Hemoglobin in the blood acts as a chemosensory signal via the mouse vomeronasal system.

The vomeronasal system plays an essential role in sensing various environmental chemical cues. Here we show that mice exposed to blood and, consequently, hemoglobin results in the activation of vomeronasal sensory neurons expressing a specific vomeronasal G protein-coupled receptor, Vmn2r88, which is mediated by the interaction site, Gly17, on hemoglobin. The hemoglobin signal reaches the medial amygdala (MeA) in both male and female mice. However, it activates the dorsal part of ventromedial hypothalamus (VMHd) only in lactating female mice. As a result, in lactating mothers, hemoglobin enhances digging and rearing behavior. Manipulation of steroidogenic factor 1 (SF1)-expressing neurons in the VMHd is sufficient to induce the hemoglobin-mediated behaviors. Our results suggest that the oxygen-carrier hemoglobin plays a role as a chemosensory signal, eliciting behavioral responses in mice in a state-dependent fashion.

Djhsp70s, especially Djhsp70c, play a key role in planarian regeneration and tissue homeostasis by regulating cell proliferation and apoptosis.

Heat shock protein 70 family (HSP70s) is one of the most conserved and important group of HSPs as molecular chaperones, which plays an important role in cytoprotection, anti-apoptosis and so on. However, the molecular mechanism of HSP70s in animal regeneration remains to be delineated. In this study, we investigate the roles of HSP70s in regeneration of planarian. The four genes, Djhsp70a, Djhsp70b, Djhsp70c, and Djhsp70d of the HSP70s, are selected from the transcriptome database, because of their high expression levels in planarians. We then study the biological roles of each gene by conducting various experimental techniques, including RNAi, RT-PCR, WISH, Whole-mount immunostaining and TUNEL. The results show: (1) External stressors, such as temperature, tissue damage and ionic liquid upregulate the expression of Djhsp70s significantly. (2) The gene expression of Djhsp70s in planarians exhibits dynamic patterns. According to the result of WISH, the Djhsp70s are mainly expressed in parenchymal tissues on both sides of the body as well as blastema. It is consistent with the data of qRT-PCR. (3) After RNA interference of Djhsp70s, the worms experience cephalic regression and lysis, body curling, stagnant regeneration and death. (4) Knockdown of Djhsp70s affect the cell proliferation and apoptosis. These results suggest that Djhsp70s are not only conserved in cytoprotection, but involved in homeostasis maintenance and regeneration process by regulating coordination of cell proliferation and apoptosis in planarians.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Evaluation of deep convolutional neural networks for in situ hybridization gene expression image representation.

High resolution in situ hybridization (ISH) images of the brain capture spatial gene expression at cellular resolution. These spatial profiles are key to understanding brain organization at the molecular level. Previously, manual qualitative scoring and informatics pipelines have been applied to ISH images to determine expression intensity and pattern. To better capture the complex patterns of gene expression in the human cerebral cortex, we applied a machine learning approach. We propose gene re-identification as a contrastive learning task to compute representations of ISH images. We train our model on an ISH dataset of ~1,000 genes obtained from postmortem samples from 42 individuals. This model reaches a gene re-identification rate of 38.3%, a 13x improvement over random chance. We find that the learned embeddings predict expression intensity and pattern. To test generalization, we generated embeddings in a second dataset that assayed the expression of 78 genes in 53 individuals. In this set of images, 60.2% of genes are re-identified, suggesting the model is robust. Importantly, this dataset assayed expression in individuals diagnosed with schizophrenia. Gene and donor-specific embeddings from the model predict schizophrenia diagnosis at levels similar to that reached with demographic information. Mutations in the most discriminative gene, Sodium Voltage-Gated Channel Beta Subunit 4 (SCN4B), may help understand cardiovascular associations with schizophrenia and its treatment. We have publicly released our source code, embeddings, and models to spur further application to spatial transcriptomics. In summary, we propose and evaluate gene re-identification as a machine learning task to represent ISH gene expression images.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.

It can be challenging to maintain tissue integrity using established histology protocols. Here, we describe a protocol composed of Hartman's fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure. For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

FDA-Approved Drug Screening for Compounds That Facilitate Hematopoietic Stem and Progenitor Cells (HSPCs) Expansion in Zebrafish.

Hematopoietic stem cells (HSCs) are a specialized subset of cells with self-renewal and multilineage differentiation potency, which are essential for their function in bone marrow or umbilical cord blood transplantation to treat blood disorders. Expanding the hematopoietic stem and progenitor cells (HSPCs) ex vivo is essential to understand the HSPCs-based therapies potency. Here, we established a screening system in zebrafish by adopting an FDA-approved drug library to identify candidates that could facilitate HSPC expansion. To date, we have screened 171 drugs of 7 categories, including antibacterial, antineoplastic, glucocorticoid, NSAIDS, vitamins, antidepressant, and antipsychotic drugs. We found 21 drugs that contributed to HSPCs expansion, 32 drugs' administration caused HSPCs diminishment and 118 drugs' treatment elicited no effect on HSPCs amplification. Among these drugs, we further investigated the vitamin drugs ergocalciferol and panthenol, taking advantage of their acceptability, limited side-effects, and easy delivery. These two drugs, in particular, efficiently expanded the HSPCs pool in a dose-dependent manner. Their application even mitigated the compromised hematopoiesis in an ikzf1-/- mutant. Taken together, our study implied that the larval zebrafish is a suitable model for drug repurposing of effective molecules (especially those already approved for clinical use) that can facilitate HSPCs expansion.

Cell Types and Synapses Expressing the SNARE Complex Regulating Proteins Complexin 1 and Complexin 2 in Mammalian Retina.

Complexins (Cplxs) 1 to 4 are components of the presynaptic compartment of chemical synapses where they regulate important steps in synaptic vesicle exocytosis. In the retina, all four Cplxs are present, and while we know a lot about Cplxs 3 and 4, little is known about Cplxs 1 and 2. Here, we performed in situ hybridization experiments and bioinformatics and exploited Cplx 1 and Cplx 2 single-knockout mice combined with immunocytochemistry and light microscopy to characterize in detail the cell type and synapse-specific distribution of Cplx 1 and Cplx 2. We found that Cplx 2 and not Cplx 1 is the main isoform expressed in normal and displaced amacrine cells and ganglion cells in mouse retinae and that amacrine cells seem to operate with a single Cplx isoform at their conventional chemical synapses. Surprising was the finding that retinal function, determined with electroretinographic recordings, was altered in Cplx 1 but not Cplx 2 single-knockout mice. In summary, the results provide an important basis for future studies on the function of Cplxs 1 and 2 in the processing of visual signals in the mammalian retina.

A protocol for whole-mount immuno-coupled hybridization chain reaction (WICHCR) in zebrafish embryos and larvae.

Characterizing mRNA and protein expression with temporal and spatial resolution is a valuable component of nearly every developmental study. Here, we describe a protocol that combines in situ hybridization chain reaction (HCR) and immunofluorescence, allowing for the detection of mRNAs and proteins simultaneously, in zebrafish embryos and larvae. This protocol expands the flexibility of multiplexed HCR by coupling it with traditional immunofluorescence detection. For complete details on the use and execution of this protocol, please refer to Choi et al. (2010, 2016, 2018) and Howard et al. (2021).

Fluorescent in situ mRNA detection in the adult mouse cochlea.

Ossification and the delicateness of the cochlear duct make histologic assessments of the mature cochlea a challenging endeavor. Treatments to soften the bone facilitate sectioning and dissection of the cochlear duct but limit in situ mRNA detection in such specimens. Here, we provide a protocol for in situ mRNA detection using hybridization chain reaction in whole-mount preparations of the adult mouse cochlea. We show examples for multi-probe detection of different mRNAs and describe combination of this method with conventional immunohistochemistry.

Optimized protocols for in situ hybridization, immunohistochemistry, and immunofluorescence on skeletal tissue.

Assessment of gene and protein expression in tissue sections is instrumental in medical research. However, this is often challenging to perform on skeletal tissues that require prolonged decalcification and have poor adhesion to slides. In this study, we optimized selected steps of in situ hybridization (ISH), immunohistochemistry (IHC), and immunofluorescence (IF) for formalin fixed and decalcified skeletal tissues. Sections from distal femur of 6-, 8- and 14- week-old rats injected with BrdU with or without a hemizygous eGFP transgene expressed under the control of a ubiquitous promotor were used. We report that proteinase K digestion is critical for the sensitivity of ISH, as concentrations that were too strong and too mild both resulted in loss of signal. In addition, intensified RNase A digestion removed nonspecific riboprobe-mRNA hybrids. Furthermore, enzymatic antigen retrieval using proteinase K provided more consistent results in IHC and can therefore be a useful alternative to heat induced epitope retrieval (HIER) for skeletal tissues where such treatment often damages the morphology. A mild proteinase K digestion also improved IF detection of GFP and worked well for double labeling IF of GFP and osteocalcin on frozen sections of formalin fixed and decalcified rat bones while maintaining morphology. In summary, this study provides strategies to improve protocols for enzymatic digestion in ISH, IHC, and IF for skeletal tissues and also demonstrates the importance of careful optimization and validation with the use of these techniques.

IL-6 expression helps distinguish Castleman's disease from IgG4-related disease in the lung.

BACKGROUND: It is difficult to distinguish between multicentric Castleman's disease (MCD) and IgG4-related lung disease (IgG4-LD), an IgG4-related disease (IgG4-RD) in the lung. METHODS: We focused on IL-6, which is elevated in MCD, to distinguish between MCD and IgG4-LD by RNAscope, a highly sensitive RNA in situ method. Six cases of MCD and four cases of IgG4-LD were selected. RESULTS: In all cases of MCD and IgG4-LD, 10 or more IgG4-positive cells were found in one high-power field. All MCD cases were inconsistent with the pathological IgG4-related comprehensive diagnostic criteria, but 2 of 6 cases had an IgG4/IgG ratio greater than 40%. In all IgG4-LD cases, histological features were consistent with the pathological IgG4-RD comprehensive diagnostic criteria. IL-6 expression was observed in all MCD and IgG4-LD cases except for one IgG4-LD biopsy. IL-6-expressing cells were mainly identified in the stroma. Sites of IL-6 expression were not characteristic and were sparse. IL-6 expression tended to be higher in MCD compared with IgG4-LD. A positive correlation was found between the IL-6 H-score and serum IL-6 level. CONCLUSION: Differences in IL-6 expression may help distinguish between MCD and IgG4-LD. In addition, the presence of high IL-6 levels may help elucidate the pathological mechanisms of IgG4-LD.

Single-Cell Imaging of m6 A Modified RNA Using m6 A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6 AISH-PLA).

N6 -methyladenosine (m6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m6 A-specific in situ hybridization mediated proximity ligation assay (m6 AISH-PLA) for cellular imaging of m6 A RNA, allowing to identify m6 A modification at specific location in RNAs and image m6 A RNA with single-cell and single-molecule resolution. Using m6 AISH-PLA, we investigated the m6 A level and subcellular location of HSP70 RNA103-m6 A in response to heat shock stress, and found an increased m6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6 AISH-PLA can serve in the study of m6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Detection of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by In Situ Hybridization (RNAscope®).

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope® to detect RVFV infection within the mosquito species, Culex pipiens, derived from the United Kingdom was demonstrated. Extensive RVFV replication was detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles.

Detection of lncRNA by LNA-Based In Situ Hybridization in Paraffin-Embedded Cancer Cell Spheroids.

Cancer cell spheroids are considered important preclinical tools to evaluate the efficacy of new drugs. In cancer cell spheroids, the cells assemble and grow in 3D structures with cell contact interactions that are partly impermeable, which leads to central hypoxia and necrosis. The cell spheroids thus possess several features identified in clinical tumors. Not only will the effect and behavior of therapeutic drugs in 3D cell spheroids be affected more similarly than in cells grown on culture plates, but molecular interactions and signaling pathways in cells are also more likely to mimic the in vivo situation. The monitoring of various biomarkers including lncRNAs in 3D cell spheroids is important to assess a potentially induced phenotype in the cells and the effects of drugs. Specifically, for lncRNAs, in situ localization can be done using locked nucleic acid (LNA) probe technology. Here we present a protocol for preparation of cell spheroids for use in LNA probe-based in situ hybridization to study lncRNA expression in paraffin embedded 3D cancer cell spheroids.