CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Developing Monoclonal Antibodies for Immunohistochemistry

The experiences of a laboratory which pioneered the application of monoclonal antibodies to diagnostic histochemistry is described. This was achieved in four key steps: (1) Monoclonal antibodies were successfully produced to replace the difficult-to-produce and limited polyclonal antibodies available for immunohistochemistry. (2) Monoclonal antibodies were produced to improve the immunoenzymatic detection of bound antibodies, using immunoperoxidase or alkaline phosphatase, increasing sensitivity and allowing the use of two chromogens when applied together. The availability of a reliable alkaline phosphatase-based detection allowed the detection of antigens in tissues with high endogenous peroxidase. (3) Methodologies were developed to unmask antigens not detected in routinely processed paraffin-embedded tissue. (4) Synthetic peptides were used as immunising antigens for the direct production of specific molecules of diagnostic interest. This was expanded to include recombinant proteins. Many reacted with fixed tissue and recognised homologous molecules in other species. In addition to these developments, the laboratory promoted the collaboration and training of researchers to spread the expertise of monoclonal production for diagnosis.

Creation of a collection of different biological sample types from elderly patients to study the relationship of clinical, systemic, tissue and cellular biomarkers of accumulation of senescent cells during aging

With aging, tissue homeostasis and their effective recovery after damage is violated. It has been shown that this may be due to the excessive accumulation of senescent (SC) cells in various tissues, which leads to the activation of chronic sterile inflammation, tissue dysfunction and, as a result, to the development of age-related diseases. To assess the contribution of SC cells to human body aging and pathogenesis of such diseases, relevant biomarkers are studied. For successful translation into clinical practice of approaches aimed at regulating the SC cell content in various tissues, it is necessary to study the relationship between the established clinical biomarkers of aging and age-related diseases, systemic aging parameters, and SC biomarkers at the tissue and cellular levels.Aim. To develop and describe action algorithms for creating a biobank of samples obtained from patients aged >65 years in order to study biomarkers of SC cell accumulation.Material and methods. To collect samples, an interaction system was built between several research, clinical and infrastructure departments of a multidisciplinary medical center. At the stage of preanalytical training, regulatory legal acts were developed, including informed consent for patients, as well as protocols for each stage of the study.Results. A roadmap was formed with action algorithms for all participants in the study, as well as with a convenient and accessible system of annotations and storage of biological samples. To date, the collection includes biological samples of 7 different types (peripheral blood serum, formalin-fixed tissue samples and formalin fixed paraffin embedded tissue specimens, samples of different cells isolated from peripheral blood, skin and adipose tissue, samples of deoxyribonucleic and ribonucleic acids, cell secretome conditioned media) obtained from 82 patients. We accumulated relevant anamnestic, clinical and laboratory data, as well as the results of experimental studies to assess the SC cell biomarkers. Using the collection, the relationship between clinical, tissue and cellular biomarkers of SC cell accumulation was studied.Conclusion. The creation of a collection of biological samples at the molecular, cellular, tissue and organism levels from one patient provides great opportunities for research in the field of personalized medicine and the study of age-related disease pathogenesis.

Lymphoma versus Carcinoma and Other Collaborations

David Mason started his research career at a time when lymphoma diagnosis was based primarily on cellular morphology, clinical symptoms and special cytochemical stains using formalin fixed tissue sections. There were occasions, however, where the morphology was unhelpful, such as in the case of anaplastic or poorly differentiated tumours, where a distinction between lymphoma and a non-haematopoietic tumour was often problematical. Accurate diagnosis became even more important with the developments in the clinical staging of lymphoma and the availability of more effective treatments. One way forward to improve diagnosis was to use immunohistochemistry to study the antigens expressed by the tumor cells.

Tissue Optimisation Strategies for High Quality Ex Vivo Diffusion Imaging

Ex vivo diffusion imaging can be used to study healthy and pathological tissue microstructure in the rodent brain with microscopic resolution, providing a link between in vivo MRI and ex vivo microscopy techniques. A major challenge for the successful acquisition of ex vivo diffusion imaging data however are changes in the relaxivity and diffusivity of brain tissue following perfusion fixation. In this study we address this question by examining the combined effects of tissue preparation factors that influence image quality, including tissue rehydration time, fixative concentration and contrast agent concentration. We present an optimisation strategy combining these factors to manipulate the T1 and T2 of fixed tissue and maximise signal-to-noise ratio (SNR) efficiency. Applying this strategy in the rat brain resulted in a doubling of SNR and an increase in SNR per unit time by 135% in grey matter and 88% in white matter. This enabled the acquisition of excellent quality high-resolution (78 μm isotropic voxel size) diffusion data in less than 4 days, with a b-value of 4000 s/mm2, 30 diffusion directions and a field of view of 40 x 13 x 18 mm, using a 9.4 Tesla scanner with a standard 39 mm volume coil and a 660 mT/m 114 mm gradient insert. It was also possible to achieve comparable data quality for a standard resolution (150 μm) diffusion dataset in 21/4 hours. In conclusion, the optimisation strategy presented here may be used to improve signal quality, increase spatial resolution and/or allow faster acquisitions in preclinical ex vivo diffusion MRI experiments.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA)

Objective Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. Material and methods Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. Results In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. Conclusion Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Targeted ExSeq -- Tissue Preparation v2

This protocol accompanies Expansion Sequencing (ExSeq), and describes the tissue preparation for Targeted ExSeq. The steps described here are a generalization of the protocols used for figures 4-6 of the paper, and represent our recommendations for future users of the technology. Fig. 1 shows the structure of the protocol schematically. There are three possible tissue preparation routes described in this protocol that are applicable to different experimental systems. Option (A): harvesting tissue from model organisms that can be transcardially perfused with PFA, followed by sectioning using a vibratome. We typically use this workflow for work on mouse brain sections (see figures 4-5 of ExSeq paper). Option (B): transcardially perfusing with PFA, followed by cryoprotection and cryosectioning. We occasionally use this protocol for work on mouse brain sections. Option (C): snap-freezing fresh tissue (i.e., human tumor biopsy samples, or freshly harvested tissue from mice), followed by cryoprotection and cryosectioning (see figures 2 and 6 of ExSeq paper). The final result of options (A), (B), and (C) is the preparation of fixed tissue sections (either on a glass slide or free-floating). The protocols then briefly converge for optional antibody staining, treatment with LabelX, a chemical that enables anchoring of RNA to the expansion microscopy (ExM) hydrogel, followed by casting of the the ExM gel. There are minor differences in these steps between free-floating and slide-mounted tissue sections, which are noted in the individual steps. The next step, digestion, is tissue-type dependent and may require some optimization for your tissue type. We provide two potential options here: (1) a gentle digestion for tissues such as mouse brain, and (2) a harsh digestion for non-brain tissues such as tumor biopies. The protocols then converge again for the rest of the process. After digestion, the gels are expanded and re-embedded within a second non-expanding hydrogel to lock in the sample size. The carboxylates within the expansion gel are then chemically passivated, enabling enzymatic reactions to be performed within the gel. The samples are now ready for library preparation. In more detail: Steps 1-4 describe the preparation of reagents for downstream steps. The protocol begins either along options (A)/(B), the Transcardial PFA perfusion path (Step 5, continuing to vibratome sectioning in Steps 6-7 for option (A), or cryotome sectioning in Steps 9-10 for option (B)), or along option (C), the Fresh Frozen path (Step 8, continuing to cryotome sectioning in Steps 9-10). The protocols then converge for optional antibody staining (Step 11), followed by LabelX anchoring (Step 12), optional sample trimming (Step 13), and formation of the expansion microscopy gel (Step 14). The details of the digestion step are tissue-type dependent (Step 15). The protocol then concludes with expansion (Step 16), re-embedding (Step 17), passivation, and optional trimming (Steps 18-19). This protocol was used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP). The tissue for this work was collected (see HTAPP-specific tissue collection protocol). The tissue sections were then frozen, cryosectioned, post-fixed, and permeabilized (following steps 9-10). No antibody staining was performed (skipping optional step 11). The sections were then treated with LabelX and gelled (steps 12-14). The gels were then digested using the robust digestion option in steps 15-16. The samples were then re-embedded, passivated, and trimmed (following steps 17-19).

HCR of raphe serotonergic neurons in fixed tissue sections v1

This is an RNA fluorescent in-situ hybridization (FISH) protocol that utilizes hybridization chain reaction technology from Molecular Instruments. The protocol fluorescently labels different mRNAs (up to 4 different mRNAs) such that they become suitable for imaging. This protocol is designed specifically for fixed mouse brain tissue sections that contain raphe serotonergic neurons, but can be applied to other regions of the mouse brain as well.

A novel herpes-like virus inducing branchial lesions in a tiger shark (Galeocerdo cuvier)

A juvenile, male tiger shark ( Galeocerdo cuvier) developed illness after capture in Florida waters and was euthanized. Gross lesions included mild skin abrasions, hepatic atrophy, and coelomic fluid. Histologically, gills contained multifocal lamellar epithelial cell necrosis and thromboses. Scattered gill and esophageal epithelial cells had large, basophilic, intracytoplasmic, and intranuclear inclusions. Ultrastructurally, lamellar epithelial cells contained arrays of intracytoplasmic viral particles and scattered intranuclear nucleocapsids. Capsulated virions were 148 ± 11 nm with an 84 ± 8 nm icosahedral nucleocapsid and an electron-dense core. Next-generation sequencing, quantitative polymerase chain reaction, and in situ hybridization performed on formalin-fixed tissue confirmed a herpes-like viral infection. The viral polymerase shared 24% to 31% protein homology with other alloherpesviruses of fish, indicating a divergent virus. This report documents the pathologic findings associated with a molecularly confirmed novel herpes-like virus in an elasmobranch.

Applications of fluorescence-guided surgery across multiple tumor types using a near-infrared labeled EGFR antibody

Background As receptor-ligand based strategies emerge for surgical imaging, the relative importance of receptor expression in different tumor types is unknown. Near-infrared (NIR) labeled epidermal growth factor receptor (EGFR) antibody, panitumumab-IRDye800, was evaluated across three cancers to demonstrate its clinical utilities and a holistic analysis framework. Methods Thirty-one patients diagnosed with high-grade glioma (HGG, n=5, NCT03510208), head and neck squamous cell carcinoma (HNSCC, n=23, NCT02415881) or lung adenocarcinoma (LAC, n=3, NCT03582124) received systemic administration of 50 mg panitumumab-IRDye800 days prior to surgery. Intraoperative NIR laparoscopic or open-field images of the surgical field were acquired and tissue mimicking phantoms were constructed to identify optimal imaging conditions. Margin distance was correlated to fluorescence on resected specimen surface. Panitumumab-IRDye800 distribution was registered to histology in fixed tissue sections. Immunohistochemistry characterized EGFR expression. Results Intraoperative NIR imaging enhanced tumor contrast against surrounding healthy tissue by 5.2-fold, 3.4-fold and 1.4-fold in HGG, HNSCC and LAC, respectively. Imaging quality was optimal at the lowest gain possible under ambient light. Ex vivo NIR fluorescence identified 78-97% of at-risk resection margins, with 72-92% sensitivity and 67-96% specificity for tumor in fixed tissue sections. Intratumoral panitumumab-IRDye800 concentration correlated with total tumoral EGFR expression (HGG > HNSCC > LAC) and delivery barrier. Cellular EGFR expression (80%) and tumor cell density (3000 cells/mm2) was highest in HGG. Conclusions In multiple tumor types, EGFR-targeting in fluorescence-guided surgery translated to enhanced macroscopic tumor contrast and successful margin assessment despite disparate tumor cell density and heterogeneous delivery of pantimumab-IRDye800.