Species formation by host shifting in avian malaria parasites

The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.Cospeciation of symbionts with their hosts has been recognized in parasites with strong vertical transmission (1, 2), viruses that spread by direct contact (3), and bacterial and viral symbionts passed from mother to offspring through the egg (4). Species formation in parasites that are transmitted between hosts by vectors is less well-understood (5, 6). Poor matching between the phylogenetic trees of vector-borne hemosporidian (malaria) parasites and their North American avian hosts suggests a predominance of host shifting compared with cospeciation (7) (reviewed in a broader context in ref. 8). Whether host shifting occurs primarily between closely related hosts and in geographic sympatry, and whether rates of host shifting followed by species formation are sufficient to explain the contemporary diversity of hemosporidian parasites, have not been determined.Many species of hemosporidian parasites have been described and named based primarily on the microscopic morphology of meronts and gametocytes in blood smears (9). The more recent discovery of hundreds of lineages based on DNA sequence variation in the mitochondrial cytochrome b gene (cyt b) (5, 10, 11) requires, however, a different species concept based on analysis of independent components of the genome (12–16). Recent estimates of the rate of molecular evolution in hemosporidian mitochondrial genes imply that the contemporary malaria parasites of vertebrates might have descended from a common ancestor within the past 20 (17) or 40 Ma (18) or, perhaps, a longer time period (19). Although an appropriate calibration for the rate of hemosporidian evolution remains unsettled (20, 21), host shifting almost certainly has been frequent, likely across great host distances at times, over the recent history of the group.Speciation in sympatry (i.e., in the absence of geographic barriers to gene flow through local host specialization) might follow host shifting if mating between parasites was assortative with respect to vertebrate host or if different hosts imposed strong disruptive selection on parasites (22). However, despite some documented feeding preferences (23–25), dipteran vectors of avian malaria parasites do not seem to be sufficiently specialized to isolate populations of parasites on different hosts (26–29). In addition, many parasite species and many parasite lineages distinguished by DNA sequence variation occur locally across broad ranges of hosts without apparent differentiation, at least in the mitochondrial cytochrome b gene (30–32) and several nuclear markers (12, 14). Alternatively, host shifting in one allopatric population of a parasite species could be followed, after sufficient host–pathogen coevolution and evolutionary differentiation to produce reproductive incompatibility, by secondary sympatry, thereby increasing local parasite diversity.Here, we examine recent nodes in an mtDNA-based phylogeny of New World hemosporidian parasites to determine the degree to which lineage formation is associated with host shifting. Although our phylogenetic reconstruction is based on a single mitochondrial gene (cyt b), phylogenies based on genes from the mitochondrial, nuclear, and apicoplast genomes are broadly consistent for the relatively recent nodes considered in this analysis (6, 12–14, 33–35). In addition, analyses of avian hemosporidian parasites based on multiple independent markers have distinguished mtDNA-defined lineages on the basis of significant linkage disequilibrium (13).We distinguish as species the lineages that differ in their mtDNA cytochrome b gene sequence (by as few as 2 nt) and, for the most part, occur in either different hosts in the same local area or the same or different hosts in different geographic areas (32, 36). In some cases, closely related lineages occur in the same host locally. Sister lineages in this analysis differ by an average of about 1% sequence divergence, although some sequences separated by as little as a single nucleotide can exhibit consistent host or geographic differences. Inference concerning the mode of species formation is based primarily on host and geographic distributions of these hemosporidian mtDNA lineages. However, the correspondence between lineages and reproductively isolated species is poorly resolved (13, 37, 38). Each node was designated as either sympatric or allopatric depending on whether the descendant lineages occurred on the same West Indian islands or in the same regions within larger continental areas. The status of closely related parasite lineages occurring locally in the same host species is ambiguous, but these lineages might reflect genetic variation within a parasite species.Previous analyses have suggested that host shifting, rather than codivergence, predominates species formation in the hemosporidian parasites of birds (5, 7). We find this most frequently to be the case in this analysis, and we discuss whether species formation by host shifting occurs primarily in sympatry or allopatry.     

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.     

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.     

Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.Toxoplasma gondii infects around a third of humans worldwide and is widely dispersed in other warm-blooded hosts. Although clinical manifestations in the brain, eye, and developing fetus receive the most attention, T. gondii is an oral pathogen and first enters the body and establishes infection in the small intestine. Infection follows consumption of cyst-containing meat or oocyst-contaminated water and produce and is associated with the development of small intestinal pathology in a variety of nonhuman hosts (1). Most notably, experimental infection of C57BL/6 mice by the oral route results in an inflammation of the small intestine that shares immunological features with inflammatory bowel disease (2). This model is useful to further our understanding of host–pathogen interactions in the intestine and of common mechanisms underpinning the development of inflammatory bowel disease (3). Nevertheless, we have limited understanding of how and in which cells infection is established in the intestine, the extent to which the parasite replicates and spreads within the intestine, and how these factors contribute to the development of pathology (2, 4–9). The ability to label living parasites fluorescently and track them in the tissues of infected hosts provides an important tool for investigating these questions (10–14).Starting in the small intestine, T. gondii must travel long distances and surmount a variety of biological barriers to establish chronic infection in the brain. These barriers include the mucus, the intestinal epithelium, and the blood–brain barrier (7, 15). Cells of the immune system are often highly motile and represent attractive transport vessels for pathogens seeking to reach and enter tissues while being protected from the external environment. Consequently, recent studies have focused on the role of immune cells in transporting parasites between tissues (4, 16–23). For example, cluster of differentiation 11b-positive (CD11b⁺) cells have been implicated in the dissemination of T. gondii through the blood and across the blood–brain barrier (4, 19). Following oral infection, it is thought that the initial invasion or traversal of intestinal epithelial cells by ingested parasites is followed by parasite replication in tissue and the transport by host cells to other tissues. However, our understanding of how the parasite enters and disseminates through the intestine itself and of the role played by immune cell populations in this initial phase of infection is extremely limited.Two-photon microscopy provides important spatial and dynamic information to further our understanding of how pathogens interact with their hosts in complex natural tissue environments (10). However, such techniques rarely have been applied to the intestine, particularly in the context of infection (14, 24–28). Here we used a physiologically relevant oral infection model in conjunction with two-photon microscopy to reveal that neutrophils in the lumen of the small intestine are motile reservoirs of live T. gondii. Our findings implicate these cells in an unexpected luminal pathway for the spread of infection.     

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).     

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.     

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.     

The pathogen Batrachochytrium dendrobatidis disturbs the frog skin microbiome during a natural epidemic and experimental infection

Symbiotic microbial communities may interact with infectious pathogens sharing a common host. The microbiome may limit pathogen infection or, conversely, an invading pathogen can disturb the microbiome. Documentation of such relationships during naturally occurring disease outbreaks is rare, and identifying causal links from field observations is difficult. This study documented the effects of an amphibian skin pathogen of global conservation concern [the chytrid fungus Batrachochytrium dendrobatidis (Bd)] on the skin-associated bacterial microbiome of the endangered frog, Rana sierrae, using a combination of population surveys and laboratory experiments. We examined covariation of pathogen infection and bacterial microbiome composition in wild frogs, demonstrating a strong and consistent correlation between Bd infection load and bacterial community composition in multiple R. sierrae populations. Despite the correlation between Bd infection load and bacterial community composition, we observed 100% mortality of postmetamorphic frogs during a Bd epizootic, suggesting that the relationship between Bd and bacterial communities was not linked to variation in resistance to mortal disease and that Bd infection altered bacterial communities. In a controlled experiment, Bd infection significantly altered the R. sierrae microbiome, demonstrating a causal relationship. The response of microbial communities to Bd infection was remarkably consistent: Several bacterial taxa showed the same response to Bd infection across multiple field populations and the laboratory experiment, indicating a somewhat predictable interaction between Bd and the microbiome. The laboratory experiment demonstrates that Bd infection causes changes to amphibian skin bacterial communities, whereas the laboratory and field results together strongly support Bd disturbance as a driver of bacterial community change during natural disease dynamics.Symbiotic interactions between microbes and multicellular organisms are ubiquitous. In recent years, research to understand the complex microbial communities living in or on multicellular organisms (termed the microbiome) has sparked fundamental changes in our understanding of the biology of metazoans (1–5). The microbiome can affect host health directly by influencing metabolism (6), development (7), inflammation (8), or behavior (9), but it may also influence host health indirectly through interactions with infectious pathogens. The microbiome may interact with pathogens through competition for resources, release of antimicrobial compounds, contact-dependent antagonism, or modulation of the host immune response (10), and an “imbalanced” microbiome may leave the host more susceptible to pathogen infection (11, 12). At the same time, an invading pathogen may disrupt the microbiome (10, 13–15). Thus, the microbiome may play a role in disease resistance, or may itself be disturbed or altered by invading pathogens. Although a wealth of recent research has described associations between microbiome composition and a variety of syndromes in both humans and animals (16–25), documentation of microbiome responses to natural epidemics of known infectious pathogens is rare.Chytridiomycosis is an emerging infectious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Bd is an aquatic fungus that infects the skin of amphibians and disrupts osmoregulation, a critical function of amphibian skin (26). Chytridiomycosis can be fatal, and the severity of disease symptoms has been linked to Bd load, which is a measure of the density of Bd cells infecting the host (27, 28). Bd has a broad host range spanning hundreds of amphibian species, and has been implicated in population extinctions and species declines worldwide (29–34). Efforts to understand and mitigate the effects of Bd have led to research examining the potential for symbiotic bacteria to increase resistance to infection by the pathogen (35, 36). Bacterial species isolated from the skin of amphibians have been shown to inhibit the growth of Bd and other fungal pathogens in culture (37–39), possibly by producing antifungal metabolites (40, 41). In a controlled laboratory experiment, inundation of Rana muscosa with the bacterium Janthinobacterium lividium protected frogs from subsequent Bd infection (42). These and other studies highlight the possible role of bacteria in resistance to chytridiomycosis, but critical questions remain. First, most research has focused on the ability of cultured bacteria to prevent Bd infection, whereas very little is known about whether Bd infection alters the diverse skin microbiome. Examining this latter concept is critical both to a basic understanding of how the microbiome interacts with pathogens and to conservation efforts because Bd-induced perturbations of the microbiome could undermine attempts to mitigate effects of Bd infection through augmentation with particular bacteria. A second knowledge gap is the paucity of comprehensive culture-independent assessments of the amphibian microbiome, which are important because the vast majority of environmental and symbiotic microbes are not readily cultured, and culture-based methods can lead to severe underestimates of diversity and biased assessment of community composition (43). Few studies have applied next-generation sequencing methods to characterize the microbial communities on amphibian skin (44–47), and, to our knowledge, none have done so in the context of Bd infection. A final challenge to understanding interactions between Bd and bacteria stems from the difficulties of drawing direct connections between laboratory and field studies. Laboratory studies are essential for definitive identification of cause and effect. However, complex natural microbiomes can be impossible to recreate in the laboratory, and field studies are needed to show whether processes identified in the laboratory are relevant in nature.We present paired laboratory and field studies using high-throughput 16S amplicon pyrosequencing both to document associations between Bd infection and the amphibian skin bacterial microbiome in nature and to deduce causal relationships in an experiment. Our work centers on the Sierra Nevada yellow-legged frog, Rana sierrae, which is severely threatened by, and has already suffered drastic declines due to, Bd (28, 48). We surveyed frogs from four distinct R. sierrae populations to test if differences in skin bacterial communities are associated with the intensity of pathogen infection. We then conducted a laboratory experiment to establish causal relationships underlying Bd-bacterial community associations. The data establish a strong effect of Bd infection on the composition of the amphibian skin bacterial microbiome that is consistent between the laboratory experiment and naturally occurring Bd dynamics in wild frog populations.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

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.     

Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis

Distinguishing tumor from normal glandular breast tissue is an important step in breast-conserving surgery. Because this distinction can be challenging in the operative setting, up to 40% of patients require an additional operation when traditional approaches are used. Here, we present a proof-of-concept study to determine the feasibility of using desorption electrospray ionization mass spectrometry imaging (DESI-MSI) for identifying and differentiating tumor from normal breast tissue. We show that tumor margins can be identified using the spatial distributions and varying intensities of different lipids. Several fatty acids, including oleic acid, were more abundant in the cancerous tissue than in normal tissues. The cancer margins delineated by the molecular images from DESI-MSI were consistent with those margins obtained from histological staining. Our findings prove the feasibility of classifying cancerous and normal breast tissues using ambient ionization MSI. The results suggest that an MS-based method could be developed for the rapid intraoperative detection of residual cancer tissue during breast-conserving surgery.Breast cancer is the most commonly diagnosed carcinoma in women in the United States and Western countries. Breast conservation surgery (BCS) has become the preferred treatment option for many women with early-stage breast cancer (1). BCS entails resection of the tumor, with a clean margin of normal tissue around it. Surgery is usually followed by radiation therapy. Results from seven large randomized prospective studies, with the largest two having over 20 y of follow-up, have shown equal survival when comparing BCS coupled with whole-breast radiation and mastectomy (2, 3).Normally, breast surgeons aim to remove a patient’s tumor, along with a rim of normal tissue that is free of cancer. Preoperative mammography, ultrasonography, or MRI may be used by the surgeon to guide adequate resection (4–6). Despite numerous improvements in imaging and surgical technique, the need for reexcision to achieve complete tumor resection in the United States typically ranges from 20–40% (7–15), and has been reported as being as high as 60% (16). The importance of reexcision is underscored by numerous studies, which have shown that incomplete resection of tumor and positive margins are associated with increased locoregional recurrence compared with negative margins (12, 17–20). Furthermore, the landmark meta-analysis performed by the Early Breast Cancer Trialists’ Collaborative Group (18, 21) directly linked local recurrence to survival, placing great emphasis on the surgeon’s role in minimizing local recurrence by obtaining adequate margins.Breast tumor reexcisions are accompanied by a number of undesirable problems: The completion of therapy is delayed, infection rates are increased, cost is increased, there can be a negative psychological impact on the patient, and there can be diminished aesthetic outcomes (22–24). The development of an intraoperative technique that allows the fast and accurate identification of residual tumor at surgical resection margins could decrease the reexcision rate, and therefore improve the care delivered to patients with cancer who are receiving BCS.To this end, multiple intraoperative methods have been explored, with various benefits as well as limitations. These methods include touch frozen section analysis (25), touch preparation cytology (26), specimen radiography (27, 28), rf spectroscopy (29, 30), Raman spectroscopy (31), radioguided occult lesion localization (32), near-IR fluorescence (33, 34), and high-frequency ultrasound (35–37). The intraoperative application of MRI, which has been successfully applied in brain surgery (38–42), is limited in its application in BCS. These limitations include MRI interpretation in the presence of acute surgical changes; lack of real-time imaging, requiring the interruption of surgery; and accurate localization of tumor based on images requiring development of fiducials (43–46).Mass spectrometry imaging (MSI) has been applied to investigate the molecular distribution of proteins, lipids, and metabolites without the use of labels (47, 48). In particular, the newly developed ambient ionization technique of desorption electrospray ionization (DESI) allows direct tissue analysis with little to no sample preparation (49, 50). Therefore, with the advantage of easy use, DESI-MSI has great potential in the application of intraoperative tumor assessment. The development of DESI-MSI enables the correlation of lipid distribution in two or three dimensions with tissue morphology (47, 51) and the distinction of cancerous from noncancerous tissues based on lipidomic information (52–54). Distinctive lipid profiles associated with different human cancers have been investigated by DESI-MSI (55–58). Moreover, the grades and subtypes of human brain tumors have been discriminated using this technique. Additionally, tumor margins have been delineated using DESI-MSI, and the results have been correlated with histopathological examination (59, 60).It has been reported that breast cancer demonstrates metabolic profiles that are distinct from those metabolic profiles found in normal breast tissue. This finding suggests a potential for using metabolite information for breast cancer diagnosis and tumor margin identification (61, 62). Here, we demonstrate an MS-based methodology for using lipidomic information to distinguish cancerous from noncancerous tissue and to delineate tumor boundaries.     

Malaria-induced changes in host odors enhance mosquito attraction

Vector-borne pathogens may alter traits of their primary hosts in ways that influence the frequency and nature of interactions between hosts and vectors. Previous work has reported enhanced mosquito attraction to host organisms infected with malaria parasites but did not address the mechanisms underlying such effects. Here we document malaria-induced changes in the odor profiles of infected mice (relative to healthy individuals) over the course of infection, as well as effects on the attractiveness of infected hosts to mosquito vectors. We observed enhanced mosquito attraction to infected mice during a key period after the subsidence of acute malaria symptoms, but during which mice remained highly infectious. This attraction corresponded to an overall elevation in the volatile emissions of infected mice observed during this period. Furthermore, data analyses—using discriminant analysis of principal components and random forest approaches—revealed clear differences in the composition of the volatile blends of infected and healthy individuals. Experimental manipulation of individual compounds that exhibited altered emission levels during the period when differential vector attraction was observed also elicited enhanced mosquito attraction, indicating that compounds being influenced by malaria infection status also mediate vector host-seeking behavior. These findings provide important insights into the cues that mediate vector attraction to hosts infected with transmissible stages of malaria parasites, as well as documenting characteristic changes in the odors of infected individuals that may have potential value as diagnostic biomarkers of infection.Parasite manipulation of hosts is a widespread phenomenon with broad significance for ecology and human health (1–5). Increased attention has recently focused on manipulation by vector-borne parasites (2, 6, 7), which may enhance their own transmission via direct effects on vector behavior (6–10) or by altering traits of their primary hosts in ways that influence vector attraction and dispersal, as well as the likelihood of pathogen acquisition by vectors during interactions with the primary host (6, 7, 10–12). In the case of pathogens vectored by insects, host odors seem particularly likely targets for manipulation, as olfactory cues play a key role in host location and discrimination by both plant- and animal-feeding insects. And a number of recent studies have documented pathogen-induced effects on volatile mediated host-vector interactions (11–16). In addition to their ecological significance, pathogen-induced changes in host-derived olfactory cues have potential applied implications for efforts to disrupt vector transmission (e.g., via the development of chemical lures or repellents), as well as for disease diagnosis. Indeed, given that a key challenge for the development of volatile-based diagnostics lies in recognizing the “signal” of disease presence against the background “noise” of genetic and environmental variation (17), it is plausible that volatile biomarkers will prove particularly valuable for detecting pathogens that actively manipulate host odors, although little work to date has explored this possibility.The current study explores potential manipulation of host odors by protozoan parasites in the genus Plasmodium responsible for causing malaria, which remains among the deadliest of human diseases and a significant hindrance to economic development in regions where it occurs (18, 19). A good deal of previous research has documented effects of the plasmodium parasites on the physiology and behavior of mosquito vectors (8, 10, 20–24). There is reason to suspect that manipulation of host odors by these parasites also influences vector behavior. For example, a provocative study found that Kenyan children harboring the transmissible (gametocyte) stage of the malaria parasite Plasmodium falciparum were more attractive to mosquitoes than uninfected children or those harboring the nontransmissible stage of the parasite (25). The cues responsible for this enhanced attraction were not identified, but parasite-induced changes in host odors seem the likeliest explanation, as the attraction occurred at a distance and was apparently not explained by variation in body heat or activity (as all of the children involved in the study were asymptomatic). A subsequent study documented preferential blood feeding by the mosquito Culex pipiens on canaries (Serinus canaria) infected with the avian malaria parasite Plasmodium relictum, but the cues mediating this preference were again not determined (26).As noted above, the identification of pathogen-induced changes in host odors that influence vector behavior has potential applied implications, and this is particularly true for malaria. Minimizing transmission by mosquito vectors is a key focus of efforts to control this devastating disease, but resistance evolution poses a continual challenge for strategies that entail suppressing vector populations (27–30). An improved understanding of the ecological mechanisms mediating vector transmission may inform the development of more effective and sustainable control strategies. Furthermore, the ability to effectively direct drug treatments and other interventions to asymptomatic carriers of infection is a key issue for controlling disease spread and likely essential for the long-range goal of malaria eradication (31). Thus, the presence of volatile biomarkers capable of distinguishing asymptomatic individuals bearing the transmissible stage of the disease—as suggested by the findings of the Kenyan field study discussed above—could potentially have great diagnostic value.With these issues in mind, we initiated laboratory studies using a mouse model and the rodent malaria parasite Plasmodium chabaudii to confirm and elucidate the role of parasite-induced volatile cues in mediating preferential vector attraction to infected individuals. Our specific goals were to assess the relative attractiveness of infected individuals to mosquito vectors (compared with healthy controls) over the course of infection and to document associated differences in the volatile profiles of healthy and infected individuals.