Use of nuclear morphometry characteristics to distinguish between normal and abnormal cervical glandular histologies.

This is a methodological study exploring the use of quantitative histopathology applied to the cervix to discriminate between normal and cancerous (consisting of adenocarcinoma and adenocarcinoma in situ) tissue samples. The goal is classifying tissue samples, which are populations of cells, from measurements on the cells. Our method uses one particular feature, the IODs-Index, to create a tissue level feature. The specific goal of this study is to find a threshold for the IODs-Index that is used to create the tissue level feature. The main statistical tool is Receiver Operating Characteristic (ROC) curve analysis. When applied to the data, our method achieved promising results with good estimated sensitivity and specificity for our data set. The optimal threshold for the IODs-Index was found to be 2.12.     

Biological activity of hamster interferon-gamma is modulated by the carboxyl-terminal tail

The Syrian golden hamster (Mesocricetus auratus) is highly susceptible to a number of intracellular pathogens. Interferon-gamma (IFN-γ), the primary macrophage-activating cytokine, plays a key role in the host defense against intracellular pathogens. The hamster IFN-γ cDNA encodes a 174 amino acid protein that has an additional 17 amino acids at the carboxyl-terminus compared to IFN-γ of mice and rats. A homologous C-terminal tail is also found in other non-murine rodents. The biological activity of hamster IFN-γ had not been investigated previously so we first demonstrated the activity of native IFN-γ in assays of IFN-γ-induced receptor signaling and antiviral activity against vesicular stomatitis virus. We then tested the hypothesis that the C-terminal tail of hamster IFN-γ could influence its biological activity. A truncated hamster IFN-γ, in which the C-terminal 17 aa were removed by insertion of a stop codon at the position corresponding to the stop codon in the mouse sequence, had approximately 10-fold greater activity than the full length protein when measured in the two bioassays. Polyclonal and monoclonal anti-hamster IFN-γ antibodies specifically inhibited this biological activity. Collectively, these data indicate that this unique structural feature influences the biological activity of hamster IFN-γ.     

The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditis elegans

The selection of a proper AUG start codon requires the base-pairing interactions between the codon on the mRNA and the anticodon of the initiator tRNA. This selection process occurs in a pre-initiation complex that includes multiple translation initiation factors and the small ribosomal subunit. To study how these initiation factors are involved in start codon recognition in multicellular organisms, we isolated mutants that allow the expression of a GFP reporter containing a non-AUG start codon. Here we describe the characterization of mutations in eif-1, which encodes the Caenorhabditis elegans translation initiation factor 1 (eIF1). Two mutations were identified, both of which are substitutions of amino acid residues that are identical in all eukaryotic eIF1 proteins. These residues are located in a structural region where the amino acid residues affected by the Saccharomyces cerevisiae eIF1 mutations are also localized. Both C. elegans mutations are dominant in conferring a non-AUG translation initiation phenotype and lead to growth arrest defects in homozygous animals. By assaying reporter constructs that have base changes at the AUG start codon, these mutants are found to allow expression from most reporters that carry single base changes within the AUG codon. This trend of non-AUG mediated initiation was also observed previously for C. elegans eIF2β mutants, indicating that these two factors play a similar role. These results support that eIF1 functions in ensuring the fidelity of AUG start codon recognition in a multicellular organism.TRANSLATION initiation is thought to be one of the most complex cellular processes in eukaryotes. It involves at least 12 translation initiation factors (eIFs) comprising over 30 polypeptides (Pestova et al. 2007). These factors bring together an initiator methionyl tRNA (Met-tRNAi), the small ribosomal subunit, and a mRNA to form a 48S initiation complex. An important role performed by this complex is to select an AUG codon to initiate translation of the mRNA. Since the first AUG at the 5′ end of most mRNAs is selected as the start site, it is believed that the initiation complex scans for an AUG start codon as it moves from the 5′-capped end of the mRNA toward the 3′ end, as proposed in the ribosomal scanning model (Kozak 1978; Kozak 1989). The recognition of the AUG start codon is mediated by the anticodon of the Met-tRNAi, and the matching base-pairing interactions between the codon of the mRNA and the anticodon determine the site of initiation (Cigan et al. 1988). These base-pairing interactions are essential, but are likely not the only components required for accurately selecting the correct AUG start codon. Numerous initiation factors along with base-pairing interactions have been shown to aid in the AUG recognition process (Pestova et al. 2007).Translation initiation factors involved in start codon selection fidelity were first identified through genetic studies performed in the yeast Saccharomyces cerevisiae. Mutant strains with a modified His4 gene that had an AUU instead of an AUG at the native start site were selected for the ability to survive on media lacking histidine (Donahue et al. 1988; Castilho-Valavicius et al. 1990). These mutants were found to be able to produce the His4 protein by using a downstream inframe UUG codon (the third codon within the His4 coding region) as the translation start site. Further analyses determined that non-AUG initiation occurred mostly from a UUG codon and not significantly from other codons (Huang et al. 1997). These mutants defined five genetic loci and were named sui1-sui5 (suppressor of initiation codon) on the basis of their ability to initiate translation at a non-AUG codon.The sui1 suppressors were found to have missense mutations in eIF1. These missense mutations showed semidominant or codominant properties in non-AUG translation initiation while deletion of the eIF1 gene led to lethality in yeast (Yoon and Donahue 1992). eIF1 is a highly conserved protein with a size of approximately 12 kDa that plays a vital role in multiple translation initiation steps. eIF1 is incorporated into a multifactor complex that includes eIF1A, eIF3, and eIF5 and stimulates the recruiting of the ternary complex (consisting of eIF2 · GTP and the charged Met-tRNAi) to the small ribosomal subunit to form the 43S pre-initiation complex (Singh et al. 2004). eIF1 acts synergistically with eIF1A to promote continuous ribosomal scanning for AUG codons by stabilizing an open conformation that allows mRNA to pass through the complex (Maag et al. 2005; Cheung et al. 2007; Passmore et al. 2007). It also mediates the assembly of the ribosomal initiation complex at the AUG start codon (Pestova et al. 1998). eIF1 dissociates from the complex upon recognition of the AUG codon and this dissociation is necessary to trigger a series of conformational changes leading to the translation elongation phase (Algire et al. 2005). Consistent with these roles, sui1 mutations reduce the affinity of eIF1 for the ribosome and cause premature release of eIF1 at non-AUG codons (Cheung et al. 2007). Other sui mutations support the involvement of four additional genes in translation initiation fidelity in yeast. Mutations have been isolated in the heterotrimeric eIF2 as SUI2 (α-subunit) (Cigan et al. 1989), SUI3 (β-subunit) (Donahue et al. 1988), and SUI4 (γ-subunit) (Huang et al. 1997), and a mutation in eIF5 corresponds to the SUI5 mutant (Huang et al. 1997).However, the genetic studies that identified these translation fidelity mutants were conducted only in yeast. It is not known if there are similar mechanisms regulating translation initiation fidelity in multicellular organisms. To address this question, we designed a genetic system to isolate C. elegans mutants that have reduced fidelity in AUG start codon selection (Zhang and Maduzia 2010). Mutants were selected on the basis of their ability to express a GFP reporter that contains a GUG codon in place of its native translation start site. Here we report the characterization of two mutants that have mutations in eIF1. Unlike yeast sui1 mutants, which preferred the UUG codon, these mutants are capable of using a subset of non-AUG codons for translation initiation. Our results are consistent with eIF1 playing a role in the fidelity of AUG codon selection, perhaps by discriminating base-pairing interactions between the codon and anticodon during start-site selection.     

Glucose delays age-dependent proteotoxicity

Nutrient availability influences an organism's life history with profound effects on metabolism and lifespan. The association between a healthy lifespan and metabolism is incompletely understood, but a central factor is glucose metabolism. Although glucose is an important cellular energy source, glucose restriction is associated with extended lifespan in simple animals and a reduced incidence of age-dependent pathologies in humans. We report here that glucose enrichment delays mutant polyglutamine, TDP-43, FUS, and amyloid-β toxicity in Caenorhabditis elegans models of neurodegeneration by reducing protein misfolding. Dysregulated metabolism is common to neurodegeneration and we show that glucose enrichment is broadly protective against proteotoxicity.     

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.     

Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

1. Download : Download high-res image (122KB)
2. Download : Download full-size image

     

Structures of P. falciparum protein kinase 7 identify an activation motif and leads for inhibitor design

Malaria is a major threat to world health. The identification of parasite targets for drug development is a priority and parasitic protein kinases suggest themselves as suitable targets as many display profound structural and functional divergences from their host counterparts. In this paper, we describe the structure of the orphan protein kinase, Plasmodium falciparum protein kinase 7 (PFPK7). Several Plasmodium protein kinases contain extensive insertions, and the structure of PFPK7 reveals how these may be accommodated as excursions from the canonical eukaryotic protein kinase fold. The constitutively active conformation of PFPK7 is stabilized by a structural motif in which the role of the conserved phosphorylated residue that assists in structuring the activation loop of many protein kinases is played by an arginine residue. We identify two series of PFPK7 ATP-competitive inhibitors and suggest further developments for the design of selective and potent PFPK7 lead compounds as potential antimalarials.