Masculinization of the X Chromosome in the Pea Aphid

Evolutionary theory predicts that sexually antagonistic mutations accumulate differentially on the X chromosome and autosomes in species with an XY sex-determination system, with effects (masculinization or feminization of the X) depending on the dominance of mutations. Organisms with alternative modes of inheritance of sex chromosomes offer interesting opportunities for studying sexual conflicts and their resolution, because expectations for the preferred genomic location of sexually antagonistic alleles may differ from standard systems. Aphids display an XX/X0 system and combine an unusual inheritance of the X chromosome with the alternation of sexual and asexual reproduction. In this study, we first investigated theoretically the accumulation of sexually antagonistic mutations on the aphid X chromosome. Our results show that i) the X is always more favourable to the spread of male-beneficial alleles than autosomes, and should thus be enriched in sexually antagonistic alleles beneficial for males, ii) sexually antagonistic mutations beneficial for asexual females accumulate preferentially on autosomes, iii) in contrast to predictions for standard systems, these qualitative results are not affected by the dominance of mutations. Under the assumption that sex-biased gene expression evolves to solve conflicts raised by the spread of sexually antagonistic alleles, one expects that male-biased genes should be enriched on the X while asexual female-biased genes should be enriched on autosomes. Using gene expression data (RNA-Seq) in males, sexual females and asexual females of the pea aphid, we confirm these theoretical predictions. Although other mechanisms than the resolution of sexual antagonism may lead to sex-biased gene expression, we argue that they could hardly explain the observed difference between X and autosomes. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms displaying an alternative mode of sex chromosome inheritance to understanding the forces shaping chromosome evolution.     

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4⁺ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4⁺ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.     

Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.     

Role of Tomato Lipoxygenase D in Wound-Induced Jasmonate Biosynthesis and Plant Immunity to Insect Herbivores

In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and pathogens.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.     

Mitochondrial respiratory chain complex activity and bioenergetic alterations in human platelets derived from pre-symptomatic and symptomatic Huntington's disease carriers

Mitochondrial dysfunction has been implicated in Huntington's disease (HD) pathogenesis. We analyzed the activity of mitochondrial complexes (Cx) I–IV, protein levels of selected Cx subunits and adenine nucleotides in platelet mitochondria from pre-symptomatic versus symptomatic HD human carriers and age-matched control individuals. Mitochondrial platelets exhibited reduced activity of citrate synthase in pre-symptomatic and Cx-I in pre-symptomatic and symptomatic HD carriers. Positive correlation between Cx activity and protein subunits was observed for Cx-I in symptomatic HD patient's mitochondria. Moreover, AMP increased in mitochondria from pre-symptomatic HD carriers. Results highlight mitochondrial changes occurring before the onset of HD clinical symptoms.     

A novel familial case of diffuse leukodystrophy related to NDUFV1 compound heterozygous mutations

NDUFV1 mutations have been related to encephalopathic phenotypes due to mitochondrial energy metabolism disturbances. In this study, we report two siblings affected by a diffuse leukodystrophy, who carry the NDUFV1 c.1156C>T (p.Arg386Cys) missense mutation and a novel 42-bp deletion. Bioinformatic and molecular analysis indicated that this deletion lead to the synthesis of mRNA molecules carrying a premature stop codon, which might be degraded by the nonsense-mediated decay system. Our results add information on the molecular basis and the phenotypic features of mitochondrial disease caused by NDUFV1 mutations.     

Biotechnological approaches to enhance the biosynthesis of ginkgolides and bilobalide in Ginkgo biloba

Ginkgo biloba is one of the oldest living tree species and its extracts or powdered leaves are one of the best selling herbal preparations. The main bioactive constituents are flavonoids and the terpene trilactones, ginkgolides and bilobalide, which are responsible for their pharmacological activity. However, there are many difficulties for ginkgo leaves supply and the chemical synthesis is far from of being applicable for commercial-scale production. G. biloba cell cultures have arisen as a useful alternative source of pharmacologically active terpene trilactones. This review sheds light on the chemistry and biosynthesis of terpene trilactones with the aim of increasing the production of these high value compounds by biotechnological approaches. Different biotechnological strategies to improve ginkgolides and bilobalide production will be discussed, including screening and selection of in vitro ginkgo cultures, cell differentiation levels of these cultures, optimization of culture conditions, feeding and elicitation strategies. Special attention will be paid in developing new methodologies to enhance ginkgo cell biomass and provide high amounts of these bioactive terpene trilactones using large-scale cell cultures.