Substantial DNA methylation differences between two major neuronal subtypes in human brain.

The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons--GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.

IRAK-4- and MyD88-dependent pathways are essential for the removal of developing autoreactive B cells in humans.

Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for interleukin-1 receptor-associated kinase 4 (IRAK-4), myeloid differentiation factor 88 (MyD88), and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal, and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8, and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive mature naive B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88-, and UNC-93B-deficient patients did not display autoreactive antibodies in their serum or develop autoimmune diseases, suggesting that IRAK-4, MyD88, and UNC-93B pathway blockade may thwart autoimmunity in humans.

Differences in DNA methylation between human neuronal and glial cells are concentrated in enhancers and non-CpG sites.

We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG-island-containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and non-neuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells.

Exercises in molecular computing.

CONSPECTUS: The successes of electronic digital logic have transformed every aspect of human life over the last half-century. The word "computer" now signifies a ubiquitous electronic device, rather than a human occupation. Yet evidently humans, large assemblies of molecules, can compute, and it has been a thrilling challenge to develop smaller, simpler, synthetic assemblies of molecules that can do useful computation. When we say that molecules compute, what we usually mean is that such molecules respond to certain inputs, for example, the presence or absence of other molecules, in a precisely defined but potentially complex fashion. The simplest way for a chemist to think about computing molecules is as sensors that can integrate the presence or absence of multiple analytes into a change in a single reporting property. Here we review several forms of molecular computing developed in our laboratories. When we began our work, combinatorial approaches to using DNA for computing were used to search for solutions to constraint satisfaction problems. We chose to work instead on logic circuits, building bottom-up from units based on catalytic nucleic acids, focusing on DNA secondary structures in the design of individual circuit elements, and reserving the combinatorial opportunities of DNA for the representation of multiple signals propagating in a large circuit. Such circuit design directly corresponds to the intuition about sensors transforming the detection of analytes into reporting properties. While this approach was unusual at the time, it has been adopted since by other groups working on biomolecular computing with different nucleic acid chemistries. We created logic gates by modularly combining deoxyribozymes (DNA-based enzymes cleaving or combining other oligonucleotides), in the role of reporting elements, with stem-loops as input detection elements. For instance, a deoxyribozyme that normally exhibits an oligonucleotide substrate recognition region is modified such that a stem-loop closes onto the substrate recognition region, making it unavailable for the substrate and thus rendering the deoxyribozyme inactive. But a conformational change can then be induced by an input oligonucleotide, complementary to the loop, to open the stem, allow the substrate to bind, and allow its cleavage to proceed, which is eventually reported via fluorescence. In this Account, several designs of this form are reviewed, along with their application in the construction of large circuits that exhibited complex logical and temporal relationships between the inputs and the outputs. Intelligent (in the sense of being capable of nontrivial information processing) theranostic (therapy + diagnostic) applications have always been the ultimate motivation for developing computing (i.e., decision-making) circuits, and we review our experiments with logic-gate elements bound to cell surfaces that evaluate the proximal presence of multiple markers on lymphocytes.

Rapid isolation of anti-idiotype aptamers for quantification of human monoclonal antibodies against SARS-CoV-2 spike protein.

Therapeutic antibodies that block viral entry have already proven to be important, first line drugs for treatments of viral infections. In the case of SARS-CoV-2, combinations of multiple therapeutic antibodies may need to be rapidly identified and formulated in a way that blocks each new, predominant variant of the virus. For efficient introduction of any new antibody combination into patients, it is important to be able to monitor patient-specific pharmacokinetics of individual antibodies, which would include the time course of their specific capacity to block the viral spike proteins. Here, we present three examples of microfluidic-based rapid isolation of companion reagents useful for establishing combination antibody therapies. These reagents are specific three-dimensional imprints of variable regions of individual human monoclonal antibodies against the -spike protein of SARS-CoV-2 virus in the form of oligonucleotide-based ligands (aptamers). We implement these anti-idiotypic aptamers as bioreceptors in graphene-based field-effect transistor sensors to accomplish label free, rapid, and sensitive detection of matching antibodies within minutes. Through this work we have demonstrated the general applicability of anti-idiotype aptamers as capture reagents in quantification of active forms of monoclonal antibodies in complex biological mixtures.

Amplification of Signal on Cell Surfaces in Molecular Cascades.

We can formulate mixtures of oligonucleotide-antibody conjugates to act as molecular cascade-based automata that analyze pairs of cell surface markers (CD markers) on individual cells in a manner consistent with the implementation of Boolean logic-for example, by producing a fluorescent label only if two markers are present. While traditional methods to characterize cells are based on transducing signals from individual cell surface markers, these cascades can be used to combine into a single signal the presence of two or even more CDs. In our original design, oligonucleotide components irreversibly flowed from one antibody to another, driven by increased hybridizations, leading to the magnitude of the final signal on each cell being determined by the surface marker that was the least abundant. This is a significant limitation to the precise labeling of narrow subpopulations, and, in order to overcome it, we changed our design to accomplish signal amplification to a more abundant cell surface marker. We show the AMPLIFY function on two examples: (1) we amplify the fluorescent label from the CD19 marker onto a fivefold more abundant CD45, and (2) we amplify broadly distributed CD45RA to a more constant marker, CD3. We expect this new function to enable the increasingly complex Boolean analysis of cell surfaces.

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen.

BACKGROUND: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. METHODS: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification. RESULTS: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein. CONCLUSION: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Overexpression of Truncated Human DISC1 Induces Appearance of Hindbrain Oligodendroglia in the Forebrain During Development.

Genetic, neuroimaging, and gene expression studies suggest a role for oligodendrocyte (OLG) dysfunction in schizophrenia (SZ). Disrupted-in-schizophrenia 1 (DISC1) is a risk gene for major psychiatric disorders, including SZ. Overexpression of mutant truncated (hDISC1), but not full-length sequence of human DISC1 in forebrain influenced OLG differentiation and proliferation of glial progenitors in the developing cerebral cortex concurrently with reduction of OLG progenitor markers in the hindbrain. We examined gene and protein expression of the molecular determinants of hindbrain OLG development and their interactions with DISC1 in mutant hDISC1 mice. We found ectopic upregulation of hindbrain glial progenitor markers (early growth response 2 [Egr2] and NK2 homeobox 2 [Nkx2-2]) in the forebrain of hDISC1 (E15) embryos. DISC1 and Nkx2-2 were coexpressed and interacted in progenitor cells. Overexpression of truncated hDISC1 impaired interactions between DISC1 and Nkx2-2, which was associated with increased differentiation of OLG and upregulation of hindbrain mature OLG markers (laminin alpha-1 [LAMA1] and myelin protein zero [MPZ]) suggesting a suppressive function of endogenous DISC1 in OLG specialization of hindbrain glial progenitors during embryogenesis. Consistent with findings in hDISC1 mice, several hindbrain OLG markers (PRX, LAMA1, and MPZ) were significantly upregulated in the superior temporal cortex of persons with SZ. These findings show a significant effect of truncated hDISC1 on glial identity cells along the rostrocaudal axis and their OLG specification. Appearance of hindbrain OLG lineage cells and their premature differentiation may affect cerebrocortical organization and contribute to the pathophysiology of SZ.

Networking particles over distance using oligonucleotide-based devices.

Microparticles covered with DNA-based computing elements that sense inputs and release oligonucleotides as outputs could be used to construct autonomous networks with increasingly complex functions. We demonstrate cascades of particles with up to three layers and a nonlinear network with an AND gate hub. In order to establish functional networks, particles do not have to be in direct physical contact.

DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age.

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based "epigenetic age" of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC.

Autonomous molecular cascades for evaluation of cell surfaces.

Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells.

Behavior of polycatalytic assemblies in a substrate-displaying matrix.

We describe polycatalytic assemblies, comprising one or two streptavidin molecules and two to six attached nucleic acid catalysts (deoxyribozymes), with phosphodiesterase activity. When exposed to a matrix covered at high densities with oligonucleotide substrates, these molecules diffuse through the matrix continuously cleaving the substrate at rates comparable to those of individual catalysts in solution. Rates of diffusion (movement), processivity, and resident times of assemblies can be controlled through the number of catalytic units and the length of substrate/product recognition regions. The assemblies were characterized at the ensemble level using surface plasmon resonance.