Antigenic cooperation among intrahost HCV variants organized into a complex network of cross-immunoreactivity

Hepatitis C virus (HCV) has the propensity to cause chronic infection. Continuous immune escape has been proposed as a mechanism of intrahost viral evolution contributing to HCV persistence. Although the pronounced genetic diversity of intrahost HCV populations supports this hypothesis, recent observations of long-term persistence of individual HCV variants, negative selection increase, and complex dynamics of viral subpopulations during infection as well as broad cross-immunoreactivity (CR) among variants are inconsistent with the immune-escape hypothesis. Here, we present a mathematical model of intrahost viral population dynamics under the condition of a complex CR network (CRN) of viral variants and examine the contribution of CR to establishing persistent HCV infection. The model suggests a mechanism of viral adaptation by antigenic cooperation (AC), with immune responses against one variant protecting other variants. AC reduces the capacity of the host’s immune system to neutralize certain viral variants. CRN structure determines specific roles for each viral variant in host adaptation, with variants eliciting broad-CR antibodies facilitating persistence of other variants immunoreacting with these antibodies. The proposed mechanism is supported by empirical observations of intrahost HCV evolution. Interference with AC is a potential strategy for interruption and prevention of chronic HCV infection.Hepatitis C virus (HCV) causes chronic infection in  ～ 70% of infected people, who become at risk for developing severe liver diseases (1). The virus establishes chronic infection by using several molecular mechanisms for averting innate immunity and attenuating effectiveness of adaptive immune responses (2). HCV is one of the most heterogeneous viruses infecting humans and exists in each infected host as a population of genetically related variants (3, 4). Substantial heterogeneity and drastic changes in genetic composition of the intrahost HCV population observed during chronic infections have been interpreted as evidence of a continuous immune escape via random mutations, thereby generating increasing genetic diversity of viral populations in infected individuals (5–7).The observed cross-immunoreactivity (CR) of HCV variants from earlier stages of infection with antibodies (Abs) from later stages and ineffectiveness of Abs to immunoreact with variants from the same stage of infection (7) seemingly support the hypothesis of immune escape as a mechanism of intrahost evolution that contributes to establishment of persistent infections. However, several recent observations are incompatible with this hypothesis. First, the intrahost HIV population diversifies and diverts continuously from acute state to chronic infection until, at the onset of immunodeficiency, it starts losing heterogeneity and eventually stops diverting (8). Surprisingly, a similar temporal pattern of diversity and diversion was observed for intrahost HCV populations (9, 10). Furthermore, for HCV, the consistent increase in negative selection during chronic infection was observed (9–13). The late-stage HCV populations were shown to remain constant and homogeneous under the strong negative selection for years, indicating a high level of intrahost adaptation (9). Certain intrahost HCV variants were observed to persist in infected hosts for up to 16 y (9, 14, 15). These observations suggest that intrahost HCV subpopulations can remain unaffected by the immune system for years over the course of infection.Second, complex dynamics of HCV populations were observed in infected hosts. The density of intrahost subpopulations was found to fluctuate significantly in the course of chronic HCV infection, with some subpopulations persisting at low frequency for years until becoming dominant or reemerging at later stages of infection after being undetectable for a long time (9, 10, 15, 16).Third, the HCV hypervariable region 1 (HVR1) contains neutralizing antigenic epitopes (17, 18). Significant genetic variation of HVR1 during chronic infection was hypothesized to facilitate escape from neutralizing antibodies (17, 18). However, recent genetic and immunological analyses showed that HVR1 antigenic diversity is extensively convergent and effectively limited, with HVR1 variants from different genotypes and subtypes being broadly cross-immunoreactive (19–21).Interactions of intrahost viral variants with the host immune system are highly complex and nonlinear (22) and were subjects of mathematical modeling with the goal to understanding the mechanisms that lead to chronic infection. Previously developed mathematical models of interaction between HIV (23–25) or HCV (22) and the immune system showed that immune escape is associated with increase in diversity of the viral population. However, the continuous immune escape predicted by these models is inconsistent with the aforementioned observations, particularly for HCV. Unlike HIV, HCV lacks the ability to induce systemic immune suppression, suggesting a different mechanism of immune adaptation. Here, we develop a model that takes into consideration broad CR among viral variants (18, 19, 26–28) and disparity between CR and neutralization (19, 29). The model predicts antigenic cooperation (AC) among HCV variants that results in protection rather than continuous escape of the HCV population from the neutralizing Ab, thus suggesting a mechanism of intrahost evolution that leads to chronic infection.     

Identification of gene ontologies linked to prefrontal–hippocampal functional coupling in the human brain

Functional interactions between the dorsolateral prefrontal cortex and hippocampus during working memory have been studied extensively as an intermediate phenotype for schizophrenia. Coupling abnormalities have been found in patients, their unaffected siblings, and carriers of common genetic variants associated with schizophrenia, but the global genetic architecture of this imaging phenotype is unclear. To achieve genome-wide hypothesis-free identification of genes and pathways associated with prefrontal–hippocampal interactions, we combined gene set enrichment analysis with whole-genome genotyping and functional magnetic resonance imaging data from 269 healthy German volunteers. We found significant enrichment of the synapse organization and biogenesis gene set. This gene set included known schizophrenia risk genes, such as neural cell adhesion molecule (NRCAM) and calcium channel, voltage-dependent, beta 2 subunit (CACNB2), as well as genes with well-defined roles in neurodevelopmental and plasticity processes that are dysfunctional in schizophrenia and have mechanistic links to prefrontal–hippocampal functional interactions. Our results demonstrate a readily generalizable approach that can be used to identify the neurogenetic basis of systems-level phenotypes. Moreover, our findings identify gene sets in which genetic variation may contribute to disease risk through altered prefrontal–hippocampal functional interactions and suggest a link to both ongoing and developmental synaptic plasticity.Imaging genetics is widely used to identify neural circuits linked to genetic risk for heritable neuropsychiatric disorders, such as schizophrenia, autism, or bipolar disorder (1). A well-established imaging genetics phenotype is functional connectivity between the right dorsolateral prefrontal cortex (DLPFC) and the left hippocampus (HC) during working memory (WM) performance (2–4). Specifically, impaired interaction of the HC and prefrontal cortex (PFC) has been proposed as a core abnormality during neurodevelopment in schizophrenia. The hippocampus provides input to the DLPFC through long-range glutamatergic connections, which have been linked to the glutamate hypothesis of the illness. Moreover, selective lesions of the hippocampus in primates and rodents have been shown to result in postpubescent changes in prefrontal regions that are consistent with neuropathological findings in schizophrenic patients (5, 6). Brain physiology during WM performance is highly heritable (7), and anomalies of prefrontal–hippocampal functional coupling during WM have been identified in schizophrenia patients (1, 2, 4, 8), their unaffected first-grade relatives (4), healthy carriers of genome-wide supported schizophrenia risk variants and subjects at risk (4, 9–12), and in genetic animal models of the disorder (13). These studies provide strong support for a role of this neural systems-level phenotype in schizophrenia pathophysiology and correspond well to current theories that conceptualize the illness as a “brain disconnection syndrome” rooted in disturbed synaptic plasticity processes (14, 15).Previous studies have characterized abnormal prefrontal–hippocampal interactions in subjects with genetic risk factors for schizophrenia (4, 9, 10, 16). In particular, genome-wide association studies (GWAS) have become a standard approach for identifying common variants that may contribute to risk phenotypes in structural and functional neuroimaging data (10, 16, 17). However, although this approach has been effective in identifying genetic risk variants for imaging phenotypes, post hoc interpretation of results is challenging. Detected risk variants often fall within intronic sequences, where a lack of prior knowledge on functionality hinders a mechanistic explanation of how they impact brain function (18).Increasing evidence suggests that common genetic risk variants for psychiatric disorders are not distributed randomly but rather lie among sets of genes with overlapping functions (19–22). Gene set enrichment analysis (GSEA) is a data analytical approach that leverages a priori knowledge to gain insight into the biological functions of genes and pathways in the analysis of genetic data (23, 24). This approach relies on analysis of sets of genes grouped by common biological characteristics, such as a shared role in particular molecular functions or metabolic pathways. GSEA can then be used to test whether genes that are more strongly associated with a phenotype of interest tend to significantly aggregate within specific biologically based “gene sets.” As an adjunct to established GWA studies and candidate gene approaches, GSEA has successfully identified genes sets with established risk genes for complex diseases such as lung cancer, Parkinson’s disease, and psychiatric disorders, yielding insight into plausible biological processes and molecular mechanisms warranting further investigation (24–26).Although in principle the same strategy can be applied to other quantitative risk-associated phenotypes (27), no prior study has attempted to identify shared biological pathways linked to individual variation in DLPFC–HC functional coupling through a combination of GSEA, whole-genome genotype data, and neuroimaging. Here we used GSEA to test the association of ontology-based gene sets derived from common genetic variants with prefrontal–hippocampal interactions in 269 healthy volunteers who performed the n-back WM task during functional magnetic resonance imaging (fMRI), a well-established paradigm to challenge DLPFC–HC interactions. Given the reviewed evidence (14, 15), we hypothesized that we would identify gene sets linked to developmental plasticity and synaptic neurotransmission, including previously identified risk genes for schizophrenia.     

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.Next-generation sequencing is transforming our understanding of human genetic variation (1) and providing profound insights into the impact of both inherited and de novo variants on human health (2, 3). At the same time, the data from these studies present serious challenges in providing information to individuals who are found to have variant forms of different proteins. To highlight these challenges, in this report we describe our experience in analyzing missense variants of the platelet αIIbβ3 integrin receptor from The Human Genome Mutation Database (HGMD), the 1000 Genomes project (1000G), the United Kingdom 10K Whole Exome Sequencing project (U.K.10KWES), the United Kingdom 10K Whole Genome Sequencing project (U.K.10KWGS), and The National Heart, Lung and Blood Institute Exome Sequencing Project (ESP); the latter four sources encompass ∼32,000 alleles derived from 16,108 individuals.The αIIbβ3 receptor has a number of virtues as a model system. First, it is required for hemostasis because platelet aggregation requires cross-linking of the activated form of αIIbβ3 by macromolecular ligands (4). Thus, defects in its biogenesis, activation, or ligand binding lead to the rare bleeding diathesis Glanzmann thrombasthenia (GT), an autosomal recessive disorder (5, 6). Patients with GT come to medical attention because of their hemorrhagic symptoms, and thus have been carefully analyzed clinically and with tests of platelet function for nearly 50 y (5, 7). The biochemical and molecular abnormalities in GT have been studied for nearly 40 y (4, 6, 8–10). In the past 10 y, high-resolution crystallography, electron microscopy, and computational studies of the αIIbβ3 receptor have provided atomic-level information on the correlation between receptor structure and function (11–21). In addition, ethnic groups with relatively high prevalence of GT have been defined that share the same genetic abnormality based on founder mutations, and the dates that some of the mutations entered the population have been estimated (22–28). An on-line registry of GT abnormalities, including patient phenotypes was developed in 1997 (29) and currently contains 51 αIIb and 43 β3 missense variants linked to the disorder (sinaicentral.mssm.edu/intranet/research/glanzmann). The frequency of GT in the general population has not been established but it has a world-wide distribution, and based on data from hematologic practices, it is rare except in areas with a high rate of consanguineous mating (30).Second, alloimmune disorders, including neonatal thrombocytopenia and posttransfusion purpura, due to amino acid substitutions in either αIIb or β3, have been characterized at the molecular biological level and correlated with mechanisms of immunologic recognition (31).Third, inherited macrothrombocytopenia and anisothrombocytopenia have been associated with heterozygous missense variants or deletions in αIIb or β3. All of these appear to induce constitutive activation of the receptor and impair proplatelet formation (32–38).Fourth, αIIbβ3 contributes to pathological platelet thrombus formation in human ischemic cardiovascular disease and αIIbβ3 is a validated target for antithrombotic therapy (39–41).Fifth, αIIbβ3 is a member of the large integrin family of receptors, which includes 24 receptors derived from 18 α- and 8 β-subunits (41, 42). These receptors are involved in important biologic processes, including development, cell migration, homing, cell survival, and adaptive immunity (41–43). More is known about the structure–function relationships of αIIbβ3 than the other members of the group, and so it serves as the paradigmatic integrin receptor (44, 45).Sixth, 3D molecular models have been built based on crystallographic and NMR data to analyze the effects of novel amino acid substitutions on receptor structure and function and the generation of alloantigens (15, 46–53). The data from these models and assessments of the severity of the amino acid change in the variants have the potential to aid in predicting whether a novel variant is likely to affect receptor function and immunogenicity (54–59).     

From the Cover: Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.Genetics contribute to the pathogenesis of Parkinson disease (PD) in two ways. Mutations in several genes can cause inherited PD (1), and risk factor variants contribute to the risk of sporadic PD (2). Some genes contribute to both mechanisms. These pleomorphic risk loci (3) include genes that encode α-synuclein and leucine-rich repeat kinase 2 (LRRK2) (4). However, risk factors for sporadic PD identified by genome-wide association studies (GWASs) (5–9) actually nominate large genomic loci with multiple candidate genes (10). These loci may include variants that change amino acids or affect disease risk through gene expression (11). Also, whether all of the genes for PD converge on a small number of biological pathways is unknown (1). It is, therefore, important to develop unbiased approaches that would resolve whether genes for PD have similar biological functions and understand the mechanism(s) of disease risk. Here, we examine one genetic cause of PD (LRRK2) and show that identifying protein interaction partners can clarify disease mechanisms.     

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.     

Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants

We compared whole-exome sequencing (WES) and whole-genome sequencing (WGS) in six unrelated individuals. In the regions targeted by WES capture (81.5% of the consensus coding genome), the mean numbers of single-nucleotide variants (SNVs) and small insertions/deletions (indels) detected per sample were 84,192 and 13,325, respectively, for WES, and 84,968 and 12,702, respectively, for WGS. For both SNVs and indels, the distributions of coverage depth, genotype quality, and minor read ratio were more uniform for WGS than for WES. After filtering, a mean of 74,398 (95.3%) high-quality (HQ) SNVs and 9,033 (70.6%) HQ indels were called by both platforms. A mean of 105 coding HQ SNVs and 32 indels was identified exclusively by WES whereas 692 HQ SNVs and 105 indels were identified exclusively by WGS. We Sanger-sequenced a random selection of these exclusive variants. For SNVs, the proportion of false-positive variants was higher for WES (78%) than for WGS (17%). The estimated mean number of real coding SNVs (656 variants, ∼3% of all coding HQ SNVs) identified by WGS and missed by WES was greater than the number of SNVs identified by WES and missed by WGS (26 variants). For indels, the proportions of false-positive variants were similar for WES (44%) and WGS (46%). Finally, WES was not reliable for the detection of copy-number variations, almost all of which extended beyond the targeted regions. Although currently more expensive, WGS is more powerful than WES for detecting potential disease-causing mutations within WES regions, particularly those due to SNVs.Whole-exome sequencing (WES) is routinely used and is gradually being optimized for the detection of rare and common genetic variants in humans (1–8). However, whole-genome sequencing (WGS) is becoming increasingly attractive as an alternative, due to its broader coverage and decreasing cost (9–11). It remains difficult to interpret variants lying outside the protein-coding regions of the genome. Diagnostic and research laboratories, whether public or private, therefore tend to search for coding variants, most of which can be detected by WES, first. Such variants can also be detected by WGS, and several studies previously compared WES and WGS for different types of variations and/or in different contexts (9, 11–16), but none of them in a really comprehensive manner. Here, we compared WES and WGS, in terms of detection rates and quality, for single-nucleotide variants (SNVs), small insertions/deletions (indels), and copy-number variants (CNVs) within the regions of the human genome covered by WES, using the most recent next-generation sequencing (NGS) technologies. We aimed to identify the most efficient and reliable approach for identifying these variants in coding regions of the genome, to define the optimal analytical filters for decreasing the frequency of false-positive variants, and to characterize the genes that were either hard to sequence by either approach or were poorly covered by WES kits.     

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.     

Brain galanin system genes interact with life stresses in depression-related phenotypes

Galanin is a stress-inducible neuropeptide and cotransmitter in serotonin and norepinephrine neurons with a possible role in stress-related disorders. Here we report that variants in genes for galanin (GAL) and its receptors (GALR1, GALR2, GALR3), despite their disparate genomic loci, conferred increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events in a European white population cohort totaling 2,361 from Manchester, United Kingdom and Budapest, Hungary. Bayesian multivariate analysis revealed a greater relevance of galanin system genes in highly stressed subjects compared with subjects with moderate or low life stress. Using the same method, the effect of the galanin system genes was stronger than the effect of the well-studied 5-HTTLPR polymorphism in the serotonin transporter gene (SLC6A4). Conventional multivariate analysis using general linear models demonstrated that interaction of galanin system genes with life stressors explained more variance (1.7%, P = 0.005) than the life stress-only model. This effect replicated in independent analysis of the Manchester and Budapest subpopulations, and in males and females. The results suggest that the galanin pathway plays an important role in the pathogenesis of depression in humans by increasing the vulnerability to early and recent psychosocial stress. Correcting abnormal galanin function in depression could prove to be a novel target for drug development. The findings further emphasize the importance of modeling environmental interaction in finding new genes for depression.Major depressive disorder (MDD) is a common and serious disease afflicting more women than men, and a leading cause of disability worldwide, associated with much suffering and major costs for society (1, 2). Environmental psychosocial stressors are important in pathogenesis, because episodes are usually preceded by adverse life events, and early childhood experiences of physical and emotional abuse and parental neglect are important vulnerability factors (3, 4). Genetic vulnerability is significant with a heritability of about 35% (5). We remain ignorant about the brain processes that translate these genetic and environmental influences into depressive symptoms or risk. A major clue is that effective antidepressant drugs act directly or indirectly to enhance neurotransmission in serotonin (5-HT) and norepinephrine monoamine pathways, proving the monoamine hypothesis of depression (6–8). Many other candidate mechanisms have been identified in anatomical, pharmacological, and behavioral studies of stress in rodents. However, the demonstration of state- or trait-related abnormalities in human monoamine or other neural systems remains frustratingly elusive, despite modern brain-imaging methods. To determine whether the neuropeptide galanin has a role in depression, we used a unique Bayesian systems-based analysis to dissect out the influence of variation in genes for the peptide and its receptors on the interaction between different psychosocial stressors and risk of depression.Current drug treatment of depression is far from satisfactory; the drugs target a limited range of monoamine mechanisms, they have an appreciable side-effect burden, and response is often partial (8, 9). In the search for better antidepressants, much attention has focused on neuropeptides and their receptors, the most diverse neurotransmitter system in the brain (8, 10–21), which includes galanin. As yet, however, there is no compelling evidence of efficacy of the neuropeptide approach or that particular peptides are involved in the pathogenesis of MDD.Galanin, a 29-aa (30 in humans) peptide (22), is widely distributed in the rodent (23, 24) and human (25–27) brain. In rat it coexists with noradrenaline (NA) in the locus coeruleus (LC) and with 5-HT in the dorsal raphe complex (28). Like other peptide cotransmitters (29), it is released when neurons fire in high-frequency bursts in response to strong behavioral and pharmacological challenge (30–32). Galanin exerts its action via three cloned receptors, GALR1, GALR2, and GALR3 (33, 34) with a broad distribution in rat (35) and primate brain (26, 36). Animal behavioral studies (31, 32, 37–41) and a single study in humans (42) suggest that galanin has a role in stress, depression-like behavior, and anxiety. In addition, there is indication from previous genetic studies on humans that the galanin system is involved in psychiatric disorders including alcoholism/addiction (43–47), panic disorder (48, 49), and chronic pain-associated depression (50). Furthermore, recent functional studies provided the first evidence that polymorphisms in a highly conserved genetic region upstream from the GAL gene regulates GAL expression in brain areas, such as the amygdala and hypothalamus, implicated in the pathogenesis of depression (51, 52).Genetic studies have the potential to identify molecular mechanisms of MDD vulnerability (53), but even mega- and meta-analyses of large genome-wide association studies (GWAS) have not identified genetic variants associated with MDD that survive genome-wide statistical correction (54, 55). Nominally significant associations will include many false-positives. Nevertheless it is noteworthy that SNPs in the gene for galanin (GAL) were among the top 10 genes whose variation was associated with MDD in a recent GWAS (55). One way to improve sensitivity is to take a system-based approach: if galanin is mechanistically involved in depression, genetic variation in the peptide and its receptors should exert similar influences, despite the fact the genes are located on entirely different chromosomes without linkage disequilibrium (LD) and with a low probability of randomly similar effects. Others have argued that improved sensitivity will come from deeper phenotyping (56) and characterization of environmental factors (3, 4, 57), because neither genetic nor environmental factors can be identified in isolation, if they modify each other’s action to a high degree. Combining these two approaches, and in view of its preclinical properties, we predicted that variation in galanin genes would strongly interact with environmental stress in determining depression vulnerability. However, including more phenotypic and environmental variables exacerbates the problem of false-positives from multiple comparisons. Consequently, analyses of gene–environment interactions involving multiple phenotypes face a similar burden as GWAS in terms of correction for multiple testing. Furthermore, the conditional nature of such interactions frequently leads to separate analysis of multiple subpopulations (i.e., to even more statistical tests). To cope with multiple hypothesis testing, we applied a Bayesian systems-based approach both at structural and parametric levels, which allows multiple correlated outcomes. This approach supported the joint exploration of the underlying mechanism at genotype, haplotype, and diplotype levels in different depression-related phenotypes, and we validated the results by conventional multivariate analysis using independent subsamples.     

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.