The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

A nonredundant role for T cell-derived interleukin 22 in antibacterial defense of colonic crypts.

Interleukin (IL)-22 is central to immune defense at barrier sites. We examined the contributions of innate lymphoid cell (ILC) and T cell-derived IL-22 during Citrobacter rodentium (C.r) infection using mice that both report Il22 expression and allow lineage-specific deletion. ILC-derived IL-22 activated STAT3 in C.r-colonized surface intestinal epithelial cells (IECs) but only temporally restrained bacterial growth. T cell-derived IL-22 induced a more robust and extensive activation of STAT3 in IECs, including IECs lining colonic crypts, and T cell-specific deficiency of IL-22 led to pathogen invasion of the crypts and increased mortality. This reflected a requirement for T cell-derived IL-22 for the expression of a host-protective transcriptomic program that included AMPs, neutrophil-recruiting chemokines, and mucin-related molecules, and it restricted IFNγ-induced proinflammatory genes. Our findings demonstrate spatiotemporal differences in the production and action of IL-22 by ILCs and T cells during infection and reveal an indispensable role for IL-22-producing T cells in the protection of the intestinal crypts.

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

Genetically targeted optical electrophysiology in intact neural circuits.

Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons. Here, we show that the recently developed "ArcLight" GEVI robustly reports both subthreshold events and action potentials in genetically targeted neurons in the intact Drosophila fruit fly brain and reveals electrical signals in neurite branches. In the same way that genetically encoded fluorescent sensors have revolutionized the study of intracellular Ca(2+) signals, ArcLight now enables optical measurement in intact neural circuits of membrane potential, the key cellular parameter that underlies neuronal information processing.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor.

Diverse innate lymphoid cell (ILC) subtypes have been defined on the basis of effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways that lead to ILC-lineage specification remain poorly characterized. Here we found that the transcriptional regulator TOX was required for the in vivo differentiation of common lymphoid progenitors into ILC lineage-restricted cells. In vitro modeling demonstrated that TOX deficiency resulted in early defects in the survival or proliferation of progenitor cells, as well as ILC differentiation at a later stage. In addition, comparative transcriptome analysis of bone marrow progenitors revealed that TOX-deficient cells failed to upregulate many genes of the ILC program, including genes that are targets of Notch, which indicated that TOX is a key determinant of early specification to the ILC lineage.

Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

High-speed low-light in vivo two-photon voltage imaging of large neuronal populations.

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates that reduce fluorescence detection to near shot-noise levels. High-photon flux excitation can overcome photon-limited shot noise, but photobleaching and photodamage restrict the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, which is voltage imaging below the shot-noise limit. This framework involved developing positive-going voltage indicators with improved spike detection (SpikeyGi and SpikeyGi2); a two-photon microscope ('SMURF') for kilohertz frame rate imaging across a 0.4 mm × 0.4 mm field of view; and a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise-limited signals. Through these combined advances, we achieved simultaneous high-speed deep-tissue imaging of more than 100 densely labeled neurons over 1 hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.

MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Identification of basal complex protein that is essential for maturation of transmission-stage malaria parasites.

Malaria remains a global driver of morbidity and mortality. To generate new antimalarials, one must elucidate the fundamental cell biology of Plasmodium falciparum, the parasite responsible for the deadliest cases of malaria. A membranous and proteinaceous scaffold called the inner membrane complex (IMC) supports the parasite during morphological changes, including segmentation of daughter cells during asexual replication and formation of transmission-stage gametocytes. The basal complex lines the edge of the IMC during segmentation and likely facilitates IMC expansion. It is unknown, however, what drives IMC expansion during gametocytogenesis. We describe the discovery of a basal complex protein, PfBLEB, which we find to be essential for gametocytogenesis. Parasites lacking PfBLEB harbor defects in IMC expansion and are unable to form mature gametocytes. This article demonstrates a role for a basal complex protein outside of asexual division, and, importantly, highlights a potential molecular target for the ablation of malaria transmission.

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.

Alkene Hydrobenzylation by a Single Catalyst That Mediates Iterative Outer-Sphere Steps.

Cross-coupling catalysts typically react and unite functionally distinct partners via sequential inner-sphere elementary steps: coordination, migratory insertion, reductive elimination, etc. Here, we report a single catalyst that cross-couples styrenes and benzyl bromides via iterative outer-sphere steps: metal-ligand-carbon interactions. Each partner forms a stabilized radical intermediate, yet heterocoupled products predominate. The system is redox-neutral and, thus, avoids exogenous oxidants, resulting in simple and scalable conditions. Numerous variations of alkene hydrobenzylation are made possible, including access to the privileged heterodibenzyl (1,2-diarylethane) motif and challenging quaternary carbon variants.

A genome-wide association study for rheumatoid arthritis replicates previous HLA and non-HLA associations in a cohort from South Africa.

The complex pathogenesis of rheumatoid arthritis (RA) is not fully understood, with few studies exploring the genomic contribution to RA in patients from Africa. We report a genome-wide association study (GWAS) of South-Eastern Bantu-Speaking South Africans (SEBSSAs) with seropositive RA (n = 531) and population controls (n = 2653). Association testing was performed using PLINK (logistic regression assuming an additive model) with sex, age, smoking and the first three principal components as covariates. The strong association with the Human Leukocyte Antigen (HLA) region, indexed by rs602457 (near HLA-DRB1), was replicated. An additional independent signal in the HLA region represented by the lead SNP rs2523593 (near the HLA-B gene; Conditional P-value = 6.4 × 10-10) was detected. Although none of the non-HLA signals reached genome-wide significance (P < 5 × 10-8), 17 genomic regions showed suggestive association (P < 5 × 10-6). The GWAS replicated two known non-HLA associations with MMEL1 (rs2843401) and ANKRD55 (rs7731626) at a threshold of P < 5 × 10-3 providing, for the first time, evidence for replication of non-HLA signals for RA in sub-Saharan African populations. Meta-analysis with summary statistics from an African-American cohort (CLEAR study) replicated three additional non-HLA signals (rs11571302, rs2558210 and rs2422345 around KRT18P39-NPM1P33, CTLA4-ICOS and AL645568.1, respectively). Analysis based on genomic regions (200 kb windows) further replicated previously reported non-HLA signals around PADI4, CD28 and LIMK1. Although allele frequencies were overall strongly correlated between the SEBSSA and the CLEAR cohort, we observed some differences in effect size estimates for associated loci. The study highlights the need for conducting larger association studies across diverse African populations to inform precision medicine-based approaches for RA in Africa.

Exploratory assessment of the effect of systemic administration of soluble glycoprotein 130 on cognitive performance and chemokine levels in a mouse model of experimental traumatic brain injury.

Uncontrolled neuroinflammation mediates traumatic brain injury (TBI) pathology and impairs recovery. Interleukin-6 (IL-6), a pleiotropic inflammatory regulator, is associated with poor clinical TBI outcomes. IL-6 operates via classical-signaling through membrane-bound IL-6 receptor (IL-6R) and trans-signaling through soluble IL-6 receptor (s)IL-6R. IL-6 trans-signaling specifically contributes to neuropathology, making it a potential precision therapeutic TBI target. Soluble glycoprotein 130 (sgp130) prevents IL-6 trans-signaling, sparing classical signaling, thus is a possible treatment. Mice received either controlled cortical impact (CCI) (6.0 ± 0.2 m/s; 2 mm; 50-60ms) or sham procedures. Vehicle (VEH) or sgp130-Fc was subcutaneously administered to sham (VEH or 1 µg) and CCI (VEH, 0.25 µg or 1 µg) mice on days 1, 4, 7, 10 and 13 post-surgery to assess effects on cognition [Morris Water Maze (MWM)] and ipsilateral hemisphere IL-6 related biomarkers (day 21 post-surgery). CCI + sgp130-Fc groups (0.25 µg and 1 µg) were combined for analysis given similar behavior/biomarker outcomes. CCI + VEH mice had longer latencies and path lengths to the platform and increased peripheral zone time versus Sham + VEH and Sham + sgp130-Fc mice, suggesting injury-induced impairments in learning and anxiety. CCI + sgp130-Fc mice had shorter platform latencies and path lengths and had decreased peripheral zone time, indicating a therapeutic benefit of sgp130-Fc after injury on learning and anxiety. Interestingly, Sham + sgp130-Fc mice had shorter platform latencies, path lengths and peripheral zone times than Sham + VEH mice, suggesting a beneficial effect of sgp130-Fc, independent of injury. CCI + VEH mice had increased brain IL-6 and decreased sgp130 levels versus Sham + VEH and Sham + sgp130-Fc mice. There was no treatment effect on IL-6, sIL6-R or sgp130 in Sham + VEH versus Sham + sgp130-Fc mice. There was also no treatment effect on IL-6 in CCI + VEH versus CCI + sgp130-Fc mice. However, CCI + sgp130-Fc mice had increased sIL-6R and sgp130 versus CCI + VEH mice, demonstrating sgp130-Fc treatment effects on brain biomarkers. Inflammatory chemokines (MIP-1ß, IP-10, MIG) were increased in CCI + VEH mice versus Sham + VEH and Sham + sgp130-Fc mice. However, CCI + sgp130-Fc mice had decreased chemokine levels versus CCI + VEH mice. IL-6 positively correlated, while sgp130 negatively correlated, with chemokine levels. Overall, we found that systemic sgp130-Fc treatment after CCI improved learning, decreased anxiety and reduced CCI-induced brain chemokines. Future studies will explore sex-specific dosing and treatment mechanisms for sgp130-Fc therapy.

Naturalistic Bladder Filling Reveals Subtypes in Overactive Bladder Syndrome That Differentially Engages Urinary Urgency-Related Brain Circuits: Results From the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN).

PURPOSE: Overactive bladder (OAB) may be attributed to dysfunction in supraspinal brain circuits. Overactive bladder participants enrolled in the LURN (Symptoms of Lower Urinary Tract Dysfunction Research Network) study reported sensations of urinary urgency during a bladder-filling paradigm while undergoing brain functional MRI to map supraspinal dysfunction. MATERIALS AND METHODS: OAB participants and controls (CONs) completed 2 resting-state functional MRI scans following consumption of 350 mL water. Scans were conducted at fuller and emptier bladder states, interleaved with voiding. Urgency ratings (0-10) were assessed. Patterns of urgency during bladder filling were investigated using latent class trajectory models. Clusters of participants encompassing each pattern (ie, subtype) were derived from aggregated groups of OAB and CON independent of diagnosis. RESULTS: Two distinct patterns of urgency trajectories were revealed: first subtype with OAB and CON who were unresponsive to bladder filling (OAB-1 and CON-1) and second highly responsive subtype predominantly containing OAB (OAB-2). OAB-2 participants scored significantly higher on urinary symptoms but not pain or psychosocial measures. Neuroimaging analyses showed change in urgency due to both bladder filling and voided volume related to multiple loci of brain network connectivity in OAB-2, and in some cases, different than OAB-1 and/or CON-1. Sensorimotor to dorsomedial/dorsolateral prefrontal connectivity mediated the relationship between stimulus (voided volume) and percept (urgency) in OAB-2. CONCLUSIONS: Our results reveal different OAB subtypes with latent class trajectory models of urgency ratings during natural bladder filling. Functional MRI revealed differences in pathophysiology between subtypes, namely sensorimotor-prefrontal connectivity is a key locus in OAB patients with higher urinary symptoms.