Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells

A new paradigm in oncology establishes a spectrum of tumorigenic potential across the heterogeneous phenotypes within a tumor. The cancer stem cell hypothesis postulates that a minute fraction of cells within a tumor, termed cancer stem cells (CSCs), have a tumor-initiating capacity that propels tumor growth. An application of this discovery is to target this critical cell population using chemotherapy; however, the process of isolating these cells is arduous, and the rarity of CSCs makes it difficult to test potential drug candidates in a robust fashion, particularly for individual patients. To address the challenge of screening drug libraries on patient-derived populations of rare cells, such as CSCs, we have developed a drug-eluting microarray, a miniaturized platform onto which a minimal quantity of cells can adhere and be exposed to unique treatment conditions. Hundreds of drug-loaded polymer islands acting as drug depots colocalized with adherent cells are surrounded by a nonfouling background, creating isolated culture environments on a solid substrate. Significant results can be obtained by testing <6% of the cells required for a typical 96-well plate. Reliability was demonstrated by an average coefficient of variation of 14% between all of the microarrays and 13% between identical conditions within a single microarray. Using the drug-eluting array, colorectal CSCs isolated from two patients exhibited unique responses to drug combinations when cultured on the drug-eluting microarray, highlighting the potential as a prognostic tool to identify personalized chemotherapeutic regimens targeting CSCs.Tumor-initiating cancer stem cells (CSCs) are being investigated as a promising therapeutic target (1). The rarity of CSCs, which constitute ∼1% of tumor cells (1, 2), limits their availability for testing, and traditional screening methods require substantial cell quantities. Industrial pharmaceutical capabilities have successfully reduced cell requirements in drug screening, but such capital-intensive facilities are typically unavailable to clinicians and pathology laboratories. The past decade has witnessed the emergence of multiple cell-based microarray platforms that address availability and cell source limitations (3–5), although these systems have inherent shortcomings. Many rely on immobilizing target molecules (6–9), limiting applicability to small molecule drug libraries, whereas others rely on robotically spotting cells (10), a technique not amenable to widespread adoption. Array platforms capable of capturing single cells have been established (11, 12), but determination of chemotherapeutic efficacy is better investigated through methods using greater cell numbers, which better capture variability in cellular responses. Furthermore, arrays of drug-loaded polymer films with an overlying cell monolayer have been developed (13), but monolayers of cells are susceptible to juxtacrine and paracrine signaling, which are particularly important for multipotent cells. In the present work, the provision of differential cell adhesion to promote seeding onto spotted drug-loaded films against a surrounding nonfouling background (i.e., a surface that resists protein adsorption and thus cell adhesion) can separate drug-eluting polymer films to create isolated culture environments. The use of programmable arraying techniques can then enable fabrication of uniquely formulated drug-eluting spots that provide prescribed drug doses and drug combinations to overlying cells for simultaneous testing on a single device.It is becoming increasingly evident in cancer treatment that simultaneously targeting multiple critical pathways using combinations of chemotherapeutic drugs can enhance outcomes (14–17). Conventional screening of chemotherapeutics uses an established panel of cancer cell lines (18) that have been derived from bulk tumors. A recently developed clinical approach involves performing in vitro chemosensitivity testing of tumor biopsy specimens to individualize treatment (19, 20). Unfortunately, benefits have been limited, with poor correlations between bulk tumor cell sensitivity and clinical efficacy. This lack of efficacy has been attributed to patient to patient variability, owing in part to intratumor heterogeneity (21–23).Tumors consist of multiple cell phenotypes. In the CSC model, a rare cell population of tumor-initiating cells perpetually self-renew and are responsible for tumor heterogeneity, metastasis, and disease recurrence (1, 24). Recent identification of unique cell surface markers that enrich tumor cell isolates for CSCs have led to novel techniques for isolating enriched colorectal CSC (CCSC) populations from patient tumor samples (25–28). For example, xenotransplantation of a single CCSC identified by high Wnt/Β-catenin signaling activity generates tumors that recapitulate the diverse phenotypic heterogeneity of the original tumor (29). Thus, identifying and isolating CCSCs out of the tumor bulk from an individual cancer patient and determining sensitivity to chemotherapeutic drugs in vitro is possible (30, 31). An approach such as the drug-eluting microarray, enabling use of low cell numbers, could potentiate personalized combination drug treatment screens for efficacy against patient-specific CSCs.     

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.     

From the Cover: Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A₂, binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A₂ and the gRNA. A₂ contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA–coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.Single-stranded (ss) RNA viruses are an abundant type of virus and infect all domains of life (1–4). One of the best-studied ssRNA virus systems is the Leviviridae, which infects Gram-negative bacteria via a variety of retractile pili (5); extensive genetic and biochemical studies have been performed on two of these phages: MS2 and Qβ (5–11). All of the Leviviridae have the same core genome, spanning 3.4–4.3 kb, encoding the maturation protein, the coat protein, and a subunit of RNA-dependent RNA replicase (SI Appendix, Fig. S1) (10). The MS2-like phages, designated true leviviruses, have a fourth gene that encodes the lysis protein, whereas the Qβ-like phages, designated alloleviviruses, have the lysis function as an additional feature of the maturation protein (called A₂ in Qβ). Qβ also encodes a minor coat protein, called A₁, arising from occasional read-through of the stop codon of the major coat protein; it has been estimated that the A₁ protein replaces 3–10 copies of the major coat protein in the virion (11) and is required for infection (12). A₁ consists of a coat domain and a read-through domain separated by a flexible linker (13). Unlike most dsDNA phages, which use specialized protein machinery to pump their genomic DNA into a capsid preassembled around a protein scaffold (14–17), ssRNA viruses, including the Leviviridae, assemble their coat proteins around the genomic RNA (gRNA), presumably because the extremely small genome does not allow for genes to encode proteins that help package the genetic material. Therefore, ssRNA viruses require direct gRNA–coat protein interactions, some of which are specific, to self-assemble the virion (18). This raises interesting questions, including how the viral RNA is selectively encapsidated over host RNAs. Addressing these questions will lead to a better understanding of the physiology of ssRNA viruses, and potentially novel therapeutics against them (19).In Leviviridae, both Qβ and MS2 have capsids with T = 3 morphology, meaning each coat protein monomer adopts one of three conformers, termed A, B, and C (20, 21). Within the viral capsid, conformers A and B form an asymmetric A/B dimer and two C conformers form a symmetric C/C dimer. The gRNAs of Qβ and MS2 form secondary structures both in vitro (22, 23) and in vivo (7). There is a specific RNA hairpin near the start of the replicase gene called the “operator,” which has a high binding affinity for the coat protein (24, 25). Although both MS2 and Qβ operators form hairpins, the coat proteins for each phage are selectively attracted to their specific operator to the point at which, if purified RNAs from both phages are mixed with the coat protein of just one phage, only the cognate RNA is encapsidated (8). In MS2, the maturation protein binds to specific regions on the 5′ and 3′ of the gRNA during capsid assembly (26).Neither the maturation protein nor the gRNA of Leviviridae was resolved in the previous high-resolution structural studies by X-ray crystallography, most likely because of the asymmetry of these two components in the stacking of symmetric particles within the crystals (20, 21). The first glimpse of RNA within the capsid came from a low-resolution structure of MS2 using single-particle cryo-EM (27). On the basis of another single-particle cryo-EM structure with icosahedral symmetry applied, it was proposed that MS2 has two concentric shells of RNA within the phage capsid (28). Subsequent work, using cryo-electron tomography and subtomogram averaging, yielded a 39-Å resolution symmetry-free density map of MS2 (29), suggesting the gRNA adopts the same conformation for each virus particle within the capsid and the maturation protein replaces a C/C dimer of the coat proteins. However, at this low resolution, it was not clear how the maturation protein, which contributes ∼1% of the molecular mass of the entire virion, interacts with the rest of the capsid or how the gRNA is organized inside the capsid shell.In this study, we report the cryo-EM structures of the canonical Allolevivirus Qβ with and without symmetry applied, at 3.7- and 7-Å resolutions, respectively. Our structures reveal features never seen before for Qβ, such as the structure of A₂, symmetry deviation of the coat proteins, organization of gRNA, and the interactions between them. These results are discussed in terms of a model for viral assembly and gRNA release in Qβ.     

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.     

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways.The first human genome sequence was determined more than 10 years ago (1, 2), and the revolution in sequencing technology has facilitated the deciphering of hundreds more human and cancer genomes since then (3, 4). The next frontier is the development of strategies for the systematic elucidation of gene function in health and disease contexts.RNAi technology has facilitated genetic approaches in mammalian cells, but the analysis of genome-wide RNAi screens remains challenging (5). Major confounding factors are false-negative results caused by insufficiently active shRNAs and false-positive results caused by off-target effects. Indeed, the challenges of off-target effects have been highlighted recently in papers by Schultz et al (6) and Adamson et al. (7), which show that these effects can be pervasive in genome-wide screens and are not robustly detected by some of the standard precautions typically used.Furthermore, even when hit genes are identified correctly, effective follow-up to uncover their function often requires intense effort. In yeast and other microorganisms, high-density genetic interaction (GI) maps have been highly successful at defining gene function, revealing functional relationships between previously uncharacterized genes and elucidating cellular pathways (8–16). GIs quantify the effect that the loss of function of one gene has on the phenotype caused by the loss of function of another gene. In GI maps, GIs are determined for a large number of pairwise combinations of genes, and genes are clustered based on the similarity of their GI patterns. The clustering typically reveals groups of genes that encode physically interacting proteins or act in a common pathway (17).In human cells, such a systematic elucidation of the functional interactions between human genes will be key to understanding how combinations of genes cause common polygenetic diseases and to developing precise therapies based on a patient’s genetic background. Additionally, GI maps can detect rare synthetic lethal gene pairs, which would be ideal drug targets for combination therapies that prevent the development of drug resistance of rapidly evolving diseases like cancer.We recently established a technology platform for constructing high-density GI maps in mammalian cells based on pooled shRNA screens and demonstrated its potential by using it to identify pathways controlling the sensitivity of human cells to the toxin ricin (18). Our two-step strategy integrates several key innovations to address the challenges to the effective application of RNAi-based approaches to study gene function in mammalian systems. A primary genome-wide screen is conducted using an ultra-complex shRNA library to identify genes of interest. A double-shRNA library targeting all pairwise combinations of hit genes from the primary screen then is used to measure genetic interactions based on a pooled experiment.Here, we describe a principled framework for collecting and analyzing data and illustrate the broad potential of our approach. We extract different quantitative, shRNA-intrinsic phenotypes, such as growth or drug sensitivity, from pooled screens. We establish a strategy for robust identification of hit genes in primary screens. Individual shRNAs targeting these hit genes are selected to construct focused single-shRNA and double-shRNA libraries. We demonstrate the use of focused libraries to compare pathways controlling sensitivity of different human cell lines to ricin and Shiga toxin. We describe our strategy for constructing a high-density GI map based on pooled screening of a double-shRNA library. We show how shRNAs with partial off-target effects can be identified and removed from GI maps. GI maps can be constructed based on different quantitative phenotypes. As described in Discussion, our strategy has several features that distinguish it from array-based approaches in which individual combinations of genes are targeted by interfering RNAs in separate wells (19(–21). Importantly, the same double-shRNA library can be screened rapidly for different phenotypes to compare context-specific GI maps. We compare a growth-based GI map and a ricin resistance-based GI map to illustrate how this strategy facilitates the dissection of functional pathways in different conditions and cellular states.     

Interplay between insulin signaling,juvenile hormone,and vitellogenin regulates maternal effects on polyphenism in ants

Polyphenism is the phenomenon in which alternative phenotypes are produced by a single genotype in response to environmental cues. An extreme case is found in social insects, in which reproductive queens and sterile workers that greatly differ in morphology and behavior can arise from a single genotype. Experimental evidence for maternal effects on caste determination, the differential larval development toward the queen or worker caste, was recently documented in Pogonomyrmex seed harvester ants, in which only colonies with a hibernated queen produce new queens. However, the proximate mechanisms behind these intergenerational effects have remained elusive. We used a combination of artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and vitellogenin quantification to investigate how the combined effect of environmental cues and hormonal signaling affects the process of caste determination in Pogonomyrmex rugosus. The results show that the interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on the production of alternative phenotypes and set vitellogenin as a likely key player in the intergenerational transmission of information. This study reveals how hibernation triggers the production of new queens in Pogonomyrmex ant colonies. More generally, it provides important information on maternal effects by showing how environmental cues experienced by one generation can translate into phenotypic variation in the next generation.Many plants and animals can express specific adaptive responses to their environment through phenotypic plasticity, whereby a given genotype can develop into different phenotypes depending on environmental conditions (1, 2). Maternal effects, through which the environmental conditions experienced by the mother are translated into phenotypic variation in the offspring (3, 4), contribute to many phenotypic traits in a wide variety of taxa (5, 6) and have important ecological and evolutionary consequences (7, 8). Investigating the mechanisms of cross-generational transmission of information underlying maternal effects is needed to better understand the optimization of phenotypes in changing environments (6) and, more generally, the evolution of life history strategies (9).In many insect species, maternal effects are known to affect polyphenism (3, 10), an extreme form of phenotypic plasticity characterized by the production of alternative and discrete phenotypes from a single genotype (1, 11–13). Such maternal effects allow adequate responses to environmental cues such as temperature, photoperiod, nutrition, and population density in many species (10). Examples of maternal effects on insect polyphenism include the production of sexual versus parthenogenetic morphs in aphids (14, 15), winged versus wingless morphs in firebugs (16), and dispersal versus solitary morphs in locusts (17, 18). The endocrine system was found to play a role in the regulation of some maternal effects on insect polyphenisms (19–21), but the nature of the physiological and genetic pathways interacting with the hormonal system to translate environmental cues into offspring polyphenism remains mostly unknown (22).The most striking example of polyphenism is found in insect societies (23), where a reproductive division of labor leads to the coexistence of fertile queens and sterile workers that greatly differ in morphology and behavior (24, 25). Even though recent studies revealed genetic influences on caste determination in social insects (reviewed in ref. 26), female caste fate is primarily influenced by environmental factors in most species studied (27–39). In ants, several studies suggested that maternal factors such as temperature or queen age may affect caste determination (40–44). However, it is only recently that the first example of maternal effects on female caste polyphenism was documented experimentally (45). Cross-fostering of eggs between hibernated and nonhibernated Pogonomyrmex colonies revealed strong maternal effects on caste production, as only eggs produced by a hibernated queen were able to develop into queens, irrespective of the hibernation status of the rest of the colony (45). Such maternal effects on the caste fate of the female offspring require that the hibernation triggers changes in the queen that affect polyphenism in the offspring. Hormones may be involved in this process in Pogonomyrmex ants, as Pogonomyrmex rugosus queen- and worker-destined eggs differed in their ecdysteroid content (45) and Pogonomyrmex barbatus mature queens treated with juvenile hormone (JH) were recently found to produce larger workers (46).Studies on the mechanisms regulating insect polyphenisms (reviewed in ref. 10) suggest that the insulin/insulin-like growth factor signaling (IIS), JH, and vitellogenin (Vg) pathways, known to regulate reproduction in adult insects (47–51), play predominant roles in modulating larval development in response to environmental cues. A well-known example illustrating the role of these pathways is the caste fate of the female brood (queen or worker) in the honey bee Apis mellifera (52–58). In this species, worker-triggered differences in larval diet induce changes in IIS that affect JH (57), possibly through the release of neuropeptides (e.g., allatostatin and allatotropin) that influence JH production by the corpus allatum, as found in Drosophila (59). Changes in JH in turn affect the production of Vg (60–62), which may be involved in the process of caste determination (62, 63). Such effects of JH on Vg production, also reported in flies (64), locusts (65), and cockroaches (66), have been proposed to involve the action of ecdysteroids (62, 67–70). IIS, JH, and Vg may also play a role in the regulation of caste differentiation of larvae in ants, as caste-specific expressions of genes involved in the IIS pathway were documented in Solenopsis invicta (71) and Diacamma sp. (72). Interestingly, caste-specific differences in IIS, JH, and Vg were also documented in adult ants and bees (48, 73–78), suggesting further roles of these pathways in the regulation of social life (74, 79).We propose that the interplay between IIS, JH, and Vg regulates maternal effects on caste polyphenism in ants by translating the environmental conditions experienced by the queen during hibernation into the production of alternative phenotypes in the offspring. Under this hypothesis, IIS would translate environmental cues into changes in JH, which would, in turn, affect the amount of Vg in queens and in eggs, thus possibly affecting the caste fate of the offspring (62, 63). This hypothesis makes four predictions. First, a pharmacological increase of JH in queens should mimic the effect of hibernation and stimulate the production of queens. Second, hibernation should affect IIS and the production of JH in queens. Third, both hibernation and a JH increase should stimulate the production of Vg in queens. Finally, Vg content should differ between queen- and worker-destined eggs. We tested these predictions by performing artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and Vg quantification in Pogonomyrmex rugosus, an ant species in which temperature-triggered changes in the queen had previously been shown to affect the relative production of queens and workers. Each of the four predictions was confirmed by our experiments, thus revealing that the interplay between IIS, JH, and Vg regulates maternal effects on caste polyphenism in P. rugosus.     

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.The genomes of eukaryotes originate from ancient endosymbiotic associations that eventually led to energy-harnessing organelles: mitochondria, common to all eukaryotes, and chloroplasts in the “green” lineage. The evolution of endosymbionts into cellular organelles was accompanied by massive gene loss, with a large proportion being transferred to the nucleus (1, 2). Nevertheless, mitochondria and chloroplasts retained a few (30–80) protein-encoding genes that play crucial roles in energy metabolism (respiration and photosynthesis). Mitochondrion and chloroplast metabolisms rely on the proper interaction of nuclear-encoded proteins and their counterparts encoded in the organelle genome. Consequently, the genes in nuclear and organellar compartments are expected to be coadapted (3).Cytonuclear coadaptation has been demonstrated by altered phenotypes observed on interspecific exchanges of cytoplasm between related species in mammals (4), yeast (5), arthropods (6), and plants, whose interspecific crosses are frequently successful (7). These alterations affect organelle function and even the organism phenotype, indicating epistasis between nuclear and cytoplasmic genes. Although cytonuclear coadaptation is generally studied at the interspecific level, the existence of intraspecific genetic diversity in organelle genomes suggests a potential for genomic coadaptation within species. A few studies have reported phenotypic effects of intraspecific cytonuclear epistasis in nonplant species (8–11). In plants, many studies have focused on cytoplasmic male sterility (CMS), an impairment of pollen production governed by nucleo-mitochondrial interactions in some hermaphroditic species (12), in particular in crops and their relatives (13). The phenotypic effects of intraspecific cytonuclear epistasis other than CMS have been reported in only a limited number of plant systems (14–17), with evidence that cytoplasmic variation contributes to local adaptation (18, 19).In recent years, several studies using reciprocal segregating populations of the model plant Arabidopsis thaliana have investigated the effect of cytonuclear epistasis on a number of laboratory-measured phenotypes such as the metabolome, defense chemistry and growth (17, 20, 21), water-use efficiency (22, 23), and seed germination (24, 25). Although some studies have reported significant effects of cytonuclear epistasis (17, 20, 21, 23, 25), others have found additive cytoplasmic effects but with weak or no cytonuclear epistasis (22). Each of these studies (with the exception of ref. 25) was, however, based on a single reciprocal cross between two natural accessions, thereby preventing the estimation of the prevalence of cytonuclear epistasis in this species. In addition, although these reports involve adaptive traits (26–30), the investigation of the effect of cytonuclear epistasis on adaptive phenotypes in field conditions is, at best, scarce in A. thaliana.Here, following the modern standards of ecological genomics (31), we explored the prevalence of cytonuclear interactions on adaptive whole-organism traits in the model plant A. thaliana in a field experiment. To do so, based on eight natural accessions of a core collection that covers a significant part of the species’ cytoplasmic and nuclear genetic diversity in A. thaliana (25, 32), we created eight series of seven cytolines. Cytolines are genotypes that combine the nuclear genome from one parent with the organelle genomes of another (33). We examined the cytolines and their parental accessions for effects of cytonuclear interactions on 28 field-measured traits related to germination, phenology, resource acquisition, plant architecture and seed dispersal, fecundity, and survival.     

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 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.     

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.Experimentally induced mutations in the genomes of model organisms have been the basis of much of our current understanding of biological mechanisms. However, traditional mutagenesis tools have significant drawbacks. Forward genetic approaches such as chemical mutagenesis lack specificity, leading to unwanted mutations at many sites in the genome. Traditional reverse genetic approaches, such as gene targeting by conventional homologous recombination, suffer from low efficiency and therefore are labor intensive. In recent years novel methods have been developed that aim to modify genomes with high precision and high efficiency by introducing double-stand breaks (DSBs) at defined loci (1). DSBs can be repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone process that frequently leads to the generation of small, mutagenic insertions and deletions (indels). HDR repairs DSBs by precisely copying sequence from a donor template, allowing specific changes to be introduced into the genome (2).The type II clustered regular interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has emerged recently as an extraordinarily powerful method for inducing site-specific DSBs in the genomes of a variety of organisms. The method exploits the RNA-guided endonuclease Cas9, which plays a key role in bacterial adaptive immune systems. Target specificity of Cas9 is encoded by a 20-nt spacer sequence in the crisprRNA, which pairs with the transactivating RNA to direct the endonuclease to the complementary target site in the DNA (3). For genome engineering, crisprRNA and transactivating RNA can be combined in a single chimeric guide RNA (gRNA), resulting in a simple two-component system for the creation of DSBs at defined sites (3). Binding of the Cas9/gRNA complex at a genomic target site is constrained only by the requirement for an adjacent short protospacer-adjacent motif (PAM), which for the commonly used Streptococcus pyogenes Cas9 is NGG (4).Several groups recently demonstrated CRISPR/Cas-mediated editing of the genome of Drosophila melanogaster (5–12), a key model organism for biological research. However, the rate of mutagenesis has varied widely both within and among different studies. Differences in the methods used to introduce Cas9 and gRNAs into the fly likely contribute significantly to different experimental outcomes. Kondo and Ueda (8) expressed both Cas9 and gRNA from transgenes stably integrated into the genome, but all other studies have used microinjection of expression plasmids or of in vitro-transcribed RNA into embryos to deliver one or both CRISPR/Cas components (5–7, 9–11). Much of the currently available evidence suggests that transgenic provision of Cas9 increases rates of germ-line transmission substantially (8, 10, 11). However, the influence of different regulatory sequences within cas9 transgenes on the rate of mutagenesis and on the location where mutations are generated within the organism has not been evaluated. The effect of different promoter sequences on the activities of gRNAs also has not been explored systematically. Therefore it is possible that suboptimal tools are being used currently for many CRISPR/Cas experiments in Drosophila.Previous studies in Drosophila have focused on the use of CRISPR/Cas to create heritable mutations in the germ line. In principle, efficient biallelic targeting within somatic cells of Drosophila would represent a powerful system to dissect the functions of genes within an organismal context. However, the feasibility of such an approach has not been explored so far.Here, we present a versatile CRISPR/Cas toolbox for Drosophila genome engineering consisting of a set of systematically evaluated transgenic Cas9 lines and gRNA-expression plasmids. We describe combinations of Cas9 and gRNA sources that can be used to induce, with high efficiency, loss-of-function mutations in nonessential or essential genes and integration of designer sequences by HDR. Finally, we show that our optimized transgenic tools permit efficient biallelic targeting in a variety of somatic tissues of the fly, allowing the characterization of mutant phenotypes directly in Cas9/gRNA-expressing animals.     

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.     

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.