Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Evolutionary insights from profiling LINE-1 activity at allelic resolution in a single human genome.

Transposable elements have created the majority of the sequence in many genomes. In mammals, LINE-1 retrotransposons have been expanding for more than 100 million years as distinct, consecutive lineages; however, the drivers of this recurrent lineage emergence and disappearance are unknown. Most human genome assemblies provide a record of this ancient evolution, but fail to resolve ongoing LINE-1 retrotranspositions. Utilizing the human CHM1 long-read-based haploid assembly, we identified and cloned all full-length, intact LINE-1s, and found 29 LINE-1s with measurable in vitro retrotransposition activity. Among individuals, these LINE-1s varied in their presence, their allelic sequences, and their activity. We found that recently retrotransposed LINE-1s tend to be active in vitro and polymorphic in the population relative to more ancient LINE-1s. However, some rare allelic forms of old LINE-1s retain activity, suggesting older lineages can persist longer than expected. Finally, in LINE-1s with in vitro activity and in vivo fitness, we identified mutations that may have increased replication in ancient genomes and may prove promising candidates for mechanistic investigations of the drivers of LINE-1 evolution and which LINE-1 sequences contribute to human disease.

Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.

The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Small-molecule inhibition of BRDT for male contraception.

A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.

Fragment correlation mass spectrometry: Determining the structures of biopolymers in a complex mixture without isolating individual components.

Fragment correlation mass spectrometry correlates ion pairs generated from the same fragmentation pathway, achieved by covariance mapping of tandem mass spectra generated with an unmodified linear ion trap without preseparation. We enable the identification of different precursors at different charge states in a complex mixture from a large isolation window, empowering an analytical approach for data-independent acquisition. The method resolves and matches isobaric fragments, internal ions, and disulfide bond fragments. We suggest that this method represents a major advance for analyzing structures of biopolymers in mixtures.

Continuous ammonia synthesis from water and nitrogen via contact electrification.

We synthesized ammonia (NH3) by bubbling nitrogen (N2) gas into bulk liquid water (200 mL) containing 50 mg polytetrafluoroethylene (PTFE) particles (~5 µm in diameter) suspended with the help of a surfactant (Tween 20, ~0.05 vol.%) at room temperature (25 °C). Electron spin resonance spectroscopy and density functional theory calculations reveal that water acts as the proton donor for the reduction of N2. Moreover, isotopic labeling of the N2 gas shows that it is the source of nitrogen in the ammonia. We propose a mechanism for ammonia generation based on the activation of N2 caused by electron transfer and reduction processes driven by contact electrification. We optimized the pH of the PTFE suspension at 6.5 to 7.0 and employed ultrasonic mixing. We found an ammonia production rate of ~420 µmol L-1 h-1 per gram of PTFE particles for the conditions described above. This rate did not change more than 10% over an 8-h period of sustained reaction.

Dependence on relative humidity in the formation of reactive oxygen species in water droplets.

Water microdroplets (7 to 11 µm average diameter, depending on flow rate) are sprayed in a closed chamber at ambient temperature, whose relative humidity (RH) is controlled. The resulting concentration of ROS (reactive oxygen species) formed in the microdroplets, measured by the amount of hydrogen peroxide (H2O2), is determined by nuclear magnetic resonance (NMR) and by spectrofluorimetric assays after the droplets are collected. The results are found to agree closely with one another. In addition, hydrated hydroxyl radical cations (•OH-H3O+) are recorded from the droplets using mass spectrometry and superoxide radical anions (•O2-) and hydroxyl radicals (•OH) by electron paramagnetic resonance spectroscopy. As the RH varies from 15 to 95%, the concentration of H2O2 shows a marked rise by a factor of about 3.5 in going from 15 to 50%, then levels off. By replacing the H2O of the sprayed water with deuterium oxide (D2O) but keeping the gas surrounding droplets with H2O, mass spectrometric analysis of the hydrated hydroxyl radical cations demonstrates that the water in the air plays a dominant role in producing H2O2 and other ROS, which accounts for the variation with RH. As RH increases, the droplet evaporation rate decreases. These two facts help us understand why viruses in droplets both survive better at low RH values, as found in indoor air in the wintertime, and are disinfected more effectively at higher RH values, as found in indoor air in the summertime, thus explaining the recognized seasonality of airborne viral infections.

Fundamental Mechanisms of Regulated Cell Death and Implications for Heart Disease.

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Respiratory Syncytial Virus Prefusion F Protein Vaccine in Older Adults.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of acute respiratory infection, lower respiratory tract disease, clinical complications, and death in older adults. There is currently no licensed vaccine against RSV infection. METHODS: In an ongoing, international, placebo-controlled, phase 3 trial, we randomly assigned, in a 1:1 ratio, adults 60 years of age or older to receive a single dose of an AS01E-adjuvanted RSV prefusion F protein-based candidate vaccine (RSVPreF3 OA) or placebo before the RSV season. The primary objective was to show vaccine efficacy of one dose of the RSVPreF3 OA vaccine against RSV-related lower respiratory tract disease, confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR), during one RSV season. The criterion for meeting the primary objective was a lower limit of the confidence interval around the efficacy estimate of more than 20%. Efficacy against severe RSV-related lower respiratory tract disease and RSV-related acute respiratory infection was assessed, and analyses according to RSV subtype (A and B) were performed. Safety was evaluated. RESULTS: A total of 24,966 participants received one dose of the RSVPreF3 OA vaccine (12,467 participants) or placebo (12,499). Over a median follow-up of 6.7 months, vaccine efficacy against RT-PCR-confirmed RSV-related lower respiratory tract disease was 82.6% (96.95% confidence interval [CI], 57.9 to 94.1), with 7 cases (1.0 per 1000 participant-years) in the vaccine group and 40 cases (5.8 per 1000 participant-years) in the placebo group. Vaccine efficacy was 94.1% (95% CI, 62.4 to 99.9) against severe RSV-related lower respiratory tract disease (assessed on the basis of clinical signs or by the investigator) and 71.7% (95% CI, 56.2 to 82.3) against RSV-related acute respiratory infection. Vaccine efficacy was similar against the RSV A and B subtypes (for RSV-related lower respiratory tract disease: 84.6% and 80.9%, respectively; for RSV-related acute respiratory infection: 71.9% and 70.6%, respectively). High vaccine efficacy was observed in various age groups and in participants with coexisting conditions. The RSVPreF3 OA vaccine was more reactogenic than placebo, but most adverse events for which reports were solicited were transient, with mild-to-moderate severity. The incidences of serious adverse events and potential immune-mediated diseases were similar in the two groups. CONCLUSIONS: A single dose of the RSVPreF3 OA vaccine had an acceptable safety profile and prevented RSV-related acute respiratory infection and lower respiratory tract disease and severe RSV-related lower respiratory tract disease in adults 60 years of age or older, regardless of RSV subtype and the presence of underlying coexisting conditions. (Funded by GlaxoSmithKline Biologicals; AReSVi-006 ClinicalTrials.gov number, NCT04886596.).

Hot spots for allosteric regulation on protein surfaces.

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and coevolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that: (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis, and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved "wiring" mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation.

Silica particles convert thiol-containing molecules to disulfides.

Synthetic amorphous silica is a common food additive and a popular cosmetic ingredient. Mesoporous silica particles are also widely studied for their potential use in drug delivery and imaging applications because of their unique properties, such as tunable pore sizes, large surfaces areas, and assumed biocompatibility. Such a nanomaterial, when consisting of pure silicon dioxide, is generally considered to be chemically inert, but in this study, we showed that oxidation yields for different compounds were facilitated by simply incubating aqueous solutions with pure silica particles. Three thiol-containing molecules, L-cysteine, glutathione, and D-penicillamine, were studied separately, and it was found that more than 95% of oxidation happened after incubating any of these compounds with mesoporous silica particles in the dark for a day at room temperature. Oxidation increased over incubation time, and more oxidation was found for particles having larger surface areas. For nonporous silica particles at submicron ranges, yields of oxidation were different based on the structures of molecules, correlating with steric hindrance while accessing surfaces. We propose that the silyloxy radical (SiO•) on silica surfaces is what facilitates oxidation. Density functional theory calculations were conducted for total energy changes for reactions between different aqueous species and silicon dioxide surfaces. These calculations identified two most plausible pathways of the lowest energy to generate SiO• radicals from water radical cations H2O•+ and hydroxyl radicals •OH, previously known to exist at water interfaces.

Making ammonia from nitrogen and water microdroplets.

Water (H2O) microdroplets are sprayed onto a magnetic iron oxide (Fe3O4) and Nafion-coated graphite mesh using compressed N2 or air as the nebulizing gas. The resulting splash of microdroplets enters a mass spectrometer and is found to contain ammonia (NH3). This gas-liquid-solid heterogeneous catalytic system synthesizes ammonia in 0.2 ms. The conversion rate reaches 32.9 ± 1.38 nmol s-1 cm-2 at room temperature without application of an external electric potential and without irradiation. Water microdroplets are the hydrogen source for N2 in contact with Fe3O4. Hydrazine (H2NNH2) is also observed as a by-product and is suspected to be an intermediate in the formation of ammonia. This one-step nitrogen-fixation strategy to produce ammonia is eco-friendly and low cost, which converts widely available starting materials into a value-added product.

Accessory cells precondition naïve T cells and regulatory T cells for cytokine-mediated proliferation.

Naïve T cells and regulatory T cells, when purified, do not proliferate to the γc-cytokines IL-2, IL-7, or IL-15, despite their expression of cognate cytokine receptors. Dendritic cells (DCs) enabled the T cell proliferation to these cytokines, through cell-to-cell contact, but independent of T cell receptor stimulation. This effect lasted after separation of T cells from DCs, enabling enhanced proliferation of the T cells in DC-depleted hosts. We propose calling this a "preconditioning effect". Interestingly, IL-2 alone was sufficient to induce phosphorylation and nuclear translocation of STAT5 in T cells, but could not activate MAPK and AKT pathways and failed to induce transcription of IL-2 target genes. "Preconditioning" was necessary to activate these two pathways and induced weak Ca2+ mobilization independent of calcium release-activated channels. When preconditioning was combined with IL-2, full activation of downstream mTOR, 4E-BP1 hyperphosphorylation, and prolonged S6 phosphorylation occurred. Collectively, accessory cells provide T cell preconditioning, a unique activation mechanism, controlling cytokine-mediated proliferation of T cells.

Contact between water vapor and silicate surface causes abiotic formation of reactive oxygen species in an anoxic atmosphere.

Spontaneous generation of reactive oxygen species (ROS) in aqueous microdroplets or at a water vapor-silicate interface is a new source of redox chemistry. However, such generation occurs with difficulty in liquid water having a large ionic strength. We report that ROS is spontaneously produced when water vapor contacts hydrogen-bonded hydroxyl groups on a silicate surface. The evolution of hydrogen-bonded species such as hydroxyl groups was investigated by using two-dimensional, time-resolved FT-IR spectroscopy. The participation of water vapor in ROS generation is confirmed by investigating the reaction of D2O vapor and hydroxyl groups on a silicate surface. We propose a reaction pathway for ROS generation based on the change of the hydrogen-bonding network and corresponding electron transfer onto the silicate surface in the water vapor-solid contact process. Our observations suggest that ROS production from water vapor-silicate contact electrification could have contributed to oxidation during the Archean Eon before the Great Oxidation Event.

Constructing and interpreting a large-scale variant effect map for an ultrarare disease gene: Comprehensive prediction of the functional impact of PSAT1 genotypes.

Reduced activity of the enzymes encoded by PHGDH, PSAT1, and PSPH causes a set of ultrarare, autosomal recessive diseases known as serine biosynthesis defects. These diseases present in a broad phenotypic spectrum: at the severe end is Neu-Laxova syndrome, in the intermediate range are infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end is childhood disease with intellectual disability. However, L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms. Therefore, knowledge of pathogenic variants can improve clinical outcomes. Here, we use a yeast-based assay to individually measure the functional impact of 1,914 SNV-accessible amino acid substitutions in PSAT. Results of our assay agree well with clinical interpretations and protein structure-function relationships, supporting the inclusion of our data as functional evidence as part of the ACMG variant interpretation guidelines. We use existing ClinVar variants, disease alleles reported in the literature and variants present as homozygotes in the primAD database to define assay ranges that could aid clinical variant interpretation for up to 98% of the tested variants. In addition to measuring the functional impact of individual variants in yeast haploid cells, we also assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results from our diploid assay successfully distinguish the genotypes of affected individuals from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements to predict the biallelic function of ~1.8 million allele combinations corresponding to potential human genotypes. Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of ultrarare diseases.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.

Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.