Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1–18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant which exhibits multiple tissue defects and harbors a ∼10 Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner (Df(Chr05:sco)z207) could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or for study of the many genes lost in the mutant.

New Functions of Low-Molecular-Weight Hyaluronic Acid on Epidermis Filaggrin Production and Degradation

Hyaluronic acid (HA) is a high-molecular-weight polysaccharide with high moisturizing power. It is composed of repeating disaccharides of N-acetyl-D-glucosamine and D-glucuronic acid. Low-molecular-weight hyaluronan (LMHA) is obtained by changing the molecular weight or modifying the functional groups of HA and is commonly used together with HA in cosmetics. The objective of this study was to determine whether LMHA promotes the synthesis of filaggrin (FLG). We also investigated whether LMHA activates FLG-degrading enzymes. Three-dimensional (3D) models of the human epidermis were cultured with LMHA. Real-time PCR was used to quantify the mRNA levels of profilaggrin (proFLG), involucrin (IVL), and FLG-degrading enzymes. FLG protein levels were measured by fluorescent antibody staining and Western blotting. The mRNA was quantified using a 3D epidermis model, and it was observed that the mRNA levels of proFLG, IVL, caspase-14 (CASP14), and bleomycin hydrolase were increased by the application of LMHA. Immunofluorescence results showed an increase in FLG proteins, and results from experiments using 3D epidermis models showed that LMHA increased the activity of CASP14. This suggests that the topical application of LMHA would result in an increase in natural moisturizing factor and promote moisturization of the stratum corneum.

Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the ParsortixTM system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.

The Normal and Fibrotic Mouse Lung in Situ Classified by High Dimensional Single Cell Analysis on Routinely Processed Tissue

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.

Differential expression in humans of the viral entry receptor ACE2 compared with the short deltaACE2 isoform lacking SARS-CoV-2 binding sites

ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent ‘Long COVID’. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.

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).

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

ONLY A SMALL FRACTION OF CELLS PRODUCE ASSEMBLED CAPSIDS DURING TRANSFECTION-BASED MANUFACTURING OF ADENO-ASSOCIATED VIRUS VECTORS

Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.