Trypanosoma brucei PUF9 Regulates mRNAs for Proteins Involved in Replicative Processes over the Cell Cycle

Many genes that are required at specific points in the cell cycle exhibit cell cycle–dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9—LIGKA, PNT1 and PNT2—were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3′ UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3′ UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase

The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology

Background: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. Results: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. Conclusions: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton

A MAP6-Related Protein Is Present in Protozoa and Is Involved in Flagellum Motility

In vertebrates the microtubule-associated proteins MAP6 and MAP6d1 stabilize cold-resistant microtubules. Cilia and flagella have cold-stable microtubules but MAP6 proteins have not been identified in these organelles. Here, we describe TbSAXO as the first MAP6-related protein to be identified in a protozoan, Trypanosoma brucei. Using a heterologous expression system, we show that TbSAXO is a microtubule stabilizing protein. Furthermore we identify the domains of the protein responsible for microtubule binding and stabilizing and show that they share homologies with the microtubule-stabilizing Mn domains of the MAP6 proteins. We demonstrate, in the flagellated parasite, that TbSAXO is an axonemal protein that plays a role in flagellum motility. Lastly we provide evidence that TbSAXO belongs to a group of MAP6-related proteins (SAXO proteins) present only in ciliated or flagellated organisms ranging from protozoa to mammals. We discuss the potential roles of the SAXO proteins in cilia and flagella function

Measurement of sin2 θlept eff using eþe− pairs from γ=Z bosons produced in pp collisions at a center-of-momentum energy of 1.96 TeV

At the Fermilab Tevatron proton-antiproton (pp¯) collider, Drell-Yan lepton pairs are produced in the process pp¯→e+e−+X through an intermediate γ∗/Z boson. The forward-backward asymmetry in the polar-angle distribution of the e− as a function of the e+e−-pair mass is used to obtain sin2θlepteff, the effective leptonic determination of the electroweak-mixing parameter sin2θW. The measurement sample, recorded by the Collider Detector at Fermilab (CDF), corresponds to 9.4  fb−1 of integrated luminosity from pp¯ collisions at a center-of-momentum energy of 1.96 TeV, and is the full CDF Run II data set. The value of sin2θlepteff is found to be 0.23248±0.00053. The combination with the previous CDF measurement based on μ+μ− pairs yields sin2θlepteff=0.23221±0.00046. This result, when interpreted within the specified context of the standard model assuming sin2θW=1−M2W/M2Z and that the W- and Z-boson masses are on-shell, yields sin2θW=0.22400±0.00045, or equivalently a W-boson mass of 80.328±0.024  GeV/c2

Nonlinear beam self-cleaning in a coupled cavity composite laser based on multimode fiber

We study a coupled cavity laser configuration where a passively Q-switched Nd:YAG microchip laser is combined with an extended cavity, including a doped multimode fiber. For appropriate coupling levels with the extended cavity, we observed that beam selfcleaning was induced in the multimode fiber thanks to nonlinear modal coupling, leading to a quasi-single mode laser output. In the regime of beam self-cleaning, laser pulse duration was reduced from 525 to 225 ps. We also observed a Q-switched mode-locked operation, where spatial self-cleaning was accompanied by far-detuned nonlinear frequency conversion in the active multimode fiber

Study of top quark production and decays involving a tau lepton at CDF and limits on a charged Higgs boson contribution

We present an analysis of top-antitop quark production and decay into a tau lepton, tau neutrino, and bottom quark using data from 9??fb-1 of integrated luminosity at the Collider Detector at Fermilab. Dilepton events, where one lepton is an energetic electron or muon and the other a hadronically decaying tau lepton, originating from proton-antiproton collisions at vs=1.96??TeV, are used. A top-antitop quark production cross section of 8.1±2.1??pb is measured, assuming standard-model top quark decays. By separately identifying for the first time the single-tau and the ditau components, we measure the branching fraction of the top quark into the tau lepton, tau neutrino, and bottom quark to be (9.6±2.8)%. The branching fraction of top quark decays into a charged Higgs boson and a bottom quark, which would imply violation of lepton universality, is limited to be less than 5.9% at a 95% confidence level [for B(H-?t¯?)=1]

Deciphering the mechanism of action of 089, a compound impairing the fungal cell cycle

Fungal infections represent an increasingly relevant clinical problem, primarily because of the increased survival of severely immune-compromised patients. Despite the availability of active and selective drugs and of well-established prophylaxis, classical antifungals are often ineffective as resistance is frequently observed. The quest for anti-fungal drugs with novel mechanisms of action is thus important. Here we show that a new compound, 089, acts by arresting fungal cells in the G2 phase of the cell cycle through targeting of SWE1, a mechanism of action unexploited by current anti-fungal drugs. The cell cycle impairment also induces a modification of fungal cell morphology which makes fungal cells recognizable by immune cells. This new class of molecules holds promise to be a valuable source of novel antifungals, allowing the clearance of pathogenic fungi by both direct killing of the fungus and enhancing the recognition of the pathogen by the host immune system.This project was supported by European Union’s Seventh Framework Programme [FP7/2007-2013] under grant agreement n° HEALTH-2010-242220 (“SYBARIS”) and by EU Framework Programme 7 Collaborative Project [242220]-JPI ENPADASI. I.S. was supported by the “Sybaris” project and by a fellowship from the Wellcome Warwick Quantitative Biomedicine Programme (Institutional Strategic Support Fund: 105627/Z/14/Z). The authors would like to thank Enrica Calura and Gavin Sherlock for support and critical comments on the analyses