Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD⁺. A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The human ALC1 (Amplified in Liver Cancer 1) protein (also referred to as CHD1L or Chromodomain-Helicase-DNA-binding protein 1-Like) is a SNF2 family chromatin remodeling enzyme that functions together with the poly(ADP-ribose) polymerase PARP1 to catalyze ATP- and NAD⁺-dependent nucleosome remodeling. The ALC1 gene is amplified in a subset of hepatocellular carcinomas, and overexpression of the ALC1 protein leads to transformation of cultured cells and appearance of spontaneous tumors in mice (1, 2). Although the precise mechanism(s) by which ALC1 overexpression contributes to tumorigenesis remains unknown, ALC1 has been implicated in multiple DNA damage repair pathways (3–6). Several recent studies have shown that ALC1 overexpression confers resistance to PARP inhibitors used in treatment of DNA repair–deficient tumors, while loss or reduction of ALC1 expression renders cells exquisitely sensitive to these drugs (7–10). Hence, understanding the functional relationships between ALC1 and PARP1 is of considerable interest.We and others initially demonstrated that ALC1 has cryptic DNA-dependent ATPase and ATP-dependent nucleosome sliding activities that are strongly activated in the presence of PARP1 and NAD⁺ (3, 11), which PARP1 and other PARPs use as substrate for synthesis of poly(ADP-ribose) (PAR) (12). ALC1 is unique among SNF2 family members in containing a macrodomain. The macrodomain, located at the enzyme’s C terminus, binds PAR chains containing three or more ADP-ribose residues (3, 11, 13–15). ALC1 macrodomain mutations that abolish PAR binding block ALC1 ATPase and nucleosome remodeling, indicating that PAR binding by the macrodomain is important for ALC1 activation. Recent studies have led to a working model for how binding of PAR to ALC1 macrodomain contributes to nucleosome remodeling. According to this model, ALC1 SNF2 ATPase domain interacts with and is held in an inactive state by the macrodomain. Upon binding of PAR to the macrodomain, this interaction is released, leading to structural changes in the ALC1 ATPase domain that relieve autoinhibition (13, 16). In subsequent steps, nucleosome binding by ALC1 stabilizes the catalytically active conformation of the ATPase, and a linker region between the ATPase and macrodomains contacts an acidic patch on nucleosomes to couple ATP hydrolysis to nucleosome sliding (17).Although it is well established that one key role of PARP1 in ALC1-dependent nucleosome remodeling is to produce PAR, it is less clear whether it makes additional contributions. Recent findings indicating that free tri-ADP ribose is sufficient to activate ALC1 ATPase activity in the absence of PARP1 (13) suggest that the role of PARP1 might be limited merely to synthesizing PAR chains. In this case, PARP1 might act simply as a bystander in ALC1-dependent nucleosome remodeling. On the other hand, PARP1 possesses both nucleosome binding and histone chaperone activities (18). This, together with our previous evidence that ALC1 and PARP1 bind cooperatively to nucleosomes to form an ALC1–PARP1–nucleosome intermediate prior to remodeling (14), makes it tempting to speculate that PARP1 might play a more active role.In the course of experiments investigating the mechanism(s) by which PARP1 contributes to ALC1-dependent nucleosome remodeling, we identified PARP1 mutants capable of activating ALC1 ATPase, but defective in supporting ALC1-catalyzed nucleosome remodeling. By investigating the properties of these and additional PARP1 mutants, we show that both the PARP1 C-terminal ADP-ribosyl transferase domain and its N-terminal region, which contains nucleosome binding activity, play important roles in ALC1-dependent nucleosome remodeling. We report these findings, which bring to light a role for PARP1 in chromatin remodeling.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

Endosomes are key platforms for transmembrane receptor and lipid sorting as well as for cell signaling. Cell surface receptors arrive from endocytic or anterograde trafficking routes and are either sent to the lysosome for degradation or recycled to other compartments, including the plasma membrane and the trans-Golgi network (1–3). Endosomal trafficking is indispensable for maintaining plasma membrane homeostasis and is tightly regulated by many pivotal protein machineries (1–3). Consequently, dysregulation of this process contributes to the development of a variety of human diseases, including Parkinson’s disease, Alzheimer’s disease, and cancer (4–6). Finally, many intracellular pathogens, including vacuolar bacteria, human papilloma virus, and SARS-CoV-2, hijack the endosomal trafficking pathways for their infection and replication (7–10).Members of the SNX-FERM subfamily have emerged as key proteins essential for sequence-dependent cargo recycling. The SNX-FERM subfamily is defined by the presence of a FERM domain, which interacts with NPxY/NxxY motifs located within the cytoplasmic tails of many transmembrane proteins, in addition to the phox homology (PX) domain, which is involved in phosphatidylinositol binding (2, 11) (Fig. 1A). The SNX-FERM subfamily encompasses three members: SNX17, SNX27, and SNX31 (2, 11). Unique to this subfamily, SNX27 also harbors a PDZ domain that can engage with PDZ-binding motifs (PDZbm) at the C-terminus of transmembrane proteins. SNX17 is a well-established regulator for endosomal trafficking and mediates trafficking of many cargoes bearing the NPxY/NxxY motif together with retriever (VPS35L, VPS26C, and VPS29), the CCC (COMMD/CCDC22/CCDC93) complex, and the actin-regulatory WASH complex (12–17). Established SNX17 cargoes include P-selectin, LDL receptor–related protein 1 (LRP1), VLDLR, and α5β1 integrins (12, 18–20). SNX31 is less studied; however, recent studies indicate that SNX31 can function analogously to SNX17 and regulate turnover and recycling of β1 integrin (21). In contrast with SNX17 and SNX31, SNX27 is most known to regulate endosomal trafficking of PDZbm-containing cargoes, such as glucose transporter GLUT1 and the β2 adrenergic receptor (22–24). SNX27 regulates endosomal trafficking through engaging with retromer (VPS35, VPS26A/B, and VPS29), a SNX–Bin/Amphiphysin/Rvs (BAR) dimer (SNX1/2 in complex with SNX5/6), and the WASH complex (23, 25–31). Both SNX17 and SNX27 have been linked with pathogenesis of multiple neurological disorders, such as Alzheimer’s disease and Down’s syndrome, emphasizing the importance of SNX-FERM–mediating endocytic recycling in development and human diseases (32–34). Interestingly, it was reported that the FERM domain of SNX27 could interact with peptides containing the NPxY/NxxY motif (35, 36), yet unlike SNX17 and SNX31, SNX27 does not promote recycling of NPxY/NxxY motif-containing cargoes.Open in a separate windowFig. 1.SNX27 specifically binds to multiple motifs centered on DLF within the N termini of SNX1/2. (A) Domain architecture of SNX-BAR and SNX-FERM proteins used in this study. PX, BAR, FERM (band4.1-ezrin-radixin-moesin), and PDZ (postsynaptic density 95-discs large-zonula occludens). (B) MBP-SNX1-N, SNX1-ΔN, and MBP pull-down of purified His-tagged SNX27 FERM. Shown is a Coomassie Blue–stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS/PAGE) gel with purified proteins (Left) and bound samples (Right). (C) Steady-state localization of different YFP-SNX1 constructs. HeLa cells were transiently transfected with mCherry-SNX27 (red) and YFP or YFP-SNX1 (green). A schematic diagram of KPI-SNX1-N is present on the bottom. (Scale bar: 10 μm.) (D) Colocalization of mCherry-SNX27 and YFP in cells in C. Each dot represents Pearson’s correlation coefficients from one cell. Data were presented as mean ± SD, and P values were calculated using one-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05; ****P < 0.0001. (E) Sequence alignment of multiple fragments from the N termini of SNX1 and SNX2 highlights a DLF motif. Red color indicates the conserved DLF or DIF residues. The NPxY/NxxY motif in P-selectin is colored in orange and used for comparison.To investigate this paradox, we utilized a holistic approach, combining biochemical, structural, and cellular studies with in vivo animal models. We show that the FERM domains of the SNX-FERM family have distinct functions: whereas the FERM domain of SNX17 recognizes the NPxY/NxxY motif, the same domain in SNX27 specifically binds to a DLF motif located within the N terminus of SNX1/2. The interaction between SNX27 and SNX1/2 is necessary for not only the recruitment of SNX27 to endosomes but also the trafficking of membrane proteins, such as GLUT1 and TRAILR1. Finally, we show that interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish, and the FERM domains of SNX17 and SNX27 are not interchangeable. Together, our data demonstrate that members of the SNX-FERM family regulate distinct endosomal trafficking routes via functionally divergent FERM domains and provide a unified answer for many different observations in the field.     

Hierarchical self-assembly of polydisperse colloidal bananas into a two-dimensional vortex phase

Self-assembly of microscopic building blocks into highly ordered and functional structures is ubiquitous in nature and found at all length scales. Hierarchical structures formed by colloidal building blocks are typically assembled from monodisperse particles interacting via engineered directional interactions. Here, we show that polydisperse colloidal bananas self-assemble into a complex and hierarchical quasi–two-dimensional structure, called the vortex phase, only due to excluded volume interactions and polydispersity in the particle curvature. Using confocal microscopy, we uncover the remarkable formation mechanism of the vortex phase and characterize its exotic structure and dynamics at the single-particle level. These results demonstrate that hierarchical self-assembly of complex materials can be solely driven by entropy and shape polydispersity of the constituting particles.

Self-assembly of microscopic building blocks is a powerful route for preparing materials with predesigned structure and engineered properties (1–7). Nature provides a fascinating range of self-assembled architectures offering insight into how structural organization can emerge at different length scales (8–13). In the biological world, for instance, tobacco mosaic virus coat proteins self-organize into sophisticated capsids around viral RNA strands (11, 14). In molecular systems, lipid molecules, such as fatty acids, form a range of self-assembled structures as relevant as cell membranes and vesicles (15, 16). At the colloidal scale, a rich variety of crystals with remarkable optical properties, such as opal and other gemstones, also assembles from a range of colloidal constituents (12, 17–20). The structural complexity of self-assembled materials is typically dictated by the combination of the type of interactions between the constituent building blocks and their shape (2, 3, 5, 6). Colloids are ideal systems to independently study the role of these key parameters, as their shape and interactions can be systematically tuned and rationally designed (5, 18, 21–23).In colloidal systems interacting solely via excluded volume interactions, the shape of the particles can already lead to the assembly of complex structures (24–28). For instance, binary colloidal crystals (25) are obtained from spherical particles, complex dodecagonal quasicrystals are formed by tetrahedrons (26), and exotic banana-shaped liquid crystals are assembled from colloidal bananas (28). Introducing complex interactions between the colloidal building blocks—on the top of their shape—leads to their assembly into hierarchical materials with structural order at multiple length scales (3, 29–31). Examples include colloidal diamond structures assembled by patchy tetrahedrons functionalized with DNA strands (20) and superlattice structures formed by octapod-like particles functionalized with hydrophobic molecules (32). The successful hierarchical self-assembly of these structures relies not only on the directionality of the particle interactions but also, on the uniformity in size of the constituent building blocks, as polydispersity typically disrupts ordering via the formation of defects (33, 34).In this work, however, we show that a colloidal suspension of polydisperse banana-shaped particles interacting only via simple excluded volume interactions (28) self-assembles into remarkably ordered concentric structures, which we term colloidal vortices. At high packing fractions, these structures form a quasi–two-dimensional (quasi-2D) hierarchical material, which we term the vortex phase. Using confocal microscopy, we uncover the formation mechanism of this tightly packed phase and characterize its exotic structure and dynamics at the single-particle level.     

Midbrain dopamine neurons arbiter OCD-like behavior

The neurobiological understanding of obsessive–compulsive disorder (OCD) includes dysregulated frontostriatal circuitry and altered monoamine transmission. Repetitive stereotyped behavior (e.g., grooming), a featured symptom in OCD, has been proposed to be associated with perturbed dopamine (DA) signaling. However, the precise brain circuits participating in DA’s control over this behavioral phenotype remain elusive. Here, we identified that DA neurons in substantia nigra pars compacta (SNc) orchestrate ventromedial striatum (VMS) microcircuits as well as lateral orbitofrontal cortex (lOFC) during self-grooming behavior. SNc–VMS and SNc–lOFC dopaminergic projections modulate grooming behaviors and striatal microcircuit function differentially. Specifically, the activity of the SNc–VMS pathway promotes grooming via D1 receptors, whereas the activity of the SNc–lOFC pathway suppresses grooming via D2 receptors. SNc DA neuron activity thus controls the OCD-like behaviors via both striatal and cortical projections as dual gating. These results support both pharmacological and brain-stimulation treatments for OCD.

Obsessive–compulsive disorder (OCD) is characterized by unwanted distressing thoughts (obsessions) and repetitive acts (compulsions), with severe disruption of daily activities (1, 2). Recent evidence suggests that various structures, such as the cortex (3–5), striatum (6–9), hypothalamus (10), hippocampus (11, 12), amygdala (13–15), and even spinal cord (16), contribute to the pathology of OCD. Imbalanced cortical–striatal activities, such as the dynamics in cortico-striato-thalamo-cortical (CSTC) circuit are recognized as the core neurobiological substrate for OCD (17–19). Specifically, hyperactivity in the medial subregion of the orbitofrontal cortex (containing medial and ventral orbital; labeled vmOFC henceforth) and ventromedial striatum (VMS) is implicated in the expression of repetitive behaviors (20–24). Rodent self-grooming can be considered as a correlate of human complex repetitive, self-directed, and sequentially patterned behaviors. Repetitive cortico-striatal stimulation that strengthens vmOFC-VMS functional connection can generate persistent OCD-like repetitive grooming behavior (25). By contrast, activating the lateral orbitofrontal cortex (lOFC)–striatal pathway prevents overexpression of both conditioned and spontaneous repetitive grooming (26). These previous studies implicate VMS’s role in encoding the grooming state of the animal, potentially through balancing its microcircuitry neural activities.Midbrain dopaminergic system is implicated in OCD-like behavior. Studies in rodents have shown that the activation of D1 receptor or D1-expressing neurons results in excessively stereotyped grooming (27, 28), whereas knocking out D1 receptor (D1R) reduces self-grooming bouts (29), suggesting that D1R signaling in the striatum may facilitate grooming. In clinical practice, dopamine antagonists have been used to augment the therapeutic effect of selective serotonin-reuptake inhibitors in patients with OCD (30). Unfortunately, the specific roles of dopamine receptors, as well as the precise brain circuits participating in dopaminergic regulations over the repetitive behavioral phenotype, remain unelucidated.Here, we show that dopamine neurons in ventral substantia nigra pars compacta (SNc) bidirectionally modulate the grooming behavior through two independent long-range circuits and distinct dopaminergic receptor profiles, respectively. Our results indicate that pharmacological and optogenetic manipulations of the dopaminergic pathway activities are sufficient to restore normal behavior.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

Active topolectrical circuits

The transfer of topological concepts from the quantum world to classical mechanical and electronic systems has opened fundamentally different approaches to protected information transmission and wave guidance. A particularly promising emergent technology is based on recently discovered topolectrical circuits that achieve robust electric signal transduction by mimicking edge currents in quantum Hall systems. In parallel, modern active matter research has shown how autonomous units driven by internal energy reservoirs can spontaneously self-organize into collective coherent dynamics. Here, we unify key ideas from these two previously disparate fields to develop design principles for active topolectrical circuits (ATCs) that can self-excite topologically protected global signal patterns. Realizing autonomous active units through nonlinear Chua diode circuits, we theoretically predict and experimentally confirm the emergence of self-organized protected edge oscillations in one- and two-dimensional ATCs. The close agreement between theory, simulations, and experiments implies that nonlinear ATCs provide a robust and versatile platform for developing high-dimensional autonomous electrical circuits with topologically protected functionalities.

Information transfer and storage in natural and man-made active systems, from sensory organs (1–3) to the internet, rely on the robust exchange of electrical signals between a large number of autonomous units that balance local energy uptake and dissipation (4, 5). Major advances in the understanding of photonic (6–9), acoustic (10–12), and mechanical (13–16) metamaterials have shown that topological protection (17–24) enables the stabilization and localization of signal propagation in passive and active (25–27) dynamical systems that violate time-reversal and/or other symmetries. Recent studies have successfully applied these ideas to realize topolectrical circuits (28) in the passive linear (29–34) and passive nonlinear (35, 36) regimes. However, despite substantial progress in the development of topological wave guides (37), lasers (38, 39), and transmission lines (40–43), the transfer of these concepts to active (44, 45) nonlinear circuits made from autonomously acting units still poses an unsolved challenge. From a broader perspective, not only does harnessing topological protection in nonlinear active circuits promise a new generation of autonomous devices, but also understanding their design and self-organization principles may offer insights into information storage and processing mechanisms in living systems, which integrate cellular activity, electrical signaling, and nonlinear feedback to coordinate essential biological functions (46, 47).Exploiting a mathematical analogy with active Brownian particle systems (26), we theoretically develop and experimentally demonstrate general design principles for active topolectrical circuits (ATCs) that achieve self-organized, self-sustained, topologically protected electric current patterns. The main building blocks of ATCs are nonlinear dissipative elements that exhibit an effectively negative resistance over a certain voltage range. Negative resistances can be realized using van der Pol (vdP) circuits (48), tunnel diodes, unijunction transistors, solid-state thyristors (49), or operational amplifiers set as negative impedance converters through current inversion (50), and the design principles described below are applicable to all these systems. Indeed, we expect them to apply to an even broader class of nonlinear systems, as similar dynamics also describe electromagnetic resonators with Kerr-type nonlinearities (51–53).     

Knot theory realizations in nematic colloids

Nematic braids are reconfigurable knots and links formed by the disclination loops that entangle colloidal particles dispersed in a nematic liquid crystal. We focus on entangled nematic disclinations in thin twisted nematic layers stabilized by 2D arrays of colloidal particles that can be controlled with laser tweezers. We take the experimentally assembled structures and demonstrate the correspondence of the knot invariants, constructed graphs, and surfaces associated with the disclination loop to the physically observable features specific to the geometry at hand. The nematic nature of the medium adds additional topological parameters to the conventional results of knot theory, which couple with the knot topology and introduce order into the phase diagram of possible structures. The crystalline order allows the simplified construction of the Jones polynomial and medial graphs, and the steps in the construction algorithm are mirrored in the physics of liquid crystals.From the invention of ropes and textiles, up to the present day, knots have played a prominent role in everyday life, essential crafts, and artistic expression. Beyond the simple tying of strings, the intriguing irreducibility of knots has led to Kelvin’s vortex model of atoms, and, subsequently, a more systematic study of knots and links in the context of knot theory (1–3). As a branch of topology, knot theory is a developing field, with many unresolved questions, including the ongoing search for an algorithm that will provide an exact identification of arbitrary knots.As knots cannot be converted one into another without the crossing of the strands––a discrete singular event––knotting topologically stabilizes the structure. In physical fields, this coexistence of discrete and continuous phenomena leads to the stabilization of geometrically and topologically nontrivial high-energy excitations (4, 5). Examples of strand-like objects in physics that can be knotted include vortices in fluids (6–9), synthetic molecules (10, 11), DNA, polymer strands and proteins (12–14), electromagnetic field lines (15, 16), zero-intensity loci in optical interference patterns (17), wave functions in topological insulators (18), cosmic strings (19), and defects in a broad selection of ordered media (20–23).Nematic liquid crystals (NLC) are liquids with a local apolar orientational order of rod-like molecules. The director field, which describes the spatial variation of the local alignment axis, supports topological point and line defects, making it an interesting medium for the observation of topological phenomena (20, 24). Defect structures in NLC and their colloidal composites (25) have been extensively studied for their potential in self-assembly and light control (26), but also to further the theoretical understanding of topological phenomena in director fields (23, 27). Objects of interest include chiral solitons (28, 29), fields around knotted particles (30–35), and knotted defects in nematic colloids (36–42). Each of these cases is unique, as the rules of knot theory interact with the rules and restrictions of each underlying material and confinement. The investigation of knotted fields is thus a specialized topic where certain theoretical aspects of knot theory emerge in a physical context.In nematic colloids––dispersions of spherical particles confined in a twisted nematic (TN) cell––disclination lines entangle arrays of particles into “nematic braids,” which can be finely controlled by laser tweezers to form various linked and knotted structures (38, 39, 43). In this paper, we focus on the diverse realizations of knot theory in such nematic colloidal structures. We complement and extend the classification and analysis of knotted disclinations from refs. 32, 38, 40 with the direct application of graph and knot theory to polarized optical micrographs. We further analyze the nematic director with constructed Pontryagin–Thom surfaces and polynomial knot invariants, which enables a comprehensive topological characterization of the knotted nematic field based on experimental data and analytical tools. We use a λ-retardation plate to observe and distinguish differently twisted domains in the optical micrograph, which correspond to medial graphs of the represented knots and contribute to the Pontryagin–Thom construction of the nematic director. Finally, we explore the organization of the space of possible configurations on a selected rectangular particle array and discuss the observed hierarchy of entangled and knotted structures.     

SEC-10 and RAB-10 coordinate basolateral recycling of clathrin-independent cargo through endosomal tubules in Caenorhabditis elegans

Despite the increasing number of regulatory proteins identified in clathrin-independent endocytic (CIE) pathways, our understanding of the exact functions of these proteins and the sequential manner in which they function remains limited. In this study, using the Caenorhabditis elegans intestine as a model, we observed a unique structure of interconnected endosomal tubules, which is required for the basolateral recycling of several CIE cargoes including hTAC, GLUT1, and DAF-4. SEC-10 is a subunit of the octameric protein complex exocyst. Depleting SEC-10 and several other exocyst components disrupted the endosomal tubules into various ring-like structures. An epistasis analysis further suggested that SEC-10 operates at the intermediate step between early endosomes and recycling endosomes. The endosomal tubules were also sensitive to inactivation of the Rab GTPase RAB-10 and disruption of microtubules. Taken together, our data suggest that SEC-10 coordinates with RAB-10 and microtubules to form the endosomal tubular network for efficient recycling of particular CIE cargoes.There has been increased interest in clathrin-independent endocytic (CIE) pathways over the past 10 y, and a growing list of endogenous plasma membrane (PM) proteins that enter cells by CIE pathways has been identified (1–3). Many CIE cargo proteins, such as the major histocompatibility complex class I (MHCI), the α-chain of the IL-2 receptor (TAC), CD59, CD44, and CD147, have been confirmed to follow the Arf6-associated CIE pathway, which is highly conserved from Caenorhabditis elegans to mammalian cells (3). Several players, including Rab10, Rab22, Rab35, Hook1, ALX-1, and RME-1/EHD-1, have been identified to modulate the recycling of CIE cargoes through different itineraries (3–8).Notably, CIE is under differential regulation on the apical and basolateral poles of polarized cells, and evidence points to a larger fraction of CIE on the basolateral pole of MDCK cells (9). Human TAC (hTAC) has been used to study canonical CIE pathway in the C. elegans intestine, which is an attractive model system for studying polarized intracellular trafficking (6, 10). Interestingly, a rapid basolateral recycling pathway has been revealed and several factors involved in the recycling of CIE cargoes have been identified, such as RAB-10, RME-1/EHD-1, ALX-1, and EHBP-1 (6–8, 11). However, it remains unclear how these proteins coordinate and regulate the trafficking of CIE cargoes through different endosomal intermediates.The exocyst is an evolutionarily conserved octameric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (12, 13). This complex was originally proposed to function in post-Golgi secretion/exocytosis by tethering exocytic vesicles with the PM (12–14). Consistent with this idea, the exocyst localizes to the PM where exocytosis actively occurs and has been implicated in many cellular trafficking processes including polarized budding in yeast (14), neurite growth in neurons (15), GLUT4 membrane insertion in fat cells (16), and cell migration (17–19). Recent studies have revealed an association between the exocyst and recycling endosome-localized proteins, such as Arf6 (20), AP1B (21), and Rab11 (22), and the presence of exocyst on multiple populations of endosomes (23). Interfering with exocyst functions affects several endocytic pathways, such as transferrin receptor (TfR) recycling in nonpolarized cells (20), and apical and basolateral recycling in polarized cells (23, 24). However, the mechanisms of how the exocyst participates in membrane recycling remain poorly understood. Attempts to examine the exact function of the exocyst in higher organisms using gene knockout methods have not been fruitful because exocyst mutations lead to embryo or larval lethality both in Drosophila and mice (25–27).Taking advantage of the powerful genetic tools available for C. elegans, we previously isolated a novel sec-10 C-terminal truncated mutation (28). The homozygous mutant is sterile but survives to adulthood. Here, using high-resolution live imaging of the C. elegans intestine, we show that sec-10 mutants display defects in basolateral recycling of particular CIE cargoes. We identified an extensive network of endosomal tubules used for efficient basolateral recycling. We propose that the concerted action of SEC-10, RAB-10, and microtubules is required to form interconnected endosomal tubules.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

From the Cover: Seasonal ITCZ migration dynamically controls the location of the (sub)tropical Atlantic biogeochemical divide

Inorganic nitrogen depletion restricts productivity in much of the low-latitude oceans, generating a selective advantage for diazotrophic organisms capable of fixing atmospheric dinitrogen (N₂). However, the abundance and activity of diazotrophs can in turn be controlled by the availability of other potentially limiting nutrients, including phosphorus (P) and iron (Fe). Here we present high-resolution data (∼0.3°) for dissolved iron, aluminum, and inorganic phosphorus that confirm the existence of a sharp north–south biogeochemical boundary in the surface nutrient concentrations of the (sub)tropical Atlantic Ocean. Combining satellite-based precipitation data with results from a previous study, we here demonstrate that wet deposition in the region of the intertropical convergence zone acts as the major dissolved iron source to surface waters. Moreover, corresponding observations of N₂ fixation and the distribution of diazotrophic Trichodesmium spp. indicate that movement in the region of elevated dissolved iron as a result of the seasonal migration of the intertropical convergence zone drives a shift in the latitudinal distribution of diazotrophy and corresponding dissolved inorganic phosphorus depletion. These conclusions are consistent with the results of an idealized numerical model of the system. The boundary between the distinct biogeochemical systems of the (sub)tropical Atlantic thus appears to be defined by the diazotrophic response to spatial–temporal variability in external Fe inputs. Consequently, in addition to demonstrating a unique seasonal cycle forced by atmospheric nutrient inputs, we suggest that the underlying biogeochemical mechanisms would likely characterize the response of oligotrophic systems to altered environmental forcing over longer timescales.Within the majority of the oligotrophic (sub)tropical regions of the surface ocean, bioavailable forms of fixed nitrogen (N) are severely depleted, restricting microbial biomass and productivity (1–3). Organisms capable of fixing atmospheric dinitrogen (N₂) thus potentially have a considerable selective advantage in these vast N-limited environments (2), with the combined activity of these diazotrophs subsequently being responsible for maintaining the oceanic fixed-N inventory and, hence, overall productivity (1, 2, 4). Although clearly not susceptible to N limitation, in common with all living organisms, oceanic diazotrophs have an absolute requirement for a wide range of other nutrient elements, including phosphorus (P) and iron (Fe) (5). Indeed, diazotrophs have an enhanced requirement for the micronutrient Fe (6–8) due to the absolute requirement for this element within nitrogenase, the catalyst responsible for N₂ fixation (1, 9, 10). It has consequently been suggested that Fe availability may play a major role in controlling oceanic N₂ fixation (1, 11).In contrast to the ubiquitous depletion of dissolved inorganic nitrogen (DIN), surface concentrations of dissolved Fe (DFe) and inorganic P (DIP) vary greatly among different regions of the oligotrophic low-latitude oceans (12–14). Marked regional differences in N₂ fixation are also observed (15) and, in combination, such observations have been argued to support the hypothesis that diazotrophy can be enhanced in regions of high-Fe inputs, resulting in subsequent drawdown of DIP (12, 13, 16). Although an ever-growing body of evidence supports the importance of Fe for diazotrophy (9, 12, 14, 17), artificial in situ tests comparable to those that unequivocally demonstrated the Fe limited status of high-nitrate low-chlorophyll (HNLC) regions (18) have yet to be performed. Indeed, the slow response timescales of some diazotrophs may ultimately render such experiments unfeasible (19, 20).The interactions among Fe, DIP, and diazotroph activity appear particularly evident in the low-latitude oligotrophic surface waters of the Atlantic Ocean (12, 17). Iron is highly insoluble and rapidly scavenged from oxic seawater (21). In consequence, the availability of this micronutrient in surface waters is often tightly coupled to external sources, including inputs of terrigenous materials via aeolian deposition (22–24). The (sub)tropical North Atlantic receives the highest deposition fluxes of aeolian dust in the global ocean (22) and is characterized by relatively high DFe (25, 26), with corresponding severe DIP depletion argued to result from significant diazotrophic activity either within or upstream of this system (12, 13). In contrast, the (sub)tropical South Atlantic gyre is characterized by relatively high DIP and very low DFe concentrations (12, 27, 28). The boundary region between these (sub)tropical gyre systems is typically characterized by high rates of N₂ fixation, which are dominated by the diazotrophic cyanobacterium Trichodesmium spp. (12, 29–31), and appear to be spatially correlated with some of the areas of highest DFe concentrations (12). Surface nutrient concentrations and diazotrophic activity thus appear to define a biogeochemical division of the Atlantic between a high DIP, low DFe (HPLFe) system in the south, and a low DIP, high DFe (LPHFe) system in the north (12).Although the biogeochemical division of the (sub)tropical Atlantic has previously been interpreted on the basis of the hypothesized Fe control of diazotrophy (12, 13), there remains the possibility that the observed spatial patterns could have other forcing factors (32). Moreover, the wider regional and global importance of Fe controls on diazotrophy in both the modern and paleo ocean remains a matter of active debate (12, 17, 33–35) with important implications for our understanding of, for example, glacial/interglacial nitrogen and carbon cycling (1, 36–38). More direct tests of the Fe–diazotrophy limitation hypothesis are thus desirable. In particular, predictable large-scale dynamic responses of marine diazotrophy and subsequent P cycling to seasonal variability in external Fe inputs has not previously been described.     

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns that can elicit a host defense response through binding to cell-surface Toll-like receptor 4 (TLR4). Systemic inflammatory response syndrome is induced by overstimulation of the innate immune response via LPSs, resulting in severe multiple organ failure, which is a major cause of death worldwide in intensive care units (1). LPS-induced dimerization of TLR4 initiates signal transduction involving the NF-κB– and MyD88-dependent and -independent pathways, thereby contributing to various inflammatory responses (2). Another set of the immune repertoire, which resides in the cytosol and comprises NLRP1, NLRP3, NAIP/NLRC4, and AIM2, is known as the inflammasome. Inflammasomes can be activated in response to a number of well-defined pathogen-derived ligands and physiological aberrations, which in turn trigger caspase-1–mediated pyroptotic death (3, 4). This process has been associated with strengthening the host defense program to eliminate intracellular bacteria.Recently, a cytosolic LPS-sensing pathway involving caspase-4/5 in humans and caspase-11 in mice was termed the noncanonical inflammasome pathway, and this pathway is independent of TLR4 (5–8). LPSs from extracellular bacteria can enter the cytoplasm and trigger caspase-4/5/11–dependent responses. LPSs can be delivered into the cytosol when LPS-containing outer membrane vesicles (OMVs) from gram-negative bacteria are taken up by the cells or when intracellular bacteria escape from the phagosomes that are damaged by host resistant factors such as guanylate-binding protein and HMGB1 or microbe-derived hemolysins (9–12). LPSs comprise three regions: lipid A, core oligosaccharide, and O-polysaccharide (also termed O-antigen). The lipid A moiety binds directly to the caspase-4/5/11 caspase activation and recruitment domain (CARD, also known as prodomain), leading to caspase oligomerization and activation (7). This event likely mimics the proximity-induced dimerization model of initiator caspase activation (13). Furthermore, caspase-4/5/11 executes downstream signaling events via gasdermin D. Activated inflammatory caspase proteolytically cleaves gasdermin D to create an N-terminal fragment that self-oligomerizes and then inserts into the cell membrane to form pores, causing lytic cell death (14–17). Various stimuli have been identified in the caspase-1–mediated canonical-inflammasome signaling pathway (3, 4), but the detailed mechanism underlying noncanonical inflammasome activation mediated by caspase-4/5/11 remains unclear.Galectins, a family of β-galactoside–binding proteins, can decode host-derived complex glycans and are involved in various biological responses (18–23). Galectins are nucleocytoplasmic proteins synthesized without a classical signal sequence, although they can be secreted through unconventional pathways (19, 21, 23, 24). Recent studies have revealed prominent roles of cytosolic galectins in host defense programs (12, 25, 26). The proposed molecular mechanisms involve the binding of galectins to host glycans exposed to the cytosolic milieu upon endosomal or phagosomal membrane damage. In addition to binding host glycans, galectins also recognize microbial glycans, particularly LPSs (27–30). However, the contribution of galectins to the host response through binding to cytosolic LPSs is unknown.Galectin-3 is an ∼30-kDa protein that contains a carbohydrate-recognition domain (CRD) connected to N-terminal proline, glycine, and tyrosine-rich tandem repeats. Upon binding to multivalent glycoconjugates through its CRD, the protein forms oligomers, which is attributable to the self-association property of its N-terminal region (31, 32). Galectin-3 binds to LPSs of various gram-negative bacteria by recognizing their carbohydrate residues (33–36).Although structural information is scarce (37), existing information suggests that ligand-induced oligomerization of caspase CARD is necessary for the activation of inflammatory caspases (7, 38). Therefore, we hypothesized that galectin-3 may be an intracellular LPS sensor that participates in LPS-induced CARD-mediated inflammatory caspase activation. Specifically, highly ordered arrays of LPS–galectin-3 complexes may amplify caspase-4/5/11 oligomerization and activation. Here, we investigated the formation of galectin-3–LPS–caspase-4/11 complexes in cell-based and cell-free systems. Our findings provide evidence regarding a role of galectin-3 as an intracellular mediator in noncanonical inflammasome activation through LPS glycan recognition.     

Metapopulations with habitat modification

Across the tree of life, organisms modify their local environment, rendering it more or less hospitable for other species. Despite the ubiquity of these processes, simple models that can be used to develop intuitions about the consequences of widespread habitat modification are lacking. Here, we extend the classic Levins metapopulation model to a setting where each of n species can colonize patches connected by dispersal, and when patches are vacated via local extinction, they retain a “memory” of the previous occupant—modeling habitat modification. While this model can exhibit a wide range of dynamics, we draw several overarching conclusions about the effects of modification and memory. In particular, we find that any number of species may potentially coexist, provided that each is at a disadvantage when colonizing patches vacated by a conspecific. This notion is made precise through a quantitative stability condition, which provides a way to unify and formalize existing conceptual models. We also show that when patch memory facilitates coexistence, it generically induces a positive relationship between diversity and robustness (tolerance of disturbance). Our simple model provides a portable, tractable framework for studying systems where species modify and react to a shared landscape.

Many interactions between species are realized indirectly, through effects on a shared environment. For example, consumers compete indirectly by altering resource availability (1, 2). However, the ways that species affect and are affected by their environment extend far beyond the consumption of resources. Across the tree of life and over a tremendous range of spatial scales, organisms make complex and sometimes substantial changes to the physical and chemical properties of their local environment (3–6). Many species also impact local biotic factors; for example, plant–soil feedbacks are often driven by changes in soil microbiome composition (4, 7–9).Numerous studies have recognized and discussed the ways that such changes can mediate interactions between species, as well as the obstacles to modeling these complex, indirect interactions (5, 7, 10–12). In some instances, the effects of environmental modification by one species on another can be accounted for implicitly in models of direct interactions (2, 13, 14) or within the well-established framework of resource competition (12, 15). But in many other cases, new modeling approaches are necessary.Because the range of ecosystems where interactions are driven by environmental modification is wide and varied, many parallel strands of theory have developed for them. Examples include “traditional” ecosystem engineers (16–20), plant–soil feedbacks (4, 7, 21), and chemically mediated interactions between microbes (5, 12). Similar dynamics underlie Janzen–Connell effects, where individuals (e.g., tropical trees) modify their local environment by supporting high densities of natural enemies (8, 22–24), and immune-mediated pathogen competition, where pathogen strains modify their hosts by inducing specific immunity (25–28). These last two examples highlight that environmental modification might be “passive,” in the sense that it is generated by the environment itself.While each of these systems has attracted careful study, it is difficult to elucidate general principles for the dynamics of environmentally mediated interactions without a simple, shared theoretical framework. Are there generic conditions for the coexistence of many species in these systems? What are typical relationships between diversity and ecosystem productivity or robustness? We especially lack theoretical expectations for high-diversity communities, as most existing models focus on the dynamics of one or two species (4, 7, 16, 17).To begin answering these questions, we introduce and analyze a flexible model for species interactions mediated by environmental modification. Two essential features of these interactions—which underlie the difficulty integrating them into standard ecological theory—are that environmental modifications are localized in space and persistent in time (10). To capture these aspects, we adopt the metapopulation framework, introduced by Levins (29), which provides a minimal model for population dynamics with distinct local and global scales. Metapopulation models underpin a productive and diverse body of theory in ecology (30, 31), including various extensions to study multispecies communities (32, 33). Here, we adopt the simplest such extension, by assuming zero-sum dynamics and an essentially horizontal community (34, 35). Our modeling framework accommodates lasting environmental modification by introducing a versatile notion of “patch memory,” in which the state of local sites depends on past occupants.In line with evidence from a range of systems, we find that patch memory can support the robust coexistence of any number of species, even in an initially homogeneous landscape. We derive quantitative conditions for species’ coexistence and show how they connect to existing conceptual models. Importantly, these conditions apply even as several model assumptions are relaxed. We also investigate an emergent relationship between species diversity and robustness, demonstrating that our modeling framework can provide insight for a variety of systems characterized by localized environmental feedbacks.     

Morphological instability and roughening of growing 3D bacterial colonies

How do growing bacterial colonies get their shapes? While colony morphogenesis is well studied in two dimensions, many bacteria grow as large colonies in three-dimensional (3D) environments, such as gels and tissues in the body or subsurface soils and sediments. Here, we describe the morphodynamics of large colonies of bacteria growing in three dimensions. Using experiments in transparent 3D granular hydrogel matrices, we show that dense colonies of four different species of bacteria generically become morphologically unstable and roughen as they consume nutrients and grow beyond a critical size—eventually adopting a characteristic branched, broccoli-like morphology independent of variations in the cell type and environmental conditions. This behavior reflects a key difference between two-dimensional (2D) and 3D colonies; while a 2D colony may access the nutrients needed for growth from the third dimension, a 3D colony inevitably becomes nutrient limited in its interior, driving a transition to unstable growth at its surface. We elucidate the onset of the instability using linear stability analysis and numerical simulations of a continuum model that treats the colony as an “active fluid” whose dynamics are driven by nutrient-dependent cellular growth. We find that when all dimensions of the colony substantially exceed the nutrient penetration length, nutrient-limited growth drives a 3D morphological instability that recapitulates essential features of the experimental observations. Our work thus provides a framework to predict and control the organization of growing colonies—as well as other forms of growing active matter, such as tumors and engineered living materials—in 3D environments.

Bacteria are known to thrive in diverse ecosystems and habitats (1–4). In nature, bacteria can be found growing on surfaces, which in addition to the ease of visualization in two dimensions, have led laboratory studies to typically focus on colony growth on two-dimensional (2D) planar surfaces. However, in many cases in nature, bacteria grow in three-dimensional (3D) habitats, such as gels and tissues inside of hosts (5–7), soils and other subsurface media (8, 9), wastewater treatment devices, and naturally occurring bodies of water (10–12). Nonetheless, despite their prevalence, the morphodynamics of bacterial colonies growing in such 3D environments remains largely unknown. Here, we ask: what determines the shape of a bacterial colony growing in three dimensions? And are there general characteristics and universal principles that span across species and specific environmental conditions?Studies of bacteria growing on 2D planar surfaces have revealed a variety of growth patterns, ranging from circular-shaped colonies (13–19) to herringbone patterns (20) and ramified, rough interfaces (13–18, 21). Some of these patterns become 3D as the colony can grow and deform into the third dimension. These morphologies are now understood to arise from friction between the growing colony and the surface and differential access to nutrients, which may also be available from the third dimension (13–18, 20, 22–26). The emergent patterns have been rationalized by incorporating these key ingredients into reaction–diffusion equations (14–16, 18, 22, 27–36), active continuum theories (19–21, 28, 31, 37–53), and agent-based models (28, 31, 38, 40, 45, 46, 54–57). Moreover, it has been suggested that these growth patterns may, in turn, influence the global function and physiology of bacterial colonies (58–60), including resistance to antibiotics (61–63) and parasites (64), resilience to environmental changes (65, 66), and genetic diversity (56, 58, 59, 67–73). However, in stark contrast to the considerable scientific effort devoted to studying 2D growth, little is known about the collective processes, the resulting morphologies, or their functional consequences for bacteria growing in three dimensions.From a physics standpoint, growing in three dimensions is fundamentally different from growing in two dimensions in terms of both nutrient access and the ability to grow and deform into an additional dimension. Consequently, we expect colony morphodynamics—the way a colony’s overall shape changes over time—to also be different. Some recent studies hint that this is indeed the case, showing how specific mechanical interactions imposed by a 3D environment can influence the morphology of growing biofilms. For instance, external fluid flows are now known to trigger the formation of streamers that stem from an initially surface-attached colony (10, 74). Also, under quiescent conditions, small (at most tens of cells across) biofilm colonies constrained in cross-linked gels adopt internally ordered structures as they grow and push outward (75), mediated by elastic stresses arising at the interface between the colony and its stiff environment. However, the behavior of larger bacterial colonies growing freely in quiescent 3D environments remains underexplored, despite the fact that they represent a fundamental building block of more complex natural colonies.Here, we combine experiments, theoretical modeling, and numerical simulations to unravel the morphodynamics of large colonies growing in 3D environments. By performing experiments with four different species of bacteria growing in transparent and easily deformed granular hydrogel matrices, we find that dense colonies growing in three dimensions undergo a morphological instability and roughen when they become nutrient limited in their interior. Independent of variations in cell type and environmental conditions, growing colonies eventually adopt a generic highly branched, broccoli-like morphology with a characteristic roughness exponent and power spectrum. Employing a continuum “active fluid” model that incorporates the coupling between nutrient diffusion, consumption, and cell growth, we trace the origin of the instability to an interplay of competition for nutrients with growth pressure–driven colony expansion. In particular, we find that these dense 3D colonies inevitably become nutrient limited in their interior, which eventually drives the periphery of the colony to become unstable and roughen. Our results thus help establish a framework to predict and control the organization of growing colonies in 3D habitats. These principles could also extend to other morphogenesis processes driven by growth in 3D environments, such as developmental processes (76, 77), tumor growth (78–82), and the expansion of engineered soft living materials (83, 84).