Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome.     

Coevolution Drives the Emergence of Complex Traits and Promotes Evolvability

The evolution of complex organismal traits is obvious as a historical fact, but the underlying causes—including the role of natural selection—are contested. Gould argued that a random walk from a necessarily simple beginning would produce the appearance of increasing complexity over time. Others contend that selection, including coevolutionary arms races, can systematically push organisms toward more complex traits. Methodological challenges have largely precluded experimental tests of these hypotheses. Using the Avida platform for digital evolution, we show that coevolution of hosts and parasites greatly increases organismal complexity relative to that otherwise achieved. As parasites evolve to counter the rise of resistant hosts, parasite populations retain a genetic record of past coevolutionary states. As a consequence, hosts differentially escape by performing progressively more complex functions. We show that coevolution''s unique feedback between host and parasite frequencies is a key process in the evolution of complexity. Strikingly, the hosts evolve genomes that are also more phenotypically evolvable, similar to the phenomenon of contingency loci observed in bacterial pathogens. Because coevolution is ubiquitous in nature, our results support a general model whereby antagonistic interactions and natural selection together favor both increased complexity and evolvability.     

Organic enrichment of sediments reduces arbuscular mycorrhizal fungi in oligotrophic lake plants

1. Arbuscular mycorrhizal fungi (AMF) commonly colonise isoetid species inhabiting oxygenated sediments in oligotrophic lakes but are usually absent in other submerged plants. We hypothesised that organic enrichment of oligotrophic lake sediments reduces AMF colonisation and hyphal growth because of sediment O₂ depletion and low carbon supply from stressed host plants. 2. We added organic matter to sediments inhabited by isoetids and measured pore‐water chemistry (dissolved O₂, inorganic carbon, Fe²⁺ and ), colonisation intensity of roots and hyphal density after 135 days of exposure. 3. Addition of organic matter reduced AMF colonisation of roots of both Lobelia dortmanna and Littorella uniflora, and high additions stressed the plants. Even small additions of organic matter almost stopped AMF colonisation of initially un‐colonised L. uniflora, though without reducing plant growth. Mean hyphal density in sediments was high (6 and 15 m cm^?3) and comparable with that in terrestrial soils (2–40 m cm^?3). Hyphal density was low in the upper 1 cm of isoetid sediments, high in the main root zone between 1 and 8 cm and positively related to root density. Hyphal surface area exceeded root surface area by 1.7–3.2 times. 4. We conclude that AMF efficiently colonise isoetids in oligotrophic sediments and form extensive hyphal networks. Small additions of organic matter to sediments induce sediment anoxia and reduce AMF colonisation of roots but cause no apparent plant stress. High organic addition induces night‐time anoxia in both the sediment and the plant tissue. Tissue anoxia reduces root growth and AMF colonisation, probably because of restricted translocation of nutrient ions and organic solutes between roots and leaves. Isoetids should rely on AMF for P uptake on nutrient‐poor mineral sediments but are capable of growing without AMF on organic sediments.     

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.     

Molecular Characterization of EGFR and EGFRvIII Signaling Networks in Human Glioblastoma Tumor Xenografts

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ∼50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.Glioblastoma multiforme (GBM)¹ is the most frequent and aggressive form of primary brain tumor (1). The current standard of care for GBM consists of surgical removal, radiotherapy, and adjuvant chemotherapy (typically temozolomide) (1). However, despite these interventions the prognosis is still poor, with mean survival time at ∼15 months following diagnosis (2). Genetic profiling of GBM tumors has been used to identify multiple distinct genetic aberrations across a diverse array of genes such as the deletion of phosphatase and tensin homolog (PTEN), p16 deletion, and mutation of TP53 (3, 4). Additionally, amplification, overexpression, and/or mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) has been identified to be a key genetic alteration in ∼50% of GBM patients (5). EGFR amplification is often accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2–7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors (6–8). EGFRvIII is characterized by the deletion of exon 2–7, resulting in an in-frame deletion of 267 amino acid residues from the extracellular domain. This deletion generates a receptor which is unable to bind ligand yet is constitutively, but weakly, active (9). Continuous low level activation leads to impaired internalization and degradation of the receptor, causing prolonged signaling (10). Expression of EGFRvIII in the absence of wtEGFR leads to the transformation of cells in vivo, drives cell proliferation in vitro, and expression of EGFRvIII correlates with poor prognosis in the clinic (6, 11, 12). EGFRvIII has been identified in GBM, lung, ovarian, and breast cancers, but has never been identified in normal tissue (13, 14). Because of the absence of this mutant receptor in normal tissue, EGFRvIII is an attractive therapeutic target. Although EGFR inhibitors, such as erlotinib and gefitinib, inhibit EGFR, EGFRvIII bearing xenograft models and cell lines are resistant to these inhibitors (15, 16). Therapeutic agents directly targeting EGFRvIII in murine GBM xenografts initially resulted in reduced tumor volume and a modest increase in survival (17). However, tumor recurrence was inevitable because of resistance by uncharacterized evasion mechanisms and adaptations (17). We propose that an improved understanding of the system-wide changes in protein expression and signaling caused by EGFRvIII expression should provide insight into specific therapeutic targets for EGFRvIII driven tumors.It is thought that EGFRvIIl enhances tumorigenicity by differential utilization (e.g. altered amplitude and kinetics and potentially novel components or pathways) of signal transduction pathways compared with ligand activated wtEGFR. Quantitative mass spectrometry has previously been applied to the identification of EGFRvIII specific phosphotyrosine signaling across four GBM cell lines expressing titrated levels of EGFRvIII relative to cells expressing the kinase-dead control (18). Cross-activation of EGFRvIII and the c-Met receptor tyrosine kinase is prevalent within these EGFRvIII overexpressing cell lines, revealing an attractive therapeutic strategy (18), which was later extended to include cross-activation of PDGFR (platelet-derived growth factor receptor) (19).Although EGFRvIII signaling has been extensively studied in GBM cell lines, the molecular mechanisms of increased tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated (20, 21). In addition, tissue culture conditions dramatically change the genetic and molecular characteristics found in primary human tumors. In particular, EGFRvIII expression is rapidly lost during generation of primary culture cells from GBM tumors. Most of the EGFRvIII-expressing cells lines are a result of stable transfection, rather than endogenous expression, of the mutant receptor (22). Additionally, the micro-environment and cellular heterogeneity of the tumor have a significant impact on the response to therapeutics, yet are poorly reflected in cell culture. As a consequence, quantification of signaling networks in glioblastoma cell lines provide a limited understanding of the signaling networks in GBM tumor samples.To overcome this limitation, the James and Sarkaria labs have generated, from patient surgical specimens, a panel of glioblastoma tumor xenografts that are maintained through serial passaging as subcutaneous xenografts in nude mice (22, 23). Maintenance of GBM tumors in this in vivo setting preserves the genetic features and phenotypes crucial to the tumorigenicity of the primary human tumors (23). With these tumor xenografts it is possible to analyze in vivo signaling networks, predict optimal therapeutic strategies based on these data, and test these predictions in a physiologically relevant system.To quantify signaling networks activated in glioblastoma tumor xenografts and determine the effect of wtEGFR or EGFRvIII expression on these networks, we applied quantitative mass spectrometry to eight human GBM xenografts expressing wtEGFR (wt) or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+) implanted into the flanks of nude mice. This analysis led to the identification and quantification of 1588 proteins (across two or more biological replicates) and 225 tyrosine phosphorylation sites on 168 proteins across eight tumor xenografts. Integration of quantitative phosphotyrosine data and protein expression profiles have uncovered the differential regulation of novel proteins and phosphotyrosine sites, which relate to the mode of action of wtEGFR and EGFRvIII overexpression in vivo. Quantification of tyrosine phosphorylation networks revealed signaling specific to each tumor xenograft. These data provide evidence for a significant amount of variation across the eight xenografts, and suggests that optimal therapeutic strategies might be specific to each tumor.     

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.     

Weight Loss,HbA1c Reduction,and Tolerability of Cetilistat in a Randomized,Placebo‐controlled Phase 2 Trial in Obese Diabetics: Comparison With Orlistat (Xenical)

The objective of this multicenter, randomized, double‐blind study was to determine the efficacy and safety of cetilistat and orlistat relative to placebo in obese patients with type 2 diabetes, on metformin. Following a 2‐week run‐in, patients were randomized to placebo, cetilistat (40, 80, or 120 mg three times daily), or orlistat 120 mg t.i.d., for 12 weeks. The primary endpoint was absolute change in body weight from baseline. Secondary endpoints included other measures of obesity and glycemic control. Similar reductions in body weight were observed in patients receiving cetilistat 80 or 120 mg t.i.d. or 120 mg t.i.d. orlistat; these reductions were significant vs. placebo (3.85 kg, P = 0.01; 4.32 kg, P = 0.0002; 3.78 kg, P = 0.008). In the 40 mg t.i.d. and placebo groups, reductions were 2.94 kg, P = 0.958 and 2.86 kg, respectively. Statistically significant reductions in glycosylated hemoglobin (HbA_1c) were noted. Cetilistat was well tolerated, and showed fewer discontinuations due to adverse events (AEs) than in the placebo and orlistat groups. Discontinuation in the orlistat group was significantly worse than in the 120 mg cetilistat and placebo groups and was entirely due to gastrointestinal (GI) AEs. Treatment with cetilistat 80 or 120 mg t.i.d., or with orlistat 120 mg t.i.d., significantly reduced body weight and improved glycemic control relative to placebo in obese diabetic patients. Cetilistat was well tolerated with the number of discontinuations due to AEs being similar to placebo.     

Phylogeography of the Trans‐Volcanic bunchgrass lizard (Sceloporus bicanthalis) across the highlands of south‐eastern Mexico

We quantify the population divergence processes that shaped population genetic structure in the Trans‐Volcanic bunchgrass lizard (Sceloporus bicanthalis) across the highlands of south‐eastern Mexico. Multilocus genetic data from nine nuclear loci and mitochondrial (mt)DNA were used to estimate the population divergence history for 47 samples of S. bicanthalis. Bayesian clustering methods partitioned S. bicanthalis into three populations: (1) a southern population in Oaxaca and southern Puebla; (2) a population in western Puebla; and (3) a northern population with a broad distribution across Hidalgo, Puebla, and Veracruz. The multilocus nuclear data and mtDNA both supported a Late Pleistocene increase in effective population size, and the nuclear data revealed low levels of unidirectional gene flow from the widespread northern population into the southern and western populations. Populations of S. bicanthalis experienced different demographic histories during the Pleistocene, and phylogeographical patterns were similar to those observed in many co‐distributed highland taxa. Although we recommend continuing to recognize S. bicanthalis as a single species, future research on the evolution of viviparity could gain novel insights by contrasting physiological and genomic patterns among the different populations located across the highlands of south‐eastern Mexico. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 852–865.     

Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4⁺ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34⁺ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.