantly enhancing sensitivity. CRISPR/Cas12a′s nuclease activity is utilized in two modes: cis cleavage generates a fluorescence signal, while trans cleavage produces an electrochemical signal, enabling dual-mode detection. The biosensor demonstrates outstanding performance, with a limit of detection (LOD) as low as 5.7 CFU mL−1 in electrochemical mode and 133.7 CFU mL−1 in fluorescence mode, showcasing excellent accuracy, stability, and sensitivity. It has been successfully applied to detecting actual samples, confirming its practical applicability. This innovative approach offers a powerful tool for the swift and precise identification of S. aureus and paves the way for developing next-generation dual-mode biosensors for various analytes. Future research will aim to simplify the detection process further, making it more accessible for use in resource-limited settings. [Display omitted] • CRISPR/Cas12a cis/trans cleavage yields fluorescence and electrochemical signals. • CHA-HCR enzyme-free isothermal amplification boosts signals, cuts costs and storage. • Dual-mode detection of S. aureus achieves satisfactory results in real samples. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance. [Display omitted] • A novel SPQC sensor based on the specific recognition of TALEs for locus-specific detection of 6mA in DNA is developed. • This SPQC sensor can locus-specific detect 6mA and distinguish single base mismatches. • The hybridization chain reaction and silver staining are utilized to enhance detection sensitivity, the LOD is 0.63 pM. • The SPQC sensor is competent for 6mA target analysis in the real cell samples. [ABSTRACT FROM AUTHOR]

Background: Percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are widely used in the treatment of coronary heart disease, but the best revascularization method for multivessel coronary artery disease (MVD) patients is still controversial. Hybrid coronary revascularization (HCR), together with CABG and PCI, have been proved to be feasible methods, but the long-term effect of HCR is not as clear as CABG.Method: By October 2020, we retrieved articles from PubMed, Web of science, EMBASE and Cochrane library databases. The main results are based on major adverse cardiovascular and cerebral events (MACCE).Result: A total of 18 articles (3 randomized controlled trials (RCTs) and 15 observational studies) were included in this meta-analysis. The outcomes of MACCE in the HCR group at perioperative, short-term (30 days to 1 year), medium-term (1 year to 5 years) and long-term (5 years and above) follow-up period were similar to those in the CABG group. The mortality rates of patients in perioperative, short-term and medium-term follow-up were similar to those in the CABG group, but lower than that in the CABG group at long-term follow-up (OR = 0.35, 95% CI 0.18-0.69, p = 0.002). The revascularization rate was higher in the HCR group during the perioperative period (OR = 3.50, 95% CI 2.07-5.94, p < 0.001), short-term (OR = 3.28, 95% CI 1.62-6.64, p < 0.001) and mid-term follow-up (OR = 2.84, 95% CI 1.64-4.92, p < 0.001).Conclusion: Our results reveal that HCR is a safe and therapeutically effective alternative in treatments for MVD patients. It has not only less short-term adverse effect, but also better long-term effect, especially in death. [ABSTRACT FROM AUTHOR]

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the airagar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. [ABSTRACT FROM AUTHOR]

Angiotensin-converting enzyme 2 (ACE2) has been identified as the cellular entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High ACE2 tissue expression and low glycine levels were suggested to increase susceptibility for SARS-CoV-2 infection and increasing circulating ACE2 has been proposed as one possible strategy to combat COVID-19. In humans, aerobic physical exercise induces an increase in plasma ACE2 in some individuals. However, it is not clear whether glycine and ACE2 levels depend on intrinsic exercise capacity or on exercise training. We used rats selectively bred for high intrinsic exercise capacity (HCR) or low exercise capacity (LCR) and tested the influence of this genetic predetermination and/or aerobic exercise on metabolites, ACE2 tissue expression and circulating ACE 2. ACE2 expression was measured in different tissues in the sedentary animals and again after 4 weeks of high-intensity aerobic exercise in both LCRs and HCRs. Sedentary HCRs exhibited significantly higher circulating ACE2 concentrations compared to LCRs, but a lower expression of ACE2 in all investigated tissues except for adipose tissue. Body weight was negatively correlated with serum ACE2 and positively correlated with ACE2 expression in the heart. Aerobic exercise caused a significant decrease in ACE2 expression in the lung, heart, muscle, and kidney both in LCRs and HCRs. Our results suggest that ACE2 expression, circulating ACE2 and glycine serum concentration are related to aerobic intrinsic exercise capacity and can be influenced with exercise. These results may support the hypothesis that physically fit individuals have a lower susceptibility for COVID-19 infection. [ABSTRACT FROM AUTHOR]

In this work, a highly sensitive biosensor for detecting cadmium ions (Cd2+) was developed based on a Cd2+‐specific DNA aptamer and a hybridization chain reaction (HCR). The Cd2+ aptamer (named S0) was used to recognize Cd2+ and trigger the HCR. Without Cd2+, S0 initiated the HCR to form long nicked dsDNA structures to quench the fluorescence. Then, Cd2+ could bind with S0 to block HCR to recover fluorescence. This biosensor had high sensitivity with a detection limit of 0.36 nM and a linear range from 0 to 10 nM. Moreover, it showed a satisfactory selectivity and recovery rates. [ABSTRACT FROM AUTHOR]

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

The central nervous system of the cephalochordate amphioxus consists of a dorsal neural tube with an anterior brain. Two decades of gene expression analyses in developing amphioxus embryos have shown that, despite apparent morphological simplicity, the amphioxus neural tube is highly regionalised at the molecular level. However, little is known about the morphogenetic mechanisms regulating the spatiotemporal emergence of cell types at distinct sites of the neural axis and how their arrangements contribute to the overall neural architecture. In vertebrates, proliferation is key to provide appropriate cell numbers of specific types to particular areas of the nervous system as development proceeds, but in amphioxus proliferation has never been studied at this level of detail, nor in the specific context of neurogenesis. Here, we describe the dynamics of cell division during the formation of the central nervous system in amphioxus embryos, and identify specific regions of the nervous system that depend on proliferation of neuronal precursors at precise time-points for their maturation. By labelling proliferating cells in vivo at specific time points in development, and inhibiting cell division during neurulation, we demonstrate that localised proliferation in the anterior cerebral vesicle is required to establish the full cell type repertoire of the frontal eye complex and the putative hypothalamic region of the amphioxus brain, while posterior proliferating progenitors, which were found here to derive from the dorsal lip of the blastopore, contribute to elongation of the caudal floor plate. Between these proliferative domains, we find that trunk nervous system differentiation is independent from cell division, in which proliferation decreases during neurulation and resumes at the early larval stage. Taken together, our results highlight the importance of proliferation as a tightly controlled mechanism for shaping and regionalising the amphioxus neural axis during development, by addition of new cells fated to particular types, or by influencing tissue geometry. [ABSTRACT FROM AUTHOR]

The central nervous system of the cephalochordate amphioxus consists of a dorsal neural tube with an anterior brain. Two decades of gene expression analyses in developing amphioxus embryos have shown that, despite apparent morphological simplicity, the amphioxus neural tube is highly regionalised at the molecular level. However, little is known about the morphogenetic mechanisms regulating the spatiotemporal emergence of cell types at distinct sites of the neural axis and how their arrangements contribute to the overall neural architecture. In vertebrates, proliferation is key to provide appropriate cell numbers of specific types to particular areas of the nervous system as development proceeds, but in amphioxus proliferation has never been studied at this level of detail, nor in the specific context of neurogenesis. Here, we describe the dynamics of cell division during the formation of the central nervous system in amphioxus embryos, and identify specific regions of the nervous system that depend on proliferation of neuronal precursors at precise time-points for their maturation. By labelling proliferating cells in vivo at specific time points in development, and inhibiting cell division during neurulation, we demonstrate that localised proliferation in the anterior cerebral vesicle is required to establish the full cell type repertoire of the frontal eye complex and the putative hypothalamic region of the amphioxus brain, while posterior proliferating progenitors, which were found here to derive from the dorsal lip of the blastopore, contribute to elongation of the caudal floor plate. Between these proliferative domains, we find that trunk nervous system differentiation is independent from cell division, in which proliferation decreases during neurulation and resumes at the early larval stage. Taken together, our results highlight the importance of proliferation as a tightly controlled mechanism for shaping and regionalising the amphioxus neural axis during development, by addition of new cells fated to particular types, or by influencing tissue geometry.

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future. [ABSTRACT FROM AUTHOR]

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

Rationale: Regular active exercise is considered therapeutic for cardiovascular disease, in part by increasing mitochondrial respiratory capacity, but a significant amount of exercise capacity is determined genetically. Animal models, demonstrating either high capacity aerobic running (HCR) or low capacity aerobic running (LCR) phenotypes, have been developed to study the intrinsic contribution, with HCR rats subsequently characterized as “disease resistant” and the LCRs as “disease prone.” Enhanced cardioprotection in HCRs has been variable and mutifactoral, but likely includes a metabolic component. These studies were conducted to determine the influence of intrinsic aerobic phenotype on cardiac mitochondrial function before and after ischemia and reperfusion.Methods: A total of 34 HCR and LCR rats were obtained from the parent colony at the University of Toledo, housed under sedentary conditions, and fed normal chow. LCR and HCR animals were randomly assigned to either control or ischemia-reperfusion (IR). On each study day, one HCR/LCR pair was anesthetized, and hearts were rapidly excised. In IR animals, the hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to global hypothermic ischemic arrest (80 min). Following the arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff perfusion system. Following reperfusion, the heart was weighed and the left ventricle (LV) was isolated. A midventricular ring was obtained to estimate infarction size [triphenyltetrazolium chloride (TTC)] and part of the remaining tissue (~150 mg) was transferred to a homogenation buffer on ice. Isolated mitochondria (MITO) samples were prepared and used to determine respiratory capacity under different metabolic conditions. In control animals, MITO were obtained and prepared similarly immediately following anesthesia and heart removal, but without IR.Results: In the control rats, both resting and maximally stimulated respiratory rates were higher (32 and 40%, respectively; p < 0.05) in HCR mitochondria compared to LCR. After IR, resting MITO respiratory rates were decreased to about 10% of control in both strains, and the augmented capacity in HCRs was absent. Maximally stimulated rates also were decreased more than 50% from control and were no longer different between phenotypes. Ca++ retention capacity and infarct size were not significantly different between HCR and LCR (49.2 ± 5.6 vs. 53.7 ± 4.9%), nor was average coronary flow during reperfusion or arrhythmogenesis. There was a significant loss of mitochondria following IR, which was coupled with decreased function in the remaining mitochondria in both strains.Conclusion: Cardiac mitochondrial capacity from HCR was significantly higher than LCR in the controls under each condition. After IR insult, the cardiac mitochondrial respiratory rates were similar between phenotypes, as was Ca++ retention capacity, infarct size, and arrhythmogenicity, despite the increased mitochondrial capacity in the HCRs before ischemia. Relatively, the loss of respiratory capacity was actually greater in HCR than LCR. These data could suggest limits in the extent to which the HCR phenotype might be “protective” against acute tissue stressors. The extent to which any of these deficits could be “rescued” by adding an active exercise component to the intrinsic phenotype is unknown.

Purpose: Previous reports have suggested that active exercise aside, intrinsic aerobic running capacity (Low = LCR, high = HCR) in otherwise sedentary animals may influence several cardiovascular health-related indicators. Relative to the HCR phenotype, the LCR phenotype is characterized by decreased endothelial reactivity, increased susceptibility to reperfusion-induced arrhythmias following short, non-infarction ischemia, and increased diet-induced insulin resistance. More broadly, the LCR phenotype has come to be characterized as a “disease prone” model, with the HCRs as “disease resistant.” Whether these effects extend to injury outcomes in an overt infarction or whether the effects are gender specific is not known. This study was designed to determine whether HCR/LCR phenotypic differences would be evident in injury responses to acute myocardial ischemia-reperfusion injury (AIR), measured as infarct size and to determine whether sex differences in infarction size were preserved with phenotypic selection.Methods: Regional myocardial AIR was induced in vivo by either 15 or 30 min ligation of the left anterior descending coronary artery, followed by 2 h of reperfusion. Global ischemia was induced in isolated hearts ex vivo using a Langendorff perfusion system and cessation of perfusion for either 15 or 30 min followed by 2 h of reperfusion. Infarct size was determined using 2, 3, 5–triphenyltetrazolium chloride (TTC) staining, and normalized to area at risk in the regional model, or whole heart in the global model. Portions of the tissue were paraffin embedded for H&E staining and histology analysis.Results: Phenotype dependent differences in infarct size were seen with 15 min occlusion/2 h reperfusion (LCR > HCR, p < 0.05) in both regional and global models. In both models, longer occlusion times (30 min/2 h) produced significantly larger infarctions in both phenotypes, but phenotypic differences were no longer present (LCR vs. HCR, p = n.s.). Sex differences in infarct size were present in each phenotype (LCR male > LCR female, p < 0.05; HCR male > HCR female, p < 0.05 regardless of length of occlusion, or ischemia model.Conclusions: There is cardioprotection afforded by high intrinsic aerobic capacity, but it is not infinite/continuous, and may be overcome with sufficient injury burden. Phenotypic selection based on endurance running capacity preserved sex differences in response to both short and longer term coronary occlusive challenges. Outcomes could not be associated with differences in system characteristics such as circulating inflammatory mediators or autonomic nervous system influences, as similar phenotypic injury patterns were seen in vivo, and in isolated crystalloid perfused heart ex vivo.

An electrochemical-DNA (E-DNA) sensor was constructed by using DNA metallization to produce an electrochemical signal reporter in situ and hybridization chain reaction (HCR) as signal amplification strategy. The cyclic voltammetry (CV) technique was used to characterize the electrochemical solid-state Ag/AgCl process. Moreover, the enzyme cleavage technique was introduced to reduce background signals and further improve recognition accuracy. On the basis of these techniques, the as-prepared E-DNA sensor exhibited superior sensing performance for trace ctDNA analysis with a detection range of 0.5 fM to 10 pM and a detection limit of 7 aM. The proposed E-DNA sensor also displayed excellent selectivity, satisfied repeatability and stability, and had good recovery, all of which supports its potential applications for future clinical sample analysis.

The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research. [Display omitted] • Construction of a novel sensing platform to realize the detection of miRNAs. • This research strategy combines CRISPR-Cas13a with HCR for detection by ECL methodology. • Highly sensitive and specific in miR-17 detection, with low detection limits (14.38 aM). • This research strategy has a very high potential for clinical and biomedical applications. [ABSTRACT FROM AUTHOR]

According to the characteristics of DNA programming, the cascaded nucleic acid amplification technology with larger output can overcome the problem of insufficient sensitivity of single nucleic acid amplification technology, and it combines the advantages of two or even multiple nucleic acid amplification technologies at the same time. In this work, a novel cascade signal amplification strategy with strand displacement amplification (SDA) and cascade hybridization chain reaction (HCR) was proposed for trace detection of hAAG and VEGF165. HAAG-induced SDA produced a large amount of S2 to open H2 on Polystyrene (PS) nanospheres, thereby triggering cascade HCR to form DNA dendritic nanostructures with rich fluorescence (FL) signal probes (565 nm). It could realize the amplification of FL signals for the detection of hAAG. Moreover, many doxorubicin (Dox) were loaded into the GC bases of DNA dendritic nanostructures, and its FL signal was effectively shielded. VEGF165 specifically bound to its aptamer to form G-quadruplex structures, which released Dox to produce a high FL signal (590 nm) for detection of VEGF165. This work developed a unique multifunctional DNA dendritic nanostructure fluorescence probe, and cleverly designed a new "On-off" switch strategy for sensitive trace detection of cancer markers. [Display omitted] • A multifunctional fluorescence probe of DNA dendritic nanostructure was developed. • A novel cascade signal amplification strategy with SDA and HCR was proposed for double targets. • A signal "On-off" switch is cleverly designed to detect dual targets by using aptamer and G-quadruplex structure. • This work shows a great contribution to simultaneous sensitive detection of cancer markers. [ABSTRACT FROM AUTHOR]

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF 4 :Yb3+, Er3+) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring. Highly sensitive detection of bacteria by fluorescent aptasensor based on DNA walking and hybridization chain reaction strategy. [Display omitted] • Highly sensitive fluorescent aptasensor was constructed by DNA walking and HCR reaction. • The sensor directly detected bacteria without complex bacterial extraction procedure. • The sensor had good stability and low detected background signal. [ABSTRACT FROM AUTHOR]

Angiotensin-converting enzyme 2 (ACE2) has been identified as the cellular entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High ACE2 tissue expression and low glycine levels were suggested to increase susceptibility for SARS-CoV-2 infection and increasing circulating ACE2 has been proposed as one possible strategy to combat COVID-19. In humans, aerobic physical exercise induces an increase in plasma ACE2 in some individuals. However, it is not clear whether glycine and ACE2 levels depend on intrinsic exercise capacity or on exercise training. We used rats selectively bred for high intrinsic exercise capacity (HCR) or low exercise capacity (LCR) and tested the influence of this genetic predetermination and/or aerobic exercise on metabolites, ACE2 tissue expression and circulating ACE 2. ACE2 expression was measured in different tissues in the sedentary animals and again after 4 weeks of high-intensity aerobic exercise in both LCRs and HCRs. Sedentary HCRs exhibited significantly higher circulating ACE2 concentrations compared to LCRs, but a lower expression of ACE2 in all investigated tissues except for adipose tissue. Body weight was negatively correlated with serum ACE2 and positively correlated with ACE2 expression in the heart. Aerobic exercise caused a significant decrease in ACE2 expression in the lung, heart, muscle, and kidney both in LCRs and HCRs. Our results suggest that ACE2 expression, circulating ACE2 and glycine serum concentration are related to aerobic intrinsic exercise capacity and can be influenced with exercise. These results may support the hypothesis that physically fit individuals have a lower susceptibility for COVID-19 infection.

For a given cloud, whether the cloud top is predominately made up of ice crystals or supercooled liquid droplets plays a large role in the clouds overall radiative effects. This study uses collocated airborne radar, lidar, and thermodynamic data from 12 high‐altitude flight legs during the Southern Ocean Clouds, Radiation, Aerosol Transport Experimental Study (SOCRATES) to characterize Southern Ocean (SO) cold sector cloud top phase (i.e., within 96 m of top) as a function of cloud top temperature (CTT). A training data set was developed to create probabilistic phase classifications based on High Spectral Resolution Lidar data and Cloud Radar data. These classifications were then used to identify dominant cloud top phase. Case studies are presented illustrating examples of supercooled liquid water at cloud top at different CTT ranges over the SO (−3°C < CTTs < −28°C). During SOCRATES, 67.4% of sampled cloud top had CTTs less than 0°C. Of the subfreezing cloud tops sampled, 91.7% had supercooled liquid water present in the top 96 m and 74.9% were classified entirely as liquid‐bearing. Liquid‐bearing cloud tops were found at CTTs as cold as −30°C. Horizontal cloud extent was also determined as a function of median cloud top height. Plain Language Summary: Low‐level clouds over the Southern Ocean have a large effect on the region's radiation budget. The radiation budget is strongly influenced by the phase (liquid or ice) of cloud tops, which is where most solar radiation is reflected, and most infrared radiation is radiated to space. For this reason, identifying the phase of cloud tops is important. In this study, airborne radar, lidar, and temperature data from 12 high‐altitude flight legs during the Southern Ocean Clouds, Radiation, Aerosol Transport Experimental Study (SOCRATES) are used to characterize Southern Ocean cloud top phase as a function of cloud top temperature. The results show that liquid is the dominant phase present in clouds over the Southern Ocean, with liquid present at cloud top temperatures as cold as −30°C. Key Points: Airborne radar, lidar, and thermodynamic data are used to determine Southern Ocean cloud top phase as a function of cloud top temperatureConsidering only clouds with subfreezing cloud top temperatures, the dominant phase at cloud top in 74.9% of clouds was liquidLiquid‐bearing cloud tops were even found at temperatures as cold as −30°C [ABSTRACT FROM AUTHOR]