Shared and Compartment-Specific Processes in Nucleus Pulposus and Annulus Fibrosus During Intervertebral Disc Degeneration.

Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFß/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours.

OBJECTIVES: CHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A on α7nAChR agonist responses, osteoarthritis (OA) severity and pain behaviours and investigated mechanisms. METHODS: Transgenic CHRFAM7A (TgCHRFAM7A) mice were used to determine the impact of CHRFAM7A on knee OA histology, pain severity in OA and other pain models, response to nAchR agonist and IL-1ß. Mouse and human cells were used for mechanistic studies. RESULTS: Transgenic (Tg) TgCHRFAM7A mice developed more severe structural damage and increased mechanical allodynia than wild type (WT) mice in the destabilisation of medial meniscus model of OA. This was associated with a decreased suppression of inflammation by α7nAchR agonist. TgCHRFAM7A mice displayed a higher basal sensitivity to pain stimuli and increased pain behaviour in the monoiodoacetate and formalin models. Dorsal root ganglia of TgCHRFAM7A mice showed increased macrophage infiltration and expression of the chemokine fractalkine and also had a compromised antinociceptive response to the α7nAchR agonist nicotine. Both native CHRNA7 and CHRFAM7A subunits were expressed in human joint tissues and the CHRFAM7A/CHRNA7 ratio was increased in OA cartilage. Human chondrocytes with two copies of CHRFAM7A had reduced anti-inflammatory responses to nicotine. CONCLUSION: CHRFAM7A is an aggravating factor for OA-associated inflammation and tissue damage and a novel genetic risk factor and therapeutic target for pain.

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents.

Senescent cell population with ZEB1 transcription factor as its main regulator promotes osteoarthritis in cartilage and meniscus.

OBJECTIVES: Single-cell level analysis of articular cartilage and meniscus tissues from human healthy and osteoarthritis (OA) knees. METHODS: Single-cell RNA sequencing (scRNA-seq) analyses were performed on articular cartilage and meniscus tissues from healthy (n=6, n=7) and OA (n=6, n=6) knees. Expression of genes of interest was validated using immunohistochemistry and RNA-seq and function was analysed by gene overexpression and depletion. RESULTS: scRNA-seq analyses of human knee articular cartilage (70 972 cells) and meniscus (78 017 cells) identified a pathogenic subset that is shared between both tissues. This cell population is expanded in OA and has strong OA and senescence gene signatures. Further, this subset has critical roles in extracellular matrix (ECM) and tenascin signalling and is the dominant sender of signals to all other cartilage and meniscus clusters and a receiver of TGFß signalling. Fibroblast activating protein (FAP) is also a dysregulated gene in this cluster and promotes ECM degradation. Regulons that are controlled by transcription factor ZEB1 are shared between the pathogenic subset in articular cartilage and meniscus. In meniscus and cartilage cells, FAP and ZEB1 promote expression of genes that contribute to OA pathogenesis, including senescence. CONCLUSIONS: These single-cell studies identified a senescent pathogenic cell cluster that is present in cartilage and meniscus and has FAP and ZEB1 as main regulators which are novel and promising therapeutic targets for OA-associated pathways in both tissues.

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

OBJECTIVES: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis. METHODS: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq). RESULTS: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of Klf2 in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2. CONCLUSIONS: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.

Extended survival versus accelerated rejection of nonhuman primate islet allografts: Effect of mesenchymal stem cell source and timing.

Mesenchymal stem cells (MSC) have been shown to be immunomodulatory, tissue regenerative, and graft promoting; however, several questions remain with regard to ideal MSC source and timing of administration. In this study, we utilized a rigorous preclinical model of allogeneic islet cell transplantation, incorporating reduced immune suppression and near to complete mismatch of major histocompatibility antigens between the diabetic cynomolgus monkey recipient and the islet donor, to evaluate both the graft promoting impact of MSC source, that is, derived from the islet recipient, the islet donor or an unrelated third party as well as the impact of timing. Co-transplant of MSC and islets on post-operative day 0, followed by additional IV MSC infusions in the first posttransplant month, resulted in prolongation of rejection free and overall islet survival and superior metabolic control for animals treated with recipient as compared to donor or third-party MSC. Immunological analyses demonstrated that infusion of MSC from either source did not prevent alloantibody formation to the islet or MSC donor; however, treatment with recipient MSC resulted in significant downregulation of memory T cells, decreased anti-donor T cell proliferation, and a trend toward increased Tregulatory:Tconventional ratios.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

BACKGROUND: Tumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR. METHODS: The novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor (EGFR) exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 (HER2) amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for EGFR and HER2, respectively. RESULTS: By removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For EGFR 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for EGFR 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all EGFR mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR. CONCLUSIONS: This novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.

Increased sensitivity using real-time dPCR for detection of SARS-CoV-2.

A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.

Feasibility and Comparison Study of Fecal Sample Collection Methods in Healthy Volunteers and Solid Organ Transplant Recipients Using 16S rRNA and Metagenomics Approaches.

The human microbiome encompasses a variety of microorganisms that change dynamically and are in close contact with the body. The microbiome influences health and homeostasis, as well as the immune system, and any significant change in this equilibrium (dysbiosis) triggers both acute and chronic health conditions. Microbiome research has surged, in part, due to advanced sequencing technologies enabling rapid, accurate, and cost-effective identification of the microbiome. A major prerequisite for stool sample collection to study the gut microbiome in longitudinal prospective studies requires standardized protocols that can be easily replicated. However, there are still significant bottlenecks to stool specimen collection that contribute to low patient retention rates in microbiome studies. These barriers are further exacerbated in solid organ transplant recipients where diarrhea is estimated to occur in up to half the patient population. We sought to test two relatively easy sample collection methods (fecal swab and wipes) and compare them to the more cumbersome "gold" standard collection method (scoop) using two different sequencing technologies (16S ribosomal RNA sequencing and shotgun metagenomics). Our comparison of the collection methods shows that both the swabs and the wipes are comparable to the scoop method in terms of bacterial abundance and diversity. The swabs, however, were closer in representation to the scoop and were easier to collect and process compared to the wipes. Potential contamination of the swab and the wipe samples by abundant skin commensals was low in our analysis. Comparison of the two sequencing technologies showed that they were complementary, and that 16S sequencing provided enough coverage to detect and differentiate between bacterial species identified in the collected samples. Our pilot study demonstrates that alternative collection methods for stool sampling are a viable option in clinical applications, such as organ transplant studies. The use of these methods may result in better patient retention recruitment rates in serial microbiome studies.

Identification of Key Determinants of Staphylococcus aureus Vaginal Colonization.

Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.

Seasonal dynamics of DNA and RNA viral bioaerosol communities in a daycare center.

BACKGROUND: Viruses play an important role in ecosystems, including the built environment (BE). While numerous studies have characterized bacterial and fungal microbiomes in the BE, few have focused on the viral microbiome (virome). Longitudinal microbiome studies provide insight into the stability and dynamics of microbial communities; however, few such studies exist for the microbiome of the BE, and most have focused on bacteria. Here, we present a longitudinal, metagenomic-based analysis of the airborne DNA and RNA virome of a children's daycare center. Specifically, we investigate how the airborne virome varies as a function of season and human occupancy, and we identify possible sources of the viruses and their hosts, mainly humans, animals, plants, and insects. RESULTS: Season strongly influenced the airborne viral community composition, and a single sample collected when the daycare center was unoccupied suggested that occupancy also influenced the community. The pattern of influence differed between DNA and RNA viromes. Human-associated viruses were much more diverse and dominant in the winter, while the summertime virome contained a high relative proportion and diversity of plant-associated viruses. CONCLUSIONS: This airborne microbiome in this building exhibited seasonality in its viral community but not its bacterial community. Human occupancy influenced both types of communities. By adding new data about the viral microbiome to complement burgeoning information about the bacterial and fungal microbiomes, this study contributes to a more complete understanding of the airborne microbiome.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.

A genome-wide survey of mutations in the Jurkat cell line.

BACKGROUND: The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some expression irregularities were observed, raising doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. RESULTS: Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. CONCLUSIONS: The Jurkat cell line harbors many mutations that are associated with cancer and contribute to Jurkat's unique characteristics. Genes with damaging mutations in the Jurkat cell line are involved in T-cell receptor signaling (PTEN, INPP5D, CTLA4, and SYK), maintenance of genome stability (TP53, BAX, and MSH2), and O-linked glycosylation (C1GALT1C1). This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.

Primer Extension, Capture, and On-Bead cDNA Ligation: An Efficient RNAseq Library Prep Method for Determining Reverse Transcription Termination Sites.

In this chapter, we describe a method for making Illumina-compatible sequencing libraries from RNA. This protocol can be used for standard RNAseq analysis for detecting differentially expressed genes. In addition, this protocol is ideally suited for adapting to RIPseq, 5'-RACE, RNA structural probing, nascent RNA sequencing, and other protocols where polymerase termination sites need to be profiled. The utilization of solid-phase bead chemistries facilitates simple workflow and efficient library yields.

Microdroplet PCR for Highly Multiplexed Targeted Bisulfite Sequencing.

Many methods exist for examining CpG DNA methylation. However, many of these are qualitative, laborious to apply to a large number of genes simultaneously, or are not easy to target to specific regions of interest. Microdroplet PCR-based bisulfite sequencing allows for quantitative single base resolution analysis of investigator selected regions of interest. Following bisulfite conversion of genomic DNA, targeted microdroplet PCR is conducted with custom primer libraries. Samples are then fragmented, concatenated, and sequenced by high-throughput sequencing. The most recent technology allows for this method to be conducted with as little as 250 ng of bisulfite-converted DNA. The primary advantage of this method is the ability to hand-select the targeted regions covered by up to 10,000 amplicons of 500-600 bp. Moreover, the nature of microdroplet PCR virtually eliminates PCR bias and allows for the amplification of all targets simultaneously in a single tube.

Allele-specific wild-type TP53 expression in the unaffected carrier parent of children with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder where an oncogenic TP53 germline mutation is passed from parent to child. Tumor protein p53 is a key tumor suppressor regulating cell cycle arrest in response to DNA damage. Paradoxically, some mutant TP53 carriers remain unaffected, while their children develop cancer within the first few years of life. To address this paradox, response to UV stress was compared in dermal fibroblasts (dFb) from an affected LFS patient vs. their unaffected carrier parent. UV induction of CDKN1A/p21, a regulatory target of p53, in LFS patient dFb was significantly reduced compared to the unaffected parent. UV exposure also induced significantly greater p53[Ser15]-phosphorylation in LFS patient dFb, a reported property of some mutant p53 variants. Taken together, these results suggested that unaffected parental dFb may express an increased proportion of wild-type vs. mutant p53. Indeed, a significantly increased ratio of wild-type to mutant TP53 allele-specific expression in the unaffected parent dFb was confirmed by RT-PCR-RFLP and RNA-seq analysis. Hence, allele-specific expression of wild-type TP53 may allow an unaffected parent to mount a response to genotoxic stress more characteristic of homozygous wild-type TP53 individuals than their affected offspring, providing protection from the oncogenesis associated with LFS.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.