Single-Cell Analysis and Tracking of Antigen-Specific T Cells: Integrating Paired Chain AIRR-Seq and Transcriptome Sequencing: A Method by the AIRR Community.

Single-cell adaptive immune receptor repertoire sequencing (scAIRR-seq) offers the possibility to access the nucleotide sequences of paired receptor chains from T-cell receptors (TCR) or B-cell receptors (BCR ). Here we describe two protocols and the downstream bioinformatic approaches that facilitate the integrated analysis of paired T-cell receptor (TR ) alpha/beta (TRA /TRB ) AIRR-seq, RNA sequencing (RNAseq), immunophenotyping, and antigen-binding information. To illustrate the methodologies with a use case, we describe how to identify, characterize, and track SARS-CoV-2-specific T cells over multiple time points following infection with the virus. The first method allows the analysis of pools of memory CD8+ cells, identifying expansions and contractions of clones of interest. The second method allows the study of rare or antigen-specific cells and allows studying their changes over time.

Genetic ancestry, admixture, and population structure in rural Dominica.

The Caribbean is a genetically diverse region with heterogeneous admixture compositions influenced by local island ecologies, migrations, colonial conflicts, and demographic histories. The Commonwealth of Dominica is a mountainous island in the Lesser Antilles historically known to harbor communities with unique patterns of migration, mixture, and isolation. This community-based population genetic study adds biological evidence to inform post-colonial narrative histories in a Dominican horticultural village. High density single nucleotide polymorphism data paired with a previously compiled genealogy provide the first genome-wide insights on genetic ancestry and population structure in Dominica. We assessed family-based clustering, inferred global ancestry, and dated recent admixture by implementing the fastSTRUCTURE clustering algorithm, modeling graph-based migration with TreeMix, assessing patterns of linkage disequilibrium decay with ALDER, and visualizing data from Dominica with Human Genome Diversity Panel references. These analyses distinguish family-based genetic structure from variation in African, European, and indigenous Amerindian admixture proportions, and analyses of linkage disequilibrium decay estimate admixture dates 5-6 generations (~160 years) ago. African ancestry accounts for the largest mixture components, followed by European and then indigenous components; however, our global ancestry inferences are consistent with previous mitochondrial, Y chromosome, and ancestry marker data from Dominica that show uniquely higher proportions of indigenous ancestry and lower proportions of African ancestry relative to known admixture in other French- and English-speaking Caribbean islands. Our genetic results support local narratives about the community's history and founding, which indicate that newly emancipated people settled in the steep, dense vegetation along Dominica's eastern coast in the mid-19th century. Strong genetic signals of post-colonial admixture and family-based structure highlight the localized impacts of colonial forces and island ecologies in this region, and more data from other groups are needed to more broadly inform on Dominica's complex history and present diversity.

Angiosomal Vascular Occlusions, Deep-Tissue Pressure Injuries, and Competing Theories: A Case Report.

ABSTRACT: Compression of the soft tissue between a support surface and a bony prominence has long been the accepted primary mechanism of pressure injury (PrI) formation, with the belief that said compression leads to capillary occlusion, ischemia, and tissue necrosis. This explanation presupposes an "outside-in" pathophysiologic process of tissue damage originating at the local capillary level. Despite advances in prevention protocols, there remains a stubbornly consistent incidence of severe PrIs including deep-tissue injuries, the latter usually evolving into stage 4 PrIs with exposed bone or tendon. This article presents just such a perioperative case with the aim of providing further evidence that these more severe PrIs may result from ischemic insults of a named vessel within specific vascular territories (labeled as angiosomes). Pressure is indeed a factor in the formation of severe PrIs, but these authors postulate that the occlusion occurred at the level of a named artery proximal to the lesion. This vascular event was likely attributable to low mean arterial pressure. The authors suggest that the terminology proposed three decades ago to call both deep-tissue injuries and stage 4 PrIs "vascular occlusion pressure injuries" should be the topic of further research and expert consensus.

Drug reaction with eosinophilia and systemic symptoms in a child with N-methyl-d-aspartate receptor encephalitis.

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a rare but potentially life-threatening disorder that can be seen as a side effect of commonly used medications, particularly those in the anti-epileptic classes. Described here is a 10-year-old boy with newly diagnosed N-methyl-d-aspartate receptor encephalitis who presented for inpatient rehabilitation and developed a sudden-onset rash with coagulopathy. Cessation of the offending agents resulted in resolution of the symptoms.

REDESIGN: RDF-based Differential Signaling Framework for Precision Medicine Analytics.

Pathway-based analysis holds promise to be instrumental in precision and personalized medicine analytics. However, the majority of pathway-based analysis methods utilize "fixed" or "rigid" data sets that limit their ability to account for complex biological inter-dependencies. Here, we present REDESIGN: RDF-based Differential Signaling Pathway informatics framework. The distinctive feature of the REDESIGN is that it is designed to run on "flexible" ontology-enabled data sets of curated signal transduction pathway maps to uncover high explanatory differential pathway mechanisms on gene-to-gene level. The experiments on two morphoproteomic cases demonstrated REDESIGN's capability to generate actionable hypotheses in precision/personalized medicine analytics.

Recapitulating and Correcting Marfan Syndrome in a Cellular Model.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in FBN1 gene, which encodes a key extracellular matrix protein FIBRILLIN-1. The haplosufficiency of FBN1 has been implicated in pathogenesis of MFS with manifestations primarily in cardiovascular, muscular, and ocular tissues. Due to limitations in animal models to study the late-onset diseases, human pluripotent stem cells (PSCs) offer a homogeneic tool for dissection of cellular and molecular pathogenic mechanism for MFS in vitro. Here, we first derived induced PSCs (iPSCs) from a MFS patient with a FBN1 mutation and corrected the mutation, thereby generating an isogenic "gain-of-function" control cells for the parental MFS iPSCs. Reversely, we knocked out FBN1 in both alleles in a wild-type (WT) human embryonic stem cell (ESC) line, which served as a loss-of-function model for MFS with the WT cells as an isogenic control. Mesenchymal stem cells derived from both FBN1-mutant iPSCs and -ESCs demonstrated reduced osteogenic differentiation and microfibril formation. We further demonstrated that vascular smooth muscle cells derived from FBN1-mutant iPSCs showed less sensitivity to carbachol as demonstrated by contractility and Ca2+ influx assay, compared to the isogenic controls cells. These findings were further supported by transcriptomic anaylsis of the cells. Therefore, this study based on both gain- and loss-of-function approaches confirmed the pathogenetic role of FBN1 mutations in these MFS-related phenotypic changes.

Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

Hypermethylation of antisense long noncoding RNAs in acute lymphoblastic leukemia.

AIM: Long noncoding RNAs serve critical regulatory functions highly specific for a tissue and its developmental stage. Antisense long ncRNA (AS-lncRNA) methylation changes in acute lymphoblastic leukemia (ALL) versus normal pre-B-cell lymphoblasts were evaluated to identify potential differential methylation in this group of genes. MATERIALS & METHODS: The methylome of ALL and normal lymphoblasts was examined by the methylated CpG island recovery assay followed by NGS. CONCLUSION: The potential effect of trans regulation by AS-lncRNA through DNA/RNA binding is significant as sequence alignment analysis of the 25 most differentially methylated AS-lncRNAs revealed 368 genes containing highly similar sequences with a median nucleotide identity of 90.8% and binding span of 122 base pairs. Regulation of biological processes and anatomical structure development were over represented. ALL classification schemes based on AS-lncRNA methylation can provide new insights into its pathogenesis and treatment.

The expression of RUNDC3B is associated with promoter methylation in lymphoid malignancies.

DNA methylation is an epigenetic modification that plays an important role in the regulation of gene expression. The function of RUNDC3B has yet to be determined, although its dysregulated expression has been associated with malignant potential of both breast and lung carcinoma. To elucidate the potential of using DNA methylation in RUNDC3B as a biomarker in lymphoid malignancies, the methylation status of six regions spanning the CpG island in the promoter region of RUNDC3B was determined in cancer cell lines. Lymphoid malignancies were found to have more prominent methylation and did not express RUNDC3B compared with myeloid malignancies and solid tumours, supporting the potential use of DNA methylation in this region as a biomarker for lymphoid malignancies. RUNDC3B contains a RUN domain in its N-terminal region that mediates interaction with Rap2, an important component of the mitogen-activated protein kinase (MAPK) cascade, which regulates cellular proliferation and differentiation. The protein sequence of RUNDC3B also contains characteristic binding sites for MAPK intermediates. Therefore, it is possible that RUNDC3B serves as a mediator between Rap2 and the MAPK signalling cascade. Three genes with MAPK-inducible expression were downregulated in a methylated leukaemia cell line (HSPA5, Jun and Fos). Jun and Fos combine to form the activating protein 1 transcription factor, and loss of this factor is associated with the dysregulation of genes involved in differentiation and proliferation. We hypothesize that the loss of RUNDC3B secondary to aberrant hypermethylation of the early growth response 3 transcription factor binding site results in dysregulated MAPK signalling and carcinogenesis in lymphoid malignancies. © 2015 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.

Targeted nanoconjugate co-delivering siRNA and tyrosine kinase inhibitor to KRAS mutant NSCLC dissociates GAB1-SHP2 post oncogene knockdown.

A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation.

Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Obesity impairs γδ T cell homeostasis and antiviral function in humans.

Obese patients are susceptible to increased morbidity and mortality associated with infectious diseases such as influenza A virus. γδ T cells and memory αß T cells play key roles in reducing viral load by rapidly producing IFN-γ and lysing infected cells. In this article we analyze the impact of obesity on T lymphocyte antiviral immunity. Obese donors exhibit a reduction in γδ T cells in the peripheral blood. The severity of obesity negatively correlates with the number of γδ T cells. The remaining γδ T cells have a skewed maturation similar to that observed in aged populations. This skewed γδ T cell population exhibits a blunted antiviral IFN-γ response. Full γδ T cell function can be restored by potent stimulation with 1-Hydroxy-2-methyl-buten-4yl 4-diphosphate (HDMAPP), suggesting that γδ T cells retain the ability to produce IFN-γ. Additionally, γδ T cells from obese donors have reduced levels of IL-2Rα. IL-2 is able to restore γδ T cell antiviral cytokine production, which suggests that γδ T cells lack key T cell specific growth factor signals. These studies make the novel finding that the γδ T cell antiviral immune response to influenza is compromised by obesity. This has important implications for the development of therapeutic strategies to improve vaccination and antiviral responses in obese patients.

Identification of a human papillomavirus-associated oncogenic miRNA panel in human oropharyngeal squamous cell carcinoma validated by bioinformatics analysis of the Cancer Genome Atlas.

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV(+) OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV(+) OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV(+/-) OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.

Genome-wide DNA methylation analysis in precursor B-cells.

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182-200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs.

Prader-Willi syndrome (PWS), a disorder of genomic imprinting, is characterized by neonatal hypotonia, hypogonadism, small hands and feet, hyperphagia and obesity in adulthood. PWS results from the loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13. We have investigated the mechanism of repression of the maternal SNORD116 cluster and 116HG. Here, we report that the zinc-finger protein ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not the active paternal alleles. Knockdown of SETDB1 in PWS-specific induced pluripotent cells (iPSCs) causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at 116HG and corresponding accumulation of the active chromatin mark histone H3 lysine 4 dimethylation (H3K4me2). We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. This observation suggests that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region.

Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two neurodevelopmental disorders most often caused by deletions of the same region of paternally inherited and maternally inherited human chromosome 15q, respectively. AS is a single gene disorder, caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, while PWS is still considered a contiguous gene disorder. Rare individuals with PWS who carry atypical microdeletions on chromosome 15q have narrowed the critical region for this disorder to a 108 kb region that includes the SNORD116 snoRNA cluster and the Imprinted in Prader-Willi (IPW) non-coding RNA. Here we report the derivation of induced pluripotent stem cells (iPSCs) from a PWS patient with an atypical microdeletion that spans the PWS critical region. We show that these iPSCs express brain-specific portions of the transcripts driven by the PWS imprinting center, including the UBE3A antisense transcript (UBE3A-ATS). Furthermore, UBE3A expression is imprinted in most of these iPSCs. These data suggest that UBE3A imprinting in neurons only requires UBE3A-ATS expression, and no other neuron-specific factors. These data also suggest that a boundary element lying within the PWS critical region prevents UBE3A-ATS expression in non-neural tissues.

Developmental exposure of fetal ovaries and fetal germ cells to endometriosis in an endometriosis model causes differential gene expression in the preimplantation embryos of the first-generation and second-generation embryos.

OBJECTIVE: To characterize multigenerational gene expression anomalies in eight-cell stage embryos associated with developmental exposure to endometriosis. DESIGN: Using an endometriosis model in rats (F0 founder generation) to evaluate gene expression in F1 (fetal exposure) and F2 (fetal germ cell exposure) generation eight-cell stage embryos. SETTING: Laboratory. ANIMAL(S): Endometriosis model in rats (Endo) and controls (Sham). INTERVENTION(S): F0 Endo and Sham rats were bred; half the pregnant rats were killed on gestational day 3 to collect F1 eight-cell stage embryos and the others gestated to term (F1 females). Adult F1 females bred; F2 eight-cell embryos collected. MAIN OUTCOME MEASURE(S): Maintenance of differential gene expression in F1 and F2 generation eight-cell embryos in endometriosis. RESULT(S): Developmental exposure to endometriosis altered the gene signaling pathways, with changes found in apoptosis, the cell cycle process, the response to oxidative stress, negative regulation of molecular function, and RNA processing. The apoptotic genes Diablo, Casp3, Parp1, Cad, and Dnaja3 were increased and the Nfkbia transcripts were decreased in F1 Endo versus F1 Sham embryos. In F2 Endo versus Sham embryos, Casp3 and Cad were statistically significantly increased, and Parp1 and Nfkbia tended to be elevated. CONCLUSION(S): Fetal and germ cell exposure to endometriosis alters apoptotic gene expression in first- and second-generation eight-cell stage embryos, supporting the hypothesis of multigenerational inheritance resulting from exposure to endometriosis in utero.

Isolation of precursor B-cell subsets from umbilical cord blood.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation. This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

The properties of genome conformation and spatial gene interaction and regulation networks of normal and malignant human cell types.

The spatial conformation of a genome plays an important role in the long-range regulation of genome-wide gene expression and methylation, but has not been extensively studied due to lack of genome conformation data. The recently developed chromosome conformation capturing techniques such as the Hi-C method empowered by next generation sequencing can generate unbiased, large-scale, high-resolution chromosomal interaction (contact) data, providing an unprecedented opportunity to investigate the spatial structure of a genome and its applications in gene regulation, genomics, epigenetics, and cell biology. In this work, we conducted a comprehensive, large-scale computational analysis of this new stream of genome conformation data generated for three different human leukemia cells or cell lines by the Hi-C technique. We developed and applied a set of bioinformatics methods to reliably generate spatial chromosomal contacts from high-throughput sequencing data and to effectively use them to study the properties of the genome structures in one-dimension (1D) and two-dimension (2D). Our analysis demonstrates that Hi-C data can be effectively applied to study tissue-specific genome conformation, chromosome-chromosome interaction, chromosomal translocations, and spatial gene-gene interaction and regulation in a three-dimensional genome of primary tumor cells. Particularly, for the first time, we constructed genome-scale spatial gene-gene interaction network, transcription factor binding site (TFBS) - TFBS interaction network, and TFBS-gene interaction network from chromosomal contact information. Remarkably, all these networks possess the properties of scale-free modular networks.