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.     

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133⁻ cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20–30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.Glioblastoma multiforme (GBM) is extremely infiltrative and difficult to treat, and most patients develop recurrence after therapy. Over the past decade, many studies have suggested that bulk GBM tumors harbor cancer stem cells (CSCs) (1, 2), a distinct subpopulation of cancer cells that are able to initiate new tumors efficiently, have long-term self-renewal capacity, and survive better against chemo- or radiotherapy (2–4). CD133 has become a widely used marker for the enrichment of GBM CSCs (GSCs) and other tumor types (5–10). However, recent studies have shown that CD133 is not specific for GSCs because CD133⁻ cells also possess tumor-initiating potential (11–13), indicating the need to identify more specific and exclusive markers for GSCs to facilitate our understanding of GSCs and therapeutic development against GBM. Several reports have proposed L1CAM, A2B5, integrin α6, MET, and CD15 as markers for GSCs (14–18). However, none of these protein markers could be used specifically to identify GSCs, and no study was reported with respect to glycans as potential markers, although glycan biosynthesis involves multiple genes and it is possible to create different structures in cancer progression. It is noted that ganglioside D2 (GD2) and ganglioside D3 (GD3) were found on the surface of neural stem cells (NSCs) and that stage-specific embryonic antigen 3 (SSEA3) and SSEA4 were found on embryonic stem cells and cancer cells (19–21), but there is no glycan marker found on the surface of GSCs.Gangliosides are sialic acid-containing glycosphingolipids (GSLs) that are most abundant in the nervous system (22). The expression levels and patterns of gangliosides during brain development shift from simple gangliosides, such as GM3 and GD3, to complex gangliosides, such as GM1, GD1a, GD1b, and GT1b (23, 24). Moreover, several unique ganglioside markers, including SSEA3, SSEA4, GD2, and GD3, have been identified in stem cells (19). GD3, a b-series ganglioside containing two sialic acids, is highly expressed in mouse and human embryonic NSCs (20, 25). In cancers, GD3 is highly accumulated in human primary melanoma tissues as well as in established melanoma cell lines (26), whereas human normal melanocytes express no or minimal levels of GD3 (27). Moreover, malignant gliomas contain higher levels of GD3, and its expression correlates with the degree of malignancy (28). GD3 is produced from the precursor GM3 by the activity of GD3 synthase (GD3S), which mediates the properties of CSCs through the c-MET signaling pathway and correlates with poor prognosis in triple-negative human breast tumors (29). These findings suggest that GD3 may play an important role in the transformation of normal cells into tumors, and imply that GD3 could be a cell surface marker for GSCs.This study was designed to identify glycan markers for the enrichment of GBM stem cells and then uses these enriched GBM stem cells to characterize tumorigenicity, their association with clinical GBM specimens, and their regulation in tumor progression. The results showed that GD2 and GD3 were positively stained on GBM neurospheres. We found that cells with high GD3 expression display functional characteristics of GSCs. Suppression of GD3S, a critical enzyme for GD3 synthesis, impeded neurosphere formation and tumor initiation. The expression of GD3S correlated with the grades of astrocytomas and mediated self-renewal through c-Met activation. Furthermore, a GD3 antibody was found to eliminate the GD3⁺ cells through complement-dependent cytotoxicity (CDC) in vitro and to suppress tumor growth in mice. These results suggest that GD3 could be a significant biomarker for GSCs, that CD3 could be combined with CD133 for the enrichment of GSCs, and that both GD3 and GD3S could be targets for the development of new therapies against GBM.     

Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers

Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical management, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that eight of 11 GBM cell lines are positive for stage-specific embryonic antigen-4 (SSEA-4), and immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4⁺ and correlated with high-grade astrocytoma. In addition, an SSEA-4–specific mAb was found to induce complement-dependent cytotoxicity against SSEA-4^hi GBM cell lines in vitro and suppressed GBM tumor growth in mice. Because SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines.Glioblastoma multiforme (GBM), accounting for 60–70% of malignant gliomas, is the most aggressive form of glioma and the most common primary brain tumor in adults (1). Despite treatment, including surgery, and chemo- or radiotherapy, the prognosis for GBM patients is poor, with a median survival rate of 14–15 mo (2). GBM is notoriously resistant to most anticancer drugs and is extremely infiltrative, hampering complete surgical resection; therefore most patients develop tumor recurrence or progression even after multiple therapies. Because of the high mortality, new therapeutic approaches, such as immunotherapy and gene therapy, have been proposed for the treatment of GBM (3).Altered glycosylation is a feature of cancer cells, and several glycan structures are well-known tumor markers (4, 5). These aberrant changes include the overall increase in the branching of N-linked glycans (6) and sialic acid content (7) and the overexpression of certain glycan epitopes, such as sialyl Lewis x (sLe^x), sialyl Tn (sTn), Lewis y (Le^y), fucosyl Gb5 (Globo H), and polysialic acid (8–10). Many tumors also exhibit increased expression of certain glycolipids, especially the gangliosides, glycosphingolipids (GSLs) with sialic acid(s) attached to the glycan chain. Gangliosides normally are observed in neural systems and are elevated in tumors, particularly the complex gangliosides associated with malignancy (11).It has been reported that human glioma biopsies show elevation of monosialylated GM3 and GM2 and their disialylated derivatives GD3 and GD2 (12–14). The increase of GD3 was most significant, but the correlation between GD3 and malignancy remains obscure (15, 16). In addition, the lacto-series gangliosides 3′-isoLM1 and 3′,6′-isoLD1 are reported to be major gangliosides in human gliomas (16–18). Because some of these glioma-associated gangliosides are rarely expressed or even are absent in normal tissues (19), they are suitable for targeted therapy (20). Hence, discovering novel glioma-associated GSLs would provide new targets for development of new therapies against gliomas.The GSLs of globo-series feature a Galα1–4Gal linkage to lactosylceramides, and this linkage is catalyzed by the lactosylceramide 4-alpha-galactosyltransferase, encoded by the A4GALT gene. Although globotriosylceramide (Gb3Cer) and globoside (Gb4Cer) constitute the basis of the P-blood group system (21), galactosyl globoside (Gb5Cer) and sialyl galactosyl globoside (sialyl Gb5Cer, SGG, MSGG), also known as “stage-specific embryonic antigen-3” (SSEA-3) and “stage-specific embryonic antigen-4” (SSEA-4) (22), respectively, are cell-surface markers widely used to define human embryonic stem cells (hESCs). Globo-series GSLs also have been observed in tumors: Globo H is overexpressed in many epithelial cancers [e.g., ovarian, gastric, prostate, lung, breast, and pancreatic cancers (23)]; SSEA-3, SSEA-4, and Globo H are expressed not only on breast cancer cells but also on breast cancer stem cells (24, 25). Moreover, high-level expression of SSEA-4 and disialosyl galactosyl globoside (disialosyl Gb5Cer) is observed in renal cell carcinoma (26), but whether globo-series GSLs are expressed in GBM is not known.In the present study, we examined the expression levels of globo-series GSLs and several tumor-associated glycans in GBM cell lines by flow cytometry. The result showed that SSEA-4, a ganglioside rarely found in normal brain tissues, was highly expressed on GBM cells and GBM specimens, as confirmed by high-performance TLC (HPTLC) immunostaining and MS. We found that anti–SSEA-4 mAb (MC813-70) could induce complement-dependent cytotoxicity in vitro and inhibit the growth of GBM in nude mice. SSEA-4 is displayed on many other types of cancers and therefore can be a target for the development of therapeutic antibodies and vaccines against SSEA-4⁺ cancers.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Optogenetic neuronal stimulation promotes functional recovery after stroke

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area’s excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity (1–4). Structural and functional remodeling of areas next to an infarct or remote regions can alter signaling within bilateral neuronal networks and thus contribute to functional recovery (3–7). Rewiring of neural connections is mediated by electrical activity, which can activate a number of plasticity mechanisms, including the release of activity-dependent neurotrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) (8–10). Both BDNF and NGF have been shown to improve recovery by enhancing axonal and dendritic sprouting (10–12).Tremendous effort has been focused on promoting recovery after stroke, including pharmacological treatment, rehabilitation (e.g., constraint-induced therapy), stem cell transplantation, and brain stimulation (1, 4, 13). Brain stimulation is a promising area of research because it allows direct activation and manipulation of the target area’s excitability (14–16). The primary motor cortex (M1) is a commonly stimulated area as it directly innervates the corticospinal tract to initiate movement (1, 7). Although electrical stimulation and transcranial magnetic stimulation show promise in promoting recovery (17, 18), these techniques are limited by imprecision and indiscriminate activation or inhibition of all cell types near the stimulated site; thus, they can produce undesired effects such as psychiatric and motor/speech problems (19–21). In addition, it has been difficult to elucidate the cell type and mechanisms driving recovery, as multiple cell types such as neurons, astrocytes, and oligodendrocytes have been shown to contribute to remodeling and recovery processes after stroke (5, 22–27).To elucidate whether activation of neurons alone can promote recovery, we used optogenetics to selectively manipulate the excitability of specific cell groups with millisecond-scale temporal precision in a manner more similar to endogenous neuronal firing patterns (21, 28, 29). This technique uses light-activated microbial proteins such as Channelrhodopsin 2 (ChR2), which depolarizes neurons when illuminated with blue light, or Halorhodopsin (NpHR), which hyperpolarizes neurons (21, 28, 29). Optogenetic approaches have been used in rodents to probe neuronal circuits for several neurological/neurodegenerative diseases, including Parkinson disease (30) and epilepsy (31). Recent studies have also used optogenetics to map functional organization after stroke (32–35). The safety and efficacy of using optogenetics in nonhuman primates has also been characterized (29, 36).In this study, we used optogenetics to selectively stimulate neurons in layer V of the ipsilesional primary motor cortex (iM1) and examine the effects of repeated neuronal stimulations in normal and stroke mice. Sensory-motor behavior tests were used to evaluate functional recovery after stroke, and plasticity-associated mechanisms, such as cerebral blood flow (CBF)/neurovascular coupling responses and activity-dependent neurotrophin expression, were investigated.     

Highly penetrative,drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.Of the ∼40,000 people diagnosed with primary brain tumors in the United States each year, an estimated 15,000 have glioblastoma multiforme (GBM), a World Health Organization grade IV malignant glioma (1). Despite considerable research efforts, the prognosis for GBM remains poor: median survival with standard-of-care therapy (surgery, systemic chemotherapy with temozolomide, and radiation) is 14.6 mo (2) and 5-y survival is 9.8% (3), with the vast majority of GBMs recurring within 2 cm of the original tumor focus (4). Histopathologically, GBM is characterized by its infiltrative nature and cellular heterogeneity, leading to a number of challenges that must be overcome by any presumptive therapy.The blood–brain barrier (BBB) is a major obstacle to treating GBM (5). It is estimated that over 98% of small-molecule drugs and ∼100% of large-molecule drugs or genes do not cross the BBB (6). Delivery of chemotherapeutics to the brain can be potentially achieved by using nanocarriers engineered for receptor-mediated transport across the BBB (7, 8), but the percentage of i.v. administered particles that enter the brain is low. It is not yet clear whether sufficient quantities of drug can be delivered by systemically administered nanoparticles to make this a useful method for treating tumors in the human brain. An alternate approach is to bypass the BBB: Clinical trials have demonstrated that the BBB can be bypassed with direct, locoregional delivery of therapeutic agents. For example, local implantation of a drug-loaded biodegradable polymer wafer (presently marketed as Gliadel), which slowly releases carmustine over a prolonged period, is a safe method for treating GBM. However, use of the Gliadel wafer results in only modest improvements in patient survival, typically 2 mo (9, 10). In prior work we showed that these wafers produce high interstitial drug concentrations in the tissue near the implant, but—because drugs move from the implant into the tissue by diffusion—penetration into tissue is limited to ∼1 mm, which could limit their efficacy (11, 12).We hypothesize that treatment of GBM can be improved by attention to three challenges: (i) enhancing the depth of penetration of locally delivered therapeutic agents, (ii) providing for long-term release of active agents, and (iii) delivering agents that are known to be effective against the cells that are most important in tumor recurrence. The first challenge can be addressed by convection-enhanced delivery (CED), in which agents are infused into the brain under a positive pressure gradient, creating bulk fluid movement in the brain interstitium (13). Recent clinical trials show that CED is safe and feasible (14–16), but CED alone is not sufficient to improve GBM treatment. For example, CED of a targeted toxin in aqueous suspension failed to show survival advantages over Gliadel wafers (14, 17). Although CED of drugs in solution results in increased penetration, most drugs have short half-lives in the brain and, as a result, they disappear soon after the infusion stops (17, 18). Loading of agents into nanocarriers—such as liposomes, micelles, dendrimers, or nanoparticles—can protect them from clearance. Significant progress has been made in CED of liposomes to the brain (19), although it is not clear that liposomes offer the advantage of long-term release. By contrast, CED of polymeric nanoparticles, such as nanoparticles made of poly(lactide-coglycolide) (PLGA), offers the possibility of controlled agent release. However, CED of PLGA nanoparticles, which are typically 100–200 nm in diameter, has been limited by the failure of particles to move by convection through the brain interstitial spaces (20–23), which are 38–64 nm in normal brain (24) and 7–100 nm in regions with tumor (25). Therefore, to overcome the first and second challenges, it is necessary to synthesize polymer nanocarriers that are much smaller than conventional particles and still capable of efficient drug loading and controlled release. We report here reliable methods for making PLGA nanoparticles with these characteristics.Drug developers have long been frustrated by the BBB, which severely limits the types of agents that can be tested for activity in the brain. We reasoned that creation of safe, versatile, brain-penetrating nanocarriers should enable direct testing of novel agents that address the complexity of GBM biology. For example, cells isolated from distinct regions of a given GBM bear grossly different expression signatures but seem to arise from a common progenitor (26): A small subpopulation of these progenitors drives tumor progression, promotes angiogenesis, and influences tumor cell migration (27–30). These cells have features of primitive neural stem cells and are called brain cancer stem cells (BCSCs) (29, 31–37). BCSCs, many of which are marked by CD133 (PROM1), are resistant to conventional drugs (28, 38), including carboplatin, cisplatin, paclitaxel, doxorubicin, vincristine, methotrexate, and temozolomide (39–42), as well as radiotherapy (29). These observations suggest that agents that affect BCSCs are more likely to lead to a cure for GBM (28, 38, 43, 44). Therefore, to illustrate the translational potential of brain-penetrating nanoparticles, we conducted a screen of ∼2,000 compounds that were previously used in Food and Drug Administration (FDA)-approved products for their ability to inhibit patient-derived BCSCs, encapsulated the best agents to emerge from the screen into brain-penetrating PLGA nanoparticles, and administered these nanocarriers by CED in a BCSC-derived xenograft model of GBM.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

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.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

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.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.