Multimodal single-cell datasets characterize antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection.

The immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we used multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after immunization with the mRNA vaccine BNT162b2. Our data indicated distinct subpopulations of CD8+ T cells, which reliably appeared 28 days after prime vaccination. Using a suite of cross-modality integration tools, we defined their transcriptome, accessible chromatin landscape and immunophenotype, and we identified unique biomarkers within each modality. We further showed that this vaccine-induced population was SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we identified these CD8+ T cell populations in scRNA-seq datasets from COVID-19 patients and found that their relative frequency and differentiation outcomes were predictive of subsequent clinical outcomes.

Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells.

Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.

New frontiers in modeling tuberous sclerosis with human stem cell-derived neurons and brain organoids.

Recent advances in human stem cell and genome engineering have enabled the generation of genetically defined human cellular models for brain disorders. These models can be established from a patient's own cells and can be genetically engineered to generate isogenic, controlled systems for mechanistic studies. Given the challenges of obtaining and working with primary human brain tissue, these models fill a critical gap in our understanding of normal and abnormal human brain development and provide an important complement to animal models. Recently, there has been major progress in modeling the neuropathophysiology of the canonical "mTORopathy" tuberous sclerosis complex (TSC) with such approaches. Studies using two- and three-dimensional cultures of human neurons and glia have provided new insights into how mutations in the TSC1 and TSC2 genes impact human neural development and function. Here we discuss recent progress in human stem cell-based modeling of TSC and highlight challenges and opportunities for further efforts in this area.

Genetically engineered human cortical spheroid models of tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC.

Widespread Translational Remodeling during Human Neuronal Differentiation.

Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identified mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3' UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation and a crucial role of 3' UTRs in driving cell-type-specific translation.

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.

Placental DNA methylation at term reflects maternal serum levels of INHA and FN1, but not PAPPA, early in pregnancy.

BACKGROUND: Early detection of pregnancies at risk of complications, such as intrauterine growth restriction (IUGR) and preeclampsia (PE), is critical for improved monitoring and preventative treatment to optimize health outcomes. We predict that levels of placental-derived proteins circulating in maternal blood reflect placental gene expression, which is associated with placental DNA methylation (DNAm) profiles. As such, placental DNAm profiling may be useful to distinguish pregnancies at risk of developing complications and correlation between DNAm and protein levels in maternal blood may give further evidence for a protein's use as a biomarker. However, few studies investigate all clinical parameters that may influence DNAm and/or protein expression, which can significantly affect the relationship between these measures. RESULTS: Candidate genes were chosen based on i) reported alterations of protein levels in maternal blood and ii) observed changes in placental DNAm (Ƨ > 0.05 and False Discovery Rate (FDR) <0.05) in pregnancies complicated by PE/IUGR. Fibronectin (FN1) enhancer DNAm and placental gene expression were inversely correlated (r = -0.88 p < 0.01). The same trend was observed between promoter DNAm and gene expression for INHBA and PAPPA, though not significant. INHBA and FN1 DNAm was associated with gestational-age corrected birth weight, while INHA levels were associated with fetal: placental weight ratio and FN1 level was associated with maternal body mass index (BMI). DNAm at the INHBA promoter in the term placenta was negatively correlated with second trimester maternal serum levels (r = -0.50 p = 0.01) and DNAm at the FN1 enhancer was negatively associated with third trimester maternal serum levels (r = -0.38, p = 0.009). However, a similar correlation was not found for PAPPA. CONCLUSIONS: These results show that establishing a correlation between altered DNAm in the term placenta and altered maternal serum levels of the corresponding protein, is affected by a number of factors. Nonetheless, the correlation between placental DNAm of INHBA/FN1 and maternal serum INHA/FN1 levels indicate that DNAm may be a useful tool to identify novel biomarkers for adverse pregnancy outcomes in some cases.

Genome-wide DNA methylation identifies trophoblast invasion-related genes: Claudin-4 and Fucosyltransferase IV control mobility via altering matrix metalloproteinase activity.

Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility.

Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Early onset pre-eclampsia is associated with altered DNA methylation of cortisol-signalling and steroidogenic genes in the placenta.

Placental cortisol is inactivated in normotensive pregnancies, but is frequently present in pre-eclampsia associated placentae. Since glucocorticoids are strongly associated with the programming of long-term health, we assessed DNA methylation of genes involved in cortisol signalling and bioavailability, and hormonal signalling in the placenta of normotensive and hypertensive pregnancies. Candidate genes/CpG sites were selected through analysis of Illumina Infinium HumanMethylation450 BeadChip array data on control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placental samples. DNA methylation was further quantified by bisulfite pyrosequencing in a larger cohort of control (n = 111) cases, in addition to EOPET (n = 19), late onset pre-eclampsia (LOPET; n = 18) and normotensive intrauterine growth restriction (nIUGR; n = 13) cases. DNA methylation (percentage points) was increased at CpG sites within genes encoding the glucocorticoid receptor (NR3C1 exon 1D promoter; +8.46%; P<0.01) and corticotropin releasing hormone (CRH) binding protein (CRHBP intron 3; +9.14%; P<0.05), and decreased within CRH (5' UTR; -4.30%; P = 0.11) in EOPET-associated placentae, but not in LOPET nor nIUGR cases, compared to controls. Differential DNA methylation was not observed among groups at the 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene promoter. Significant hypomethylation was observed in pre-eclampsia but not nIUGR placentae for steroidogenic genes, including CYP11A1 (exon1; EOPET; -9.66%; P<0.00001, and LOPET; -5.77%; P<0.001), 3ß-hydroxy-delta-5-steroid dehydrogenase type 1 (HSD3B1 exon 2; EOPET; -12.49%; P<0.00001, and LOPET; -6.88%; P<0.001), TEA domain family member 3 (TEAD3 intron 1; EOPET; -12.56%; P<0.00001) and CYP19 (placental-specific exon 1.1 promoter; EOPET; -10.62%, P<0.0001). These data represent dysregulation of the placental epigenome in pre-eclampsia related to genes involved in maintaining the hormonal environment during pregnancy and highlights particular susceptibility in the early onset syndrome.

The human placenta methylome.

Tissue-specific DNA methylation is found at promoters, enhancers, and CpG islands but also over larger genomic regions. In most human tissues, the vast majority of the genome is highly methylated (>70%). Recently, sequencing of bisulfite-treated DNA (MethylC-seq) has revealed large partially methylated domains (PMDs) in some human cell lines. PMDs cover up to 40% of the genome and are associated with gene repression and inactive chromatin marks. However, to date, only cultured cells and cancers have shown evidence for PMDs. Here, we performed MethylC-seq in full-term human placenta and demonstrate it is the first known normal tissue showing clear evidence of PMDs. We found that PMDs cover 37% of the placental genome, are stable throughout gestation and between individuals, and can be observed with lower sensitivity in Illumina 450K Infinium data. RNA-seq analysis confirmed that genes in PMDs are repressed in placenta. Using a hidden Markov model to map placental PMDs genome-wide and compare them to PMDs in other cell lines, we found that genes within placental PMDs have tissue-specific functions. For regulatory regions, methylation levels in promoter CpG islands are actually higher for genes within placental PMDs, despite the lower overall methylation of surrounding regions. Similar to PMDs, polycomb-regulated regions are hypomethylated but smaller and distinct from PMDs, with some being hypermethylated in placenta compared with other tissues. These results suggest that PMDs are a developmentally dynamic feature of the methylome that are relevant for understanding both normal development and cancer and may be of use as epigenetic biomarkers.

Hypoxia alters the epigenetic profile in cultured human placental trophoblasts.

The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension influences placental function, and is associated with preeclampsia, a condition displaying altered development of placental trophoblast. We hypothesized that oxygen tension affects villous trophoblast by modulation of gene expression through DNA methylation. We used the Infinium HumanMethylation450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under < 1%, 8% and 20% oxygen levels. We found no effect of oxygen tension on average DNA methylation for either cell phenotype, but a set of loci became hypermethylated in cytotrophoblasts exposed for 24 h to < 1% oxygen, as compared with those exposed to 8% or 20% oxygen. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally relevant genes including CD59, CFB, GRAM3 and ZNF217. Intriguingly, 70 out of the 147 CpGs that became hypermethylated in < 1% oxygen overlapped with CpG sites that became hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast.

Hypomethylation of the LEP gene in placenta and elevated maternal leptin concentration in early onset pre-eclampsia.

In pre-eclampsia, placental leptin is up-regulated and leptin is elevated in maternal plasma. To investigate potential epigenetic regulation of the leptin (LEP) gene in normal and complicated pregnancy, DNA methylation was assessed at multiple reported regulatory regions in placentae from control pregnancies (n=111), and those complicated by early onset pre-eclampsia (EOPET; arising <34 weeks; n=19), late onset pre-eclampsia (LOPET; arising ≥34 weeks; n=18) and normotensive intrauterine growth restriction (nIUGR; n=13). The LEP promoter was hypomethylated in EOPET, but not LOPET or nIUGR placentae, particularly at CpG sites downstream of the transcription start site (-10.1%; P<0.0001). Maternal plasma leptin was elevated in EOPET and LOPET (P<0.05), but not nIUGR, compared with controls. EOPET cases showed a trend towards biallelic LEP expression rather than skewed allelic expression observed in control placentae, suggesting that loss of normal monoallelic expression at the LEP locus is associated with hypomethylation, leading to increased overall LEP expression.

Absence of SYCP3 mutations in women with recurrent miscarriage with at least one trisomic miscarriage.

Mutations within the coding regions of the synaptonemal complex gene SYCP3 have previously been reported in women with recurrent miscarriage. The present study found no mutations in any of the coding exons or the intron/exon boundaries among 50 recurrent miscarriage patients with at least one documented trisomic miscarriage, suggesting that mutations in SYCP3 do not contribute significantly to risk for recurrent miscarriage through maternal meiotic nondisjunction.

Context-specific and/or context-free challenges and opportunities in writing scholarly reviews in health care management: a conceptual note.

Challenges and opportunities arise from the significantly different perspectives of context-specific versus context-free researchers and the literatures they contribute to. Reviews of one type or the other or both types of literatures may provide different understandings of the state of the art in a particular area of health care management. Suggestions for writing quality reviews are also included along with suggested topics for future reviews.

Medical group affiliations: interorganizational relationships and organizational performance.

The hyperturbulent health care environment is causing health care organizations to create interorganizational relationships (IORs). This article reports on a study of 686 medical groups that assessed how 11 types of IORs affected 7 dimensions of organizational performance. Organizational performance was ascertained through self-reported questions about performance relative to local market competitors. Respondents believed that, to varying degrees, all IORs lead to a competitive advantage over local competitors in all seven performance categories. There was no consistent pattern for either loose or tight linkages to be associated with superior performance. Consequently, loose linkages may be preferable to tighter linkages (i.e., membership in a fully integrated delivery system) that require higher levels of resource commitment.