CountASAP: A Lightweight, Easy to Use Python Package for Processing ASAPseq Data.

Declining sequencing costs coupled with the increasing availability of easy-to-use kits for the isolation of DNA and RNA transcripts from single cells have driven a rapid proliferation of studies centered around genomic and transcriptomic data. Simultaneously, a wealth of new techniques have been developed that utilize single cell technologies to interrogate a broad range of cell-biological processes. One recently developed technique, transposase-accessible chromatin with sequencing (ATAC) with select antigen profiling by sequencing (ASAPseq), provides a combination of chromatin accessibility assessments with measurements of cell-surface marker expression levels. While software exists for the characterization of these datasets, there currently exists no tool explicitly designed to reformat ASAP surface marker FASTQ data into a count matrix which can then be used for these downstream analyses. To address this, we created CountASAP, an easy-to-use Python package purposefully designed to transform FASTQ files from ASAP experiments into count matrices compatible with commonly-used downstream bioinformatic analysis packages. CountASAP takes advantage of the independence of the relevant data structures to perform fully parallelized matches of each sequenced read to user-supplied input ASAP oligos and unique cell-identifier sequences.

Modeling recapitulates the heterogeneous outcomes of SARS-CoV-2 infection and quantifies the differences in the innate immune and CD8 T-cell responses between patients experiencing mild and severe symptoms.

SARS-CoV-2 infection results in highly heterogeneous outcomes, from cure without symptoms to acute respiratory distress and death. Empirical evidence points to the prominent roles of innate immune and CD8 T-cell responses in determining the outcomes. However, how these immune arms act in concert to elicit the outcomes remains unclear. Here, we developed a mathematical model of within-host SARS-CoV-2 infection that incorporates the essential features of the innate immune and CD8 T-cell responses. Remarkably, by varying the strengths and timings of the two immune arms, the model recapitulated the entire spectrum of outcomes realized. Furthermore, model predictions offered plausible explanations of several confounding clinical observations, including the occurrence of multiple peaks in viral load, viral recrudescence after symptom loss, and prolonged viral positivity. We applied the model to analyze published datasets of longitudinal viral load measurements from patients exhibiting diverse outcomes. The model provided excellent fits to the data. The best-fit parameter estimates indicated a nearly 80-fold stronger innate immune response and an over 200-fold more sensitive CD8 T-cell response in patients with mild compared to severe infection. These estimates provide quantitative insights into the likely origins of the dramatic inter-patient variability in the outcomes of SARS-CoV-2 infection. The insights have implications for interventions aimed at preventing severe disease and for understanding the differences between viral variants.

An epithelial Nfkb2 pathway exacerbates intestinal inflammation by supplementing latent RelA dimers to the canonical NF-κB module.

Aberrant inflammation, such as that associated with inflammatory bowel disease (IBD), is fueled by the inordinate activity of RelA/NF-κB factors. As such, the canonical NF-κB module mediates controlled nuclear activation of RelA dimers from the latent cytoplasmic complexes. What provokes pathological RelA activity in the colitogenic gut remains unclear. The noncanonical NF-κB pathway typically promotes immune organogenesis involving Nfkb2 gene products. Because NF-κB pathways are intertwined, we asked whether noncanonical signaling aggravated inflammatory RelA activity. Our investigation revealed frequent engagement of the noncanonical pathway in human IBD. In a mouse model of experimental colitis, we established that Nfkb2-mediated regulations escalated the RelA-driven proinflammatory gene response in intestinal epithelial cells, exacerbating the infiltration of inflammatory cells and colon pathologies. Our mechanistic studies clarified that cell-autonomous Nfkb2 signaling supplemented latent NF-κB dimers, leading to a hyperactive canonical RelA response in the inflamed colon. In sum, the regulation of latent NF-κB dimers appears to link noncanonical Nfkb2 signaling to RelA-driven inflammatory pathologies and may provide for therapeutic targets.

Detecting One-Dimensional Dipolar Bosonic Crystal Orders via Full Distribution Functions.

We explore the ground states of a few dipolar bosons in optical lattices with incommensurate filling. The competition of kinetic, potential, and interaction energies leads to the emergence of a variety of crystal state orders with characteristic one- and two-body densities. We probe the transitions between these orders and construct the emergent state diagram as a function of the dipolar interaction strength and the lattice depth. We show that the crystal state orders can be observed using the full distribution functions of the particle number extracted from simulated single-shot images.

Immune Differentiation Regulator p100 Tunes NF-κB Responses to TNF.

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose primary physiological function involves coordinating inflammatory and adaptive immune responses. However, uncontrolled TNF signaling causes aberrant inflammation and has been implicated in several human ailments. Therefore, an understanding of the molecular mechanisms underlying dynamical and gene controls of TNF signaling bear significance for human health. As such, TNF engages the canonical nuclear factor kappa B (NF-κB) pathway to activate RelA:p50 heterodimers, which induce expression of specific immune response genes. Brief and chronic TNF stimulation produces transient and long-lasting NF-κB activities, respectively. Negative feedback regulators of the canonical pathway, including IκBα, are thought to ensure transient RelA:p50 responses to short-lived TNF signals. The non-canonical NF-κB pathway mediates RelB activity during immune differentiation involving p100. We uncovered an unexpected role of p100 in TNF signaling. Brief TNF stimulation of p100-deficient cells triggered an additional late NF-κB activity consisting of RelB:p50 heterodimers, which modified the TNF-induced gene-expression program. In p100-deficient cells subjected to brief TNF stimulation, RelB:p50 not only sustained the expression of a subset of RelA-target immune response genes but also activated additional genes that were not normally induced by TNF in WT mouse embryonic fibroblasts (MEFs) and were related to immune differentiation and metabolic processes. Despite this RelB-mediated distinct gene control, however, RelA and RelB bound to mostly overlapping chromatin sites in p100-deficient cells. Repeated TNF pulses strengthened this RelB:p50 activity, which was supported by NF-κB-driven RelB synthesis. Finally, brief TNF stimulation elicited late-acting expressions of NF-κB target pro-survival genes in p100-deficient myeloma cells. In sum, our study suggests that the immune-differentiation regulator p100 enforces specificity of TNF signaling and that varied p100 levels may provide for modifying TNF responses in diverse physiological and pathological settings.

A TNF-p100 pathway subverts noncanonical NF-κB signaling in inflamed secondary lymphoid organs.

Lymphotoxin-beta receptor (LTßR) present on stromal cells engages the noncanonical NF-κB pathway to mediate RelB-dependent expressions of homeostatic chemokines, which direct steady-state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF-κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection-inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non-infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTßR-stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF-mediated inhibitions in inflamed SLOs of immunized Nfkb2-/- mice. In sum, we reveal that an inhibitory TNF-p100 pathway modulates the adaptive compartment during immune responses.

Late-phase synthesis of IκBα insulates the TLR4-activated canonical NF-κB pathway from noncanonical NF-κB signaling in macrophages.

The nuclear factor κB (NF-κB) transcription factors coordinate the inflammatory immune response during microbial infection. Pathogenic substances engage canonical NF-κB signaling through the heterodimer RelA:p50, which is subjected to rapid negative feedback by inhibitor of κBα (IκBα). The noncanonical NF-κB pathway is required for the differentiation of immune cells; however, cross-talk between both pathways can occur. Concomitantly activated noncanonical signaling generates p52 from the p100 precursor. The synthesis of p100 is induced by canonical signaling, leading to the formation of the late-acting RelA:p52 heterodimer. This cross-talk prolongs inflammatory RelA activity in epithelial cells to ensure pathogen clearance. We found that the Toll-like receptor 4 (TLR4)-activated canonical NF-κB signaling pathway is insulated from lymphotoxin ß receptor (LTßR)-induced noncanonical signaling in mouse macrophage cell lines. Combined computational and biochemical studies indicated that the extent of NF-κB-responsive expression of Nfkbia, which encodes IκBα, inversely correlated with cross-talk. The Nfkbia promoter showed enhanced responsiveness to NF-κB activation in macrophages compared to that in fibroblasts. We found that this hyperresponsive promoter engaged the RelA:p52 dimer generated during costimulation of macrophages through TLR4 and LTßR to trigger synthesis of IκBα at late time points, which prevented the late-acting RelA cross-talk response. Together, these data suggest that, despite the presence of identical signaling networks in cells of diverse lineages, emergent cross-talk between signaling pathways is subject to cell type-specific regulation. We propose that the insulation of canonical and noncanonical NF-κB pathways limits the deleterious effects of macrophage-mediated inflammation.

Real-time electro-diffusion method to discriminate carbon nanomaterials.

We report both the experimental and theoretical insights of differential electro-diffusion behavior of carbon nanomaterials (e.g. single wall, multiwall carbon nanotubes, and graphene). We thus discriminate one from the other in a soft gel system. The differential mobility of such material depends on their intrinsic properties, both extend and rate of migration bearing the discriminatory signature. The mobility analysis is made by a real time monitoring of the respective bands.

Stimulus-selective crosstalk via the NF-κB signaling system reinforces innate immune response to alleviate gut infection.

Tissue microenvironment functions as an important determinant of the inflammatory response elicited by the resident cells. Yet, the underlying molecular mechanisms remain obscure. Our systems-level analyses identified a duration code that instructs stimulus specific crosstalk between TLR4-activated canonical NF-κB pathway and lymphotoxin-ß receptor (LTßR) induced non-canonical NF-κB signaling. Indeed, LTßR costimulation synergistically enhanced the late RelA/NF-κB response to TLR4 prolonging NF-κB target gene-expressions. Concomitant LTßR signal targeted TLR4-induced newly synthesized p100, encoded by Nfkb2, for processing into p52 that not only neutralized p100 mediated inhibitions, but potently generated RelA:p52/NF-κB activity in a positive feedback loop. Finally, Nfkb2 connected lymphotoxin signal within the intestinal niche in reinforcing epithelial innate inflammatory RelA/NF-κB response to Citrobacter rodentium infection, while Nfkb2(-/-) mice succumbed to gut infections owing to stromal defects. In sum, our results suggest that signal integration via the pleiotropic NF-κB system enables tissue microenvironment derived cues in calibrating physiological responses.