Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores

Centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) directs kinetochore assembly through a hierarchical binding of CENPs, starting with CENP-C and CENP-T. Centromeres are also the chromosomal regions where cohesion, mediated by cohesin, is most prominently maintained in mitosis. While most cohesin dissociates from chromosome arms in prophase, Shugoshin 1 (Sgo1) prevents this process at centromeres. Centromeric localization of Sgo1 depends on histone H2A phosphorylation by the kinase Bub1, but whether additional interactions with kinetochore components are required for Sgo1 recruitment is unclear. Using the Xenopus egg cell-free system, we here show that both CENP-C and CENP-T can independently drive centromeric accumulation of Sgo1 through recruitment of Bub1 to the KNL1, MIS12, NDC80 (KMN) network. The spindle assembly checkpoint (SAC) kinase Mps1 is also required for this pathway even in the absence of checkpoint signaling. Sgo1 recruitment is abolished in chromosomes lacking kinetochore components other than CENP-A. However, forced targeting of Bub1 to centromeres is sufficient to restore Sgo1 localization under this condition.     

Sensitivity Analysis of Reactive Ecological Dynamics

Ecological systems with asymptotically stable equilibria may exhibit significant transient dynamics following perturbations. In some cases, these transient dynamics include the possibility of excursions away from the equilibrium before the eventual return; systems that exhibit such amplification of perturbations are called reactive. Reactivity is a common property of ecological systems, and the amplification can be large and long-lasting. The transient response of a reactive ecosystem depends on the parameters of the underlying model. To investigate this dependence, we develop sensitivity analyses for indices of transient dynamics (reactivity, the amplification envelope, and the optimal perturbation) in both continuous- and discrete-time models written in matrix form. The sensitivity calculations require expressions, some of them new, for the derivatives of equilibria, eigenvalues, singular values, and singular vectors, obtained using matrix calculus. Sensitivity analysis provides a quantitative framework for investigating the mechanisms leading to transient growth. We apply the methodology to a predator-prey model and a size-structured food web model. The results suggest predator-driven and prey-driven mechanisms for transient amplification resulting from multispecies interactions.     

Fine mapping of the root-knot nematode resistance gene <Emphasis Type="Italic">Me1</Emphasis> in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and development of markers tightly linked to <Emphasis Type="Italic">Me1</Emphasis>

Root-knot nematodes (RKNs) can severely damage crops, including peppers, worldwide. The application of resistance genes identified in the Capsicum annuum genome may represent a safe and economically relevant strategy for controlling RKNs. Among the Me genes (Me1, Me3, Me7, and N) that have been mapped to a cluster on chromosome P9, Me1 confers a heat-stable and broad-spectrum resistance that is difficult for virulent RKNs to overcome. In this study, we developed several closely linked kompetitive allele-specific PCR (KASPar) markers, simple sequence repeat (SSR) markers, sequence characterized amplified region (SCAR) markers, and high-resolution melting (HRM) markers for the mapping of RKN-resistance genes. Analyses of 948 individuals (BC₁ and F₂ progenies) revealed that Me1 was located between SCAR marker 16880-1-V2 and HRM marker 16830-H-V2, with 13 and 0 recombination events with Me1, respectively. These markers were localized to a 132-kb interval, which included six genes. The development of several PCR-based markers closely linked to Me1 will be useful for the marker-assisted selection of RKN resistance in pepper cultivars. Among these markers, 16830-H-V2 and 16830-CAPS are present in the CA09g16830 gene, which is predicted to be a putative late blight resistance protein homolog R1A-3 gene. This gene appears to be a suitable Me1 candidate gene.     

In vivo evaluation of the antimutagenic and antigenotoxic effects of β-glucan extracted from Saccharomyces cerevisiae in acute treatment with multiple doses

Ample evidence suggests that cancer is triggered by mutagenic damage and diets or supplements capable of reducing such incidences can be related to the prevention of neoplasy development or to an improvement in life quality of patients who undergo chemotherapy. This research aimed to evaluate the antimutagenic and antigenotoxic activity of β-glucan. We set up 8 experimental groups: control (Group 1), cyclophosphamide (Group 2), Groups 3–5 to assess the effect of β-glucan administration, and Groups 6–8 to evaluate the association between cyclophosphamide and β-glucan. The intraperitonial concentrations of β-glucan used were 100, 150 and 200 mg/kg. Micronucleus and comet assays showed that within the first week of treatment β-glucan presented a damage reduction rate between 100–62.04% and 94.34–59.52% for mutagenic and genotoxic damages, respectively. This activity decreased as the treatment was extended. During the sixth week of treatment antimutagenicity rates were reduced to 59.51–39.83% and antigenotoxicity was not effective. This leads to the conclusion that the efficacy of β-glucan in preventing DNA damage is limited when treatment is extended, and that its use as a chemotherapeutic adjuvant need to be better clarified.     

The rust transferred proteins—a new family of effector proteins exhibiting protease inhibitor function

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.     

Heterozygous mutations in TREX1 cause familial chilblain lupus and dominant Aicardi-Goutieres syndrome

TREX1 constitutes the major 3'-->5' DNA exonuclease activity measured in mammalian cells. Recently, biallelic mutations in TREX1 have been shown to cause Aicardi-Goutieres syndrome at the AGS1 locus. Interestingly, Aicardi-Goutieres syndrome shows overlap with systemic lupus erythematosus at both clinical and pathological levels. Here, we report a heterozygous TREX1 mutation causing familial chilblain lupus. Additionally, we describe a de novo heterozygous mutation, affecting a critical catalytic residue in TREX1, that results in typical Aicardi-Goutieres syndrome.     

Study of solid-phase synthesis and purification strategies for the preparation of polyglutamine peptides

Many neurodegenerative diseases are related to an abnormal expansion of the CAG trinucleotide that produces polyglutamine segments in several proteins. However, the pathogenesis of these neurodegenerative states is not yet well understood. Thus, to evaluate the molecular mechanisms leading to those diseases, suitable research tools such as synthetic polyglutamine peptides are required. The synthesis and purification of such peptides are usually difficult because of poor solubility, which leads to low coupling and/or deblocking reactivity. After exploring many synthesis, solubilization and purification approaches, a protocol allowing the production of polyglutamines in good yield and high purity was developed. With this protocol, peptides of 10-30 glutamine residues were synthesized using a linear solid-phase strategy combined with a maximal side-chain protection scheme using fluorenylmethyloxycarbonyl (Fmoc) chemistry. After cleavage of the peptide from the polymeric support, the crude material was treated with glacial acetic acid and lyophilized. This treatment significantly improved the solubility of the polyglutamine peptides thus allowing their dissolution in aqueous conditions and purification through reverse-phase high performance liquid chromatography. These solubilization and purification conditions led to the formation of N-pyroglutamyl peptide derivatives that were easily isolated. These N-pyroglutamylated compounds also appear as useful research tools because data from the literature suggest that N-terminal modification of polyglutamine segments might play a role in their pathogenic properties.