Local sex-ratio dynamics: a model for the dioecious liverwort Marchantia inflexa

In many dioecious bryophyte species, population sex ratios range from all female to all male. The focal species of the present study, the liverwort Marchantia inflexa, forms patches on rock and bark surfaces, and these differ widely in sex ratio at a rainforest field site in Trinidad. This analysis – to our knowledge the first modeling study of sex-ratio dynamics in a dioecious clonal organism – addresses abundances of male and female M. inflexa through time within an individual patch. We represent the life history of this species using seven different stages (non-reproductive, asexually reproductive, sexually reproductive males, non-reproductive, asexually reproductive, unfertilized and fertilized sexual females) and express their dynamics using ordinary differential equations. Some of the stages become more abundant as thalli extend over the substrate and may overgrow each other to capture space. Our simple representation of dynamics within the patch failed to stabilize the sex ratio: females gradually eliminated males at low to moderate disturbance frequency and males eliminated females at high disturbance frequency. This pattern did not hinge on whether sexual propagules could germinate within the patch, but asexual reproduction (via gemmae dispersed within the patch) played an important role. This suggests that the maintenance of sex in these populations may hinge on metapopulation structure and dynamics. Though sexual reproduction appears to be unimportant within patches, spores provide the primary means of recolonizing patches eliminated by large-scale disturbances. We found that shortly after the patch was fully occupied, the production of these wind-dispersed spores was maximized, but spore production declined thereafter as the sex ratio became increasingly biased toward one sex or the other. Much additional modeling and empirical work is needed to link within-patch dynamics across patches and account for dynamics at the metapopulation level.     

Differential Mismatch Recognition Specificities of Eukaryotic MutS Homologs,MutSα and MutSβ

In eukaryotes, the recognition of the DNA postreplication errors and initiation of the mismatch repair is carried out by two MutS homologs: MutSα and MutSβ. MutSα recognizes base mismatches and 1 to 2 unpaired nucleotides whereas MutSβ recognizes longer insertion-deletion loops (IDLs) with 1 to 15 unpaired nucleotides as well as certain mismatches. Results from molecular dynamics simulations of native MutSβ:IDL-containing DNA and MutSα:mismatch DNA complexes as well as complexes with swapped DNA substrates provide mechanistic insight into how the differential substrate specificities are achieved by MutSα and MutSβ, respectively. Our simulations results suggest more extensive interactions between MutSβ and IDL-DNA and between MutSα and mismatch-containing DNA that suggest corresponding differences in stability. Furthermore, our simulations suggest more expanded mechanistic details involving a different degree of bending when DNA is bound to either MutSα or MutSβ and a more likely opening of the clamp domains when noncognate substrates are bound. The simulation results also provide detailed information on key residues in MutSβ and MutSα that are likely involved in recognizing IDL-DNA and mismatch-containing DNA, respectively.     

A Novel Ribosomopathy Caused by Dysfunction of RPL10 Disrupts Neurodevelopment and Causes X-Linked Microcephaly in Humans

Neurodevelopmental defects in humans represent a clinically heterogeneous group of disorders. Here, we report the genetic and functional dissection of a multigenerational pedigree with an X-linked syndromic disorder hallmarked by microcephaly, growth retardation, and seizures. Using an X-linked intellectual disability (XLID) next-generation sequencing diagnostic panel, we identified a novel missense mutation in the gene encoding 60S ribosomal protein L10 (RPL10), a locus associated previously with autism spectrum disorders (ASD); the p.K78E change segregated with disease under an X-linked recessive paradigm while, consistent with causality, carrier females exhibited skewed X inactivation. To examine the functional consequences of the p.K78E change, we modeled RPL10 dysfunction in zebrafish. We show that endogenous rpl10 expression is augmented in anterior structures, and that suppression decreases head size in developing morphant embryos, concomitant with reduced bulk translation and increased apoptosis in the brain. Subsequently, using in vivo complementation, we demonstrate that p.K78E is a loss-of-function variant. Together, our findings suggest that a mutation within the conserved N-terminal end of RPL10, a protein in close proximity to the peptidyl transferase active site of the 60S ribosomal subunit, causes severe defects in brain formation and function.     

Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination

Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr²⁰) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr²⁷. Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr²⁰ caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr²⁷ mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr²⁰ is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXXΦ (where X is any amino acid and Φ is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr²⁰ both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr²⁰ may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3.     

The Bacteriostatic Protein Lipocalin 2 Is Induced in the Central Nervous System of Mice with West Nile Virus Encephalitis

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.     

Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r², 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.     

Analysis of mRNA recognition by human thymidylate synthase

Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2′-deoxyuridine-5′-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site.