Dendritic cell SIRT1–HIF1α axis programs the differentiation of CD4+ T cells through IL-12 and TGF-β1

The differentiation of naive CD4⁺ T cells into distinct lineages plays critical roles in mediating adaptive immunity or maintaining immune tolerance. In addition to being a first line of defense, the innate immune system also actively instructs adaptive immunity through antigen presentation and immunoregulatory cytokine production. Here we found that sirtuin 1 (SIRT1), a type III histone deacetylase, plays an essential role in mediating proinflammatory signaling in dendritic cells (DCs), consequentially modulating the balance of proinflammatory T helper type 1 (T_H1) cells and antiinflammatory Foxp3⁺ regulatory T cells (T_reg cells). Genetic deletion of SIRT1 in DCs restrained the generation of T_reg cells while driving T_H1 development, resulting in an enhanced T-cell–mediated inflammation against microbial responses. Beyond this finding, SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)-dependent pathway, orchestrating the reciprocal T_H1 and T_reg lineage commitment through DC-derived IL-12 and TGF-β1. Our studies implicates a DC-based SIRT1–HIF1α metabolic checkpoint in controlling T-cell lineage specification.CD4⁺ T cells are essential components of the adaptive immune system that regulate immune responses against foreign antigen. Upon antigen recognition, naive CD4⁺ T cells undergo activation and expansion, and, depending on inflammatory contexts and cytokine milieus, differentiate into functional and phenotypic T helper (T_H) subsets characterized by distinct cytokine production profile and function (1–3). T_H1 cells produce IFN-γ and elicit cellular immunity in responding to intracellular pathogens; T_H2 cells produce IL-4 and IL-5 and promote humoral immunity in responding to extracellular bacteria and helminthes; and T_H17 cells produce IL-17 and mediate antifungal defense and inflammation (4, 5). Additionally, regulatory T cells, often known as “induced regulatory T cells” (iT_reg cells), which act in synergy with naturally occurring T_reg cells (nT_reg cells), produce IL-10 and TGF-β1 and dampen immune responses elicited from T_H1, T_H2, and T_H17 (6–9).Dendritic cells (DCs), an essential component in the innate immune system, play a critical role in initiating front-line primary immune responses and directing subsequent pathogen-specific adaptive immune responses (2). In addition to presenting antigens and modulating cell surface costimulatory molecules, DC-derived cytokines and chemokines can result in either a proinflammatory or antiinflammatory environment, engaging distinct T-cell differentiation programs on naive CD4⁺ T cells (1, 10–16). For example, DC-producing IL-12 can support T_H1 development, whereas DC-producing IL-10 or TGF-β1 can support T_reg development. Recent studies from us and others have shown that innate signaling in DCs mediated by G protein-coupled receptor S1P1, the mitogen-activated kinases (MAPKs), and Wnt–β-catenin plays important roles in stimulating adaptive immune responses through directing native CD4⁺ T-cell differentiation (17–20). However, other critical signaling components in DCs that may play a role in shaping T-cell lineage engagement remain to be identified.SIRT1 is a mammalian homolog of the yeast NAD⁺-dependent protein deacetylase Sirt2, and plays a role in a variety of essential biological processes, including cell cycle progression, apoptosis, cell survival, gene silencing, heterochromatin formation, tumorigenesis, metabolism, and development (21, 22). SIRT1 has also been implicated in regulating immune responses. In T cells, SIRT1 is required to maintain T-cell tolerance (23, 24) and also play a role in inhibiting the function of T_reg cells in allograft survival (25). In myeloid cells, SIRT1 limits the inflammatory process by inhibiting the expression of proinflammatory cytokines (26, 27), while promoting DC maturation and T_H2 response in airway allergy (28). However, whether SIRT1 is involved in bridging the innate immune signal to adaptive immune response remains unexplored.Here, we found that SIRT1 plays a critical role in determining the T-cell lineage fate by directing DC-derived cytokine production, which links innate and adaptive immune modulation. Largely through a HIF1α–dependent signaling pathway, SIRT1 is required for the reciprocal production of IL-12 and TGF-β1 production in DCs as well as the expression of IL-12Rβ2 and TGF-βR2 in responding T cells, resulting in a differential lineage engagement of T_H1 and iT_reg in the microbial-induced inflammation.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

The antiinflammatory activity of intravenous immunoglobulin (IVIG) is dependent on the presence of sialic acid in the core IgG fragment crystallizable domain (Fc) glycan, resulting in increased conformational flexibility of the C_H2 domain with corresponding modulation of Fc receptor (FcR) binding specificity from type I to type II receptors. Sialylated IgG Fc (sFc) increases the activation threshold of innate effector cells to immune complexes by stimulating the up-regulation of the inhibitory receptor FcγRIIB. We have found that the structural alterations induced by sialylation can be mimicked by specific amino acid modifications to the C_H2 domain. An IgG Fc variant with a point mutation at position 241 (F→A) exhibits antiinflammatory activity even in the absence of sialylation. F241A and sFc protect mice from arthritis in the K/BxN-induced model and, in the T cell-mediated experimental autoimmune encephalomyelitis (EAE) mouse model, suppress disease by specifically activating regulatory T cells (T_reg cells). Protection by these antiinflammatory Fcs in both antibody- and T cell-mediated autoimmune diseases required type II FcRs and the induction of IL-33. These results further clarify the mechanism of action of IVIG in both antibody- and T cell-mediated inflammatory diseases and demonstrate that Fc variants that mimic the structural alterations induced by sialylation, such as F241A, can be promising therapeutic candidates for the treatment of various autoimmune disorders.Intravenous immunoglobulin (IVIG), although initially developed as an Ig replacement therapy in patients with hypogammaglobulinemia (1), has gained widespread use for its immunomodulatory activities. It is an approved therapeutic for the treatment of autoimmune disorders such as immunothrombocytopenia (ITP), chronic inflammatory demyelinating polyneuropathy, Kawasaki’s disease, and Guillain-Barre syndrome (2, 3), and is used in a growing number of autoimmune and inflammatory disorders. Its antiinflammatory activity has been shown to result from the presence of a specific glycan, the α2,6-sialylated, complex biantennary structure present on the C_H2 domain of the fragment crystallizable domain (Fc) and found in a small proportion of heterogeneous antibody preparations in IVIG (4). Sialylation of the Fc glycan on the C_H2 domain results in IgGs that can engage type II Fc receptors (FcRs) such as specific ICAM-3 grabbing non-integrin-related 1 (SIGN-R1), dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), and CD23 (5–8), while reducing their binding affinity to type I FcRs (9–11). Studies in mouse models of serum-induced arthritis, antibody-dependent ITP, nephrotoxic nephritis, and autoimmune blistering diseases confirmed the antiinflammatory activity of the sialylated Fc, whether from IVIG or generated from recombinantly expressed IgG1 (5, 9, 12, 13). Moreover, increasing the percentage of sialylated Fc fragments either in IVIG or recombinant expressed IgG1 resulted in an enhanced therapeutic potency of these preparations (6, 9, 12, 14). Elucidation of the mechanism by which sialylated IgG Fc (sFc) induces an antiinflammatory response was first reported in murine models of arthritis, demonstrating that selective binding of sialylated Fc to type II FcRs resulted in the production of interleukin (IL) 33 by regulatory macrophages, which in turn stimulated IL-4 secretion from basophils. IL-4 induced the up-regulation of the inhibitory receptor FcγRIIB on effector macrophages, thereby increasing the activation threshold of these cells and suppressing inflammation (15, 16). Subsequent studies have confirmed that IVIG treatment of human populations resulted in both increased serum IL-33 levels and FcγRIIB expression on lymphoid and myeloid cells, consistent with murine data (17–19).Crystallographic and biophysical studies on sialylated and asialylated IgG Fc fragments have provided insights into the structural basis for the antiinflammatory activity of sialylated Fc. Sialylation of the complex, biantennary glycan of the IgG Fc results in increased conformational flexibility of the C_H2 domain (20), thereby sampling the closed conformations of the C_H2 domain required for type II FcR binding (11). In contrast, asialylated Fc structures uniformly result in open Fc conformations, consistent with their binding specificity for type I FcRs (21). Glycan interactions with amino acid residues of the C_H2 domain are disrupted upon sialylation, providing a basis for the observed conformational changes seen in the protein structure and consistent with a model proposed for the binding specificity of sialylated Fc for type II FcRs (11). Based on these observations, we generated a series of Fc variants targeting the amino acids of the C_H2 domain that interact with the glycan, with the goal of determining their impact on type II FcR binding and the resulting antiinflammatory activity. Both gain- and loss-of-function mutants were examined in this study. The identification of a gain-of-function variant, which could mimic the conformational state induced by sialylation, without requiring this specific carbohydrate modification, may potentially simplify the development of antiinflammatory IgG Fc for therapeutic use (20). We succeeded in identifying a mutation (F241A) predicted to increase mobility of the α1,3-arm, and which replicates the antiinflammatory activity of sialylated Fc even in the absence of sialylation. We have characterized this variant, in comparison with sialylated Fc, in both antibody and T-cell models of autoimmune inflammation.Although the basis for IVIG protection in antibody-mediated models of inflammation has been extensively studied, as summarized above (9, 12, 13, 16), recent studies have demonstrated that IVIG can also protect in classical T cell-mediated autoimmune disorders, such as experimental autoimmune encephalomyelitis (EAE) (22), as well as in a model of airway hyperresponsiveness (23, 24). This therapeutic effect of IVIG is proposed to result from the activation and expansion of regulatory T cells (T_reg cells), thus suppressing T-cell responses by IFN-γ–secreting T_H1 and IL-17–secreting T_H17 cells (25, 26). We therefore sought to investigate whether the T_reg-cell activation and expansion were also the result of sialylated Fcs or the F241A variant. Using F241A and sialylated and asialylated IVIG, we investigated the mechanisms of action of their immunomodulatory effects on T_reg-cell activation and suppression of T cell-dependent autoimmunity. We demonstrate that the sialylation of IVIG is critically required for T_reg-cell activation and expansion and that the F241A variant is sufficient to suppress T cell-dependent inflammation in the EAE mouse model. Furthermore, both sFc as well as F241A stimulate the production of IL-33 that in turn activates T_reg cells through the ST2 receptor, contributing to the suppression of T cell-mediated autoimmune responses.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33_95–270, IL-33_107–270, and IL-33_109–270, were 30-fold more potent than full-length human IL-33_1–270 for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33–dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66–111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33–mediated responses in allergic asthma and other inflammatory diseases.Interleukin-33 (IL-33), previously known as nuclear factor from high endothelial venules or NF-HEV (1, 2), is an IL-1 family cytokine (3) that signals through the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2 (4, 5) and induces expression of cytokines and chemokines in various immune cell types, including mast cells, basophils, eosinophils, Th2 lymphocytes, invariant natural killer T, and natural killer cells (3, 4, 6–8). Studies in IL-33–deficient mice indicate that IL-33 plays important roles in type-2 innate immunity and innate-type allergic airway inflammation (9–13). Indeed, IL-33 is a key activator of the recently described group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes) (14–17). These cells control eosinophil homeostasis in blood and adipose tissue (18, 19) and produce extremely high amounts of the type-2 cytokines IL-5 and IL-13 in response to IL-33 (14–16). ILC2s also play important roles in allergic airway inflammation (20–24), atopic skin disease (25–28), helminth infection in the intestine (11, 12, 14–16), and influenza virus infection in the lungs (29, 30).Based on animal model studies and analyses of diseased tissues from patients, IL-33 has been proposed as a candidate therapeutic target for several important diseases, including asthma and other allergic diseases, rheumatoid arthritis, inflammatory bowel diseases, and cardiovascular diseases (4, 6). IL-33 is likely to play a critical role in asthma because the IL33 and IL1RL1/ST2 genes have been reproducibly identified as major susceptibility loci in several independent large-scale genome-wide association studies of human asthma (31, 32).Despite these important advances into the roles of IL-33, very little is known yet about the mechanisms regulating its activity. Full-length human IL-33 is a 270 amino acid protein localized in the nucleus of endothelial and epithelial cells in blood vessels and epithelial barrier tissues (1, 2, 33, 34), which associates with chromatin (2) and histones H2A-H2B, through a short chromatin-binding motif located in its N-terminal part (amino acids 40–58) (35). IL-33 can be released in the extracellular space upon cellular damage or necrotic cell death (36, 37), and it was thus proposed to function as an alarmin (alarm signal or endogenous danger signal), which alerts the immune system to tissue injury following trauma or infection (33, 36, 37).Proteases have been shown to regulate IL-33 activity. Full-length IL-33_1–270 is biologically active but processing by caspases after residue Asp₁₇₈ in the IL-1–like cytokine domain results in its inactivation (36, 37). In contrast, inflammatory proteases from neutrophils, cathepsin G, and elastase, process full-length IL-33 into mature forms that contain an intact IL-1–like cytokine domain and that have an increased biological activity compared with full-length IL-33_1–270 (38). Although neutrophils have been implicated in virus-induced exacerbations of asthma, they are unlikely to be involved in the processing of IL-33 during allergic inflammation. We therefore investigated the possibility that other cell types may be involved in this process. Mast cells, which are widely recognized for their roles as effector cells in allergic disorders, were good candidates because they interact directly with ILC2s in vivo (26) and they are strategically positioned close to vessel walls and epithelial surfaces exposed to the environment (39), the major sites of IL-33 expression (33, 34). We now demonstrate that activated human mast cells and purified mast cell proteases, tryptase and chymase, generate mature forms of IL-33 (IL-33_95–270, IL-33_107–270, and IL-33_109–270), which are ∼30-fold more potent than full-length IL-33_1–270 for activation of ILC2s ex vivo. These IL-33 mature forms are also potent inducers of ILC2s, eosinophils, and type-2 cytokines in vivo. Our study suggests that release of the C-terminal IL-1–like cytokine domain, through proteolytic maturation within the central cleavage/activation domain (amino acids 66–111), is important for full biological activity of IL-33.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome

Haploinsufficiency of the voltage-gated sodium channel Na_V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na_V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na_V1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.Dravet syndrome (DS), also referred to as “severe myoclonic epilepsy in infancy,” is a rare genetic epileptic encephalopathy characterized by frequent intractable seizures, severe cognitive deficits, and premature death (1–3). DS is caused by loss-of-function mutations in SCN1A, the gene encoding type I voltage-gated sodium channel Na_V1.1, which usually arise de novo in the affected individuals (4–7). Like DS patients, mice with heterozygous loss-of-function mutations in Scn1a exhibit ataxia, sleep disorder, cognitive deficit, autistic-like behavior, and premature death (8–14). Like DS patients, DS mice first become susceptible to seizures caused by elevation of body temperature and subsequently experience spontaneous myoclonic and generalized tonic-clonic seizures (11). Global deletion of Na_V1.1 impairs Na⁺ currents and action potential (AP) firing in GABAergic-inhibitory interneurons (8–10), and specific deletion of Na_V1.1 in forebrain interneurons is sufficient to cause DS in mice (13, 15). These data suggest that the loss of interneuron excitability and resulting disinhibition of neural circuits cause DS, but the functional role of different subtypes of interneurons in the cerebral cortex in DS remains unknown.Neocortical GABAergic interneurons shape cortical output and display great diversity in morphology and function (16, 17). The expression of parvalbumin (PV) and somatostatin (SST) defines two large, nonoverlapping groups of interneurons (16, 18, 19). In layer V of the cerebral cortex, PV-expressing fast-spiking interneurons and SST-expressing Martinotti cells each account for ∼40% of interneurons, and these interneurons are the major inhibitory regulators of cortical network activity (17, 20). Layer V PV interneurons make synapses on the soma and proximal dendrites of pyramidal neurons (18, 19), where they mediate fast and powerful inhibition (21, 22). Selective heterozygous deletion of Scn1a in neocortical PV interneurons increases susceptibility to chemically induced seizures (23), spontaneous seizures, and premature death (24), indicating that this cell type may contribute to Scn1a deficits. However, selective deletion of Scn1a in neocortical PV interneurons failed to reproduce the effects of DS fully, suggesting the involvement of other subtypes of interneurons in this disease (23, 24). Layer V Martinotti cells have ascending axons that arborize in layer I and spread horizontally to neighboring cortical columns, making synapses on apical dendrites of pyramidal neurons (17, 25, 26). They generate frequency-dependent disynaptic inhibition (FDDI) that dampens excitability of neighboring layer V pyramidal cells (27–29), contributing to maintenance of the balance of excitation and inhibition in the neocortex. However, the functional roles of Martinotti cells and FDDI in DS are unknown.Because layer V forms the principal output pathway of the neocortex, reduction in inhibitory input to layer V pyramidal cells would have major functional consequences by increasing excitatory output from all cortical circuits. However, the effects of the DS mutation on interneurons and neural circuits that provide inhibitory input to layer V pyramidal cells have not been determined. Here we show that the intrinsic excitability of layer V fast-spiking PV interneurons and SST Martinotti cells and the FDDI mediated by Martinotti cells are reduced dramatically in DS mice, leading to an imbalance in the excitation/inhibition ratio. Our results suggest that loss of Na_V1.1 in these two major types of interneurons may contribute synergistically to increased cortical excitability, epileptogenesis, and cognitive deficits in DS.     

Emergence of spatial structure in the tumor microenvironment due to the Warburg effect

Drastic metabolic alterations, such as the Warburg effect, are found in most if not all types of malignant tumors. Emerging evidence shows that cancer cells benefit from these alterations, but little is known about how they affect noncancerous stromal cells within the tumor microenvironment. Here we show that cancer cells are better adapted to metabolic changes in the microenvironment, leading to the emergence of spatial structure. A clear example of tumor spatial structure is the localization of tumor-associated macrophages (TAMs), one of the most common stromal cell types found in tumors. TAMs are enriched in well-perfused areas, such as perivascular and cortical regions, where they are known to potentiate tumor growth and invasion. However, the mechanisms of TAM localization are not completely understood. Computational modeling predicts that gradients—of nutrients, gases, and metabolic by-products such as lactate—emerge due to altered cell metabolism within poorly perfused tumors, creating ischemic regions of the tumor microenvironment where TAMs struggle to survive. We tested our modeling prediction in a coculture system that mimics the tumor microenvironment. Using this experimental approach, we showed that a combination of metabolite gradients and differential sensitivity to lactic acid is sufficient for the emergence of macrophage localization patterns in vitro. This suggests that cancer metabolic changes create a microenvironment where tumor cells thrive over other cells. Understanding differences in tumor-stroma sensitivity to these alterations may open therapeutic avenues against cancer.Cancer cells in tumors display pronounced metabolic alterations (1–10). The genetic and biochemical mechanisms behind these changes are under intensive investigation, but the question of how metabolic changes affect noncancerous cells in the tumor microenvironment remains largely unanswered. The Warburg effect—or oxidative glycolysis, a process whereby cells exhibit a high glycolytic rate even in the presence of oxygen—is arguably the best-known metabolic alteration in cancer (1). Due to a lower yield of glucose to ATP associated with glycolysis, the Warburg effect was initially viewed as a detrimental aberration (1, 5). However, it is now clear that ATP is not a limiting resource for cell growth (4, 9) and that glycolytic alterations increase glucose and glutamine uptake, enhance reductive power, and favor anabolism by retaining carbon-rich macromolecules (4, 7, 9). Thus, rather than being detrimental, metabolic alterations in tumor cells can be required to sustain the high proliferation rate that characterizes malignant cancers (4, 7, 9). In fact, similar metabolic changes occur in healthy processes with rapid population growth such as pluripotent stem-cell proliferation (11), T-cell activation (12), embryonic development (13), and wound healing (14), suggesting that cancer cells have co-opted conserved metabolic processes used by rapidly proliferating cells (4, 7, 9).Despite their beneficial effect for cell proliferation, metabolic changes have dramatic consequences on the extracellular milieu. Alterations in tumor metabolism were first identified by studying how cancer cells alter their culture media (1, 5). Chaotic vascularization can be a feature in tumors in vivo, which intensifies the effect of cancer cells on their microenvironment and causes damaging processes such as acidosis, hypoxia, and nutrient deprivation (15, 16). Thus, cancer cells must balance the benefits of an altered metabolism with its potentially toxic extracellular consequences.Cancer is a disease of clonal evolution where different cell lineages compete (17, 18). Mathematical models in the literature suggest that metabolic modifications can be advantageous for lineages competing within tumors (16, 19–21). Nonetheless, how stromal cells within tumors cope with these changes has been largely neglected. Thus, it is possible that a toxic microenvironment created by metabolic alterations may be a mechanism for cancer cells to gain a selective advantage.We focused our study on how tumor metabolism affects macrophages. Tumor-associated macrophages (TAMs) are one of the most common stromal cell types found within tumors, and their number is directly correlated with poor patient prognosis in the majority of cancers analyzed to date (22–26). TAMs are well adapted to, and recruited toward, low-oxygen-tension regions (22, 27, 28). However, TAMs in vivo are also enriched in well-perfused regions of the tumor—such as the invasive edge and perivascular areas—where they potentiate cancer progression and invasion (29–31). Other tumor-associated stromal cells, live, or even dying cancer cells are known to recruit macrophages to the tumor (32–34). Nonetheless, why resident and recruited macrophages do not infiltrate the tumor homogenously remains poorly understood. An intriguing hypothesis then is that TAMs may be precluded from poorly perfused regions because metabolic alterations generate a toxic environment where only adapted tumor cells can survive.Here we show that metabolically altered microenvironments can indeed provide cancer cells with a selective advantage. In particular, these cancer cells are more resistant than macrophages to high levels of lactic acid produced by their glycolytic metabolism. We combine computational modeling with a custom-made cell culture system that allows the emergence of spatially graded microenvironments ranging from well-perfused to ischemic regions. With this approach we show that differential sensitivity to lactic acid between cancer cells and macrophages is sufficient to generate localization patterns that resemble in vivo observations.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

From the Cover: PNAS Plus: Cell-cycle regulation of formin-mediated actin cable assembly

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Eukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5–14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16–19) or cellular extracts (20–25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified proteins differs from that of F-actin assembled by the Arp2/3 complex in the full complexity of cytoplasmic extracts (19, 26–28).Formin-based actin filament assembly using purified proteins also has been reported (29, 30). However, reconstitution of formin-derived actin cables under the more physiological conditions represented by cell extracts has not yet been reported.The actin nucleation activity of formin proteins is regulated by an inhibitory interaction between the N- and C-terminal domains, which can be released when GTP-bound Rho protein binds to the formin N-terminal domain, allowing access of the C terminus (FH1-COOH) to actin filament barbed ends (31–40). In yeast, the formin Bni1 N terminus also has an inhibitory effect on actin nucleation through binding to the C terminus (41).Interestingly, several recent reports provided evidence for cell-cycle regulation of F-actin dynamics in oocytes and early embryos (42–45). However, which specific types of actin structures are regulated by the cell cycle and what kind of nucleation factors and actin interacting-proteins are involved remain to be determined.Here, we report a reconstitution of actin cables in yeast extracts from microbeads derivatized with Bni1 FH1-COOH, identifying the proteins involved, increasing the inventory of the proteins that regulate actin cable dynamics and establishing that the actin cable reconstitution in cytoplasmic extracts is cell-cycle regulated.     

Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP–tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3′-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H₂O₂-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.Exposure to ionizing irradiation (IR) has dire consequences for the cell, because it causes the formation of radicals and reactive oxygen species (ROS) that can oxidize and damage cellular components including proteins and DNA (1), whereas protection from IR-induced radical-mediated protein oxidation can lead to significant radio resistance (2). At the DNA level, IR leads to single-stranded and double-stranded DNA breaks (DSBs), with the latter being a severe threat to cellular survival (3). To cope with DSBs that may arise physiologically and/or by genotoxic environmental impacts such as IR, the cell repairs DSBs by two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR), which predominate in the G1 and G2 phase of the cell cycle, respectively, and underlie cell-cycle-dependent sensitivities to IR exposure (4, 5). In the G2 phase and in the first meiotic prophase, DSB repair is mediated by HR, in yeast meiosis addressing ∼150 DSBs (6, 7) that are formed by the Topo2-related endonuclease/transesterase enzyme SPO11 (8). Absence of DSBs and the resulting compromised spore viability (9) can be partially rescued by ionizing irradiation (10). In all, yeast cells exhibit a high resistance to IR (11–13) which is also true for cells in prophase I (10, 14). In the meiosis of numerous species, programmed Spo11-induced DSBs are instrumental for homologous chromosome search and pairing and provide the substrate for HR, generating two outcomes: noncrossovers (NCO) and crossovers (CO) that allow for homolog segregation in the meiosis I division (reviewed by refs. 15 and 16). For CO to occur, homologous chromosomes need to encounter and pair lengthwise (synapse) during first meiotic prophase (see ref. 17). Homolog pairing occurs after completion of premeiotic DNA replication. Live cell studies in the synaptic meiosis of the yeast Saccharomyces cerevisiae (2n = 32) have shown that meiotic telomeres (18), chromosomes, and bivalents undergo a striking mobility throughout the entire prophase I (19–21), which contrasts with the relative immobility of pachytene bivalents in mammalian prophase I (22). It has been found that rapid and continuous telomere and chromosome movements in budding yeast meiocytes depend on actin polymerization (18–20) and an intact meiotic telomere complex (21, 23, 24). Besides a general mobility of chromosomes throughout prophase I, single bivalents are capable to rapidly move away and return to the motile chromosome mass, a behavior termed “maverick” formation (19) or rapid chromosome movements (20, 21).Exposure to ionizing radiation induces a plethora of physicochemical effects in the irradiated cells including DNA damage (1, 3). Extensive research addressing the adverse effects of IR exposure using yeast as a model system had largely been directed toward mutation induction, DSB repair, and cell cycle effects (e.g., 11–13, 25, 26). Meiotic yeast cells exposed to 50–80 krad (500–800 Gy) X or γ irradiation have been shown to exhibit a profound reduction in cell survival, particularly when exposed in the G1 cell cycle phase that lacks a sister chromatid for repair (4). Irradiated meiotic yeast cells exhibit mutations and chromosome missegregation at meiosis I, leading to reduced sporulation (5, 10, 27). While previous studies addressed late deterministic effects in irradiated yeast cells such as DNA repair, mutations, and cell survival, we were interested in the immediate consequences of IR exposure on motile meiotic chromosomes. Bivalent mobility can be expected to promote chromosomal rearrangements, if it continues after the formation of ectopic unregulated DSBs. Chromosomal translocations have, for instance, been observed after irradiation of mitotic budding yeast cells (28) and of meiotic prophase cells of mice (29). Furthermore, meiotic chromosome mobility has been proposed to be involved in regulating (adverse) chromosomal interactions (30). To study the consequences of IR exposure on meiotic chromosome mobility we followed live bivalent movements in X-irradiated and nonirradiated yeast cells expressing the GFP-tagged version of the synaptonemal complex protein ZIP1 (19) undergoing sporulation.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.