Targeted Deletion of the Gene Encoding the La Autoantigen (Sj?gren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss

La antigen (Sjögren''s syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren''s syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1^Cre La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.     

Purification and characterization of aminoglycoside phosphotransferase APH(6)-Id,a streptomycin-inactivating enzyme

As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of ³²P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K _m(streptomycin) of 0.38 ± 0.13 mM, K _m(ATP) of 1.03 ± 0.1 mM, V _max of 3.2 ± 1.1 μmol/min/mg, and k _cat of 1.7 ± 0.6 s^?1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.     

Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides a platform for structure-based drug design

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.     

IDEPI: Rapid Prediction of HIV-1 Antibody Epitopes and Other Phenotypic Features from Sequence Data Using a Flexible Machine Learning Platform

Since its identification in 1983, HIV-1 has been the focus of a research effort unprecedented in scope and difficulty, whose ultimate goals — a cure and a vaccine – remain elusive. One of the fundamental challenges in accomplishing these goals is the tremendous genetic variability of the virus, with some genes differing at as many as 40% of nucleotide positions among circulating strains. Because of this, the genetic bases of many viral phenotypes, most notably the susceptibility to neutralization by a particular antibody, are difficult to identify computationally. Drawing upon open-source general-purpose machine learning algorithms and libraries, we have developed a software package IDEPI (IDentify EPItopes) for learning genotype-to-phenotype predictive models from sequences with known phenotypes. IDEPI can apply learned models to classify sequences of unknown phenotypes, and also identify specific sequence features which contribute to a particular phenotype. We demonstrate that IDEPI achieves performance similar to or better than that of previously published approaches on four well-studied problems: finding the epitopes of broadly neutralizing antibodies (bNab), determining coreceptor tropism of the virus, identifying compartment-specific genetic signatures of the virus, and deducing drug-resistance associated mutations. The cross-platform Python source code (released under the GPL 3.0 license), documentation, issue tracking, and a pre-configured virtual machine for IDEPI can be found at https://github.com/veg/idepi.

This is a PLOS Computational Biology Software Article

     

The millipede genus Orthomorpha Bollman, 1893 in Laos (Diplopoda,Polydesmida, Paradoxosomatidae), with descriptions of new species

The genus Orthomorpha is currently represented in Laos by nine species, including three, O. paviei Brölemann, 1896, O. communis Likhitrakarn, Golovatch & Panha, 2011 and O. cambodjana (Attems, 1953), which are new to the fauna of the country, and further three new to science: O. suberectoides sp. n., O. gladiata sp. n. and O. sutchariti sp. n.     

A vertebrate homolog of the cell cycle regulator Dbf4 is an inhibitor of Wnt signaling required for heart development

Early stages of vertebrate heart development have been linked to Wnt signaling. Here we show in both gain- and loss-of-function experiments that XDbf4, a known regulator of Cdc7 kinase, is an inhibitor of the canonical Wnt signaling pathway. Depletion of endogenous XDbf4 protein did not disturb gastrulation movements or early organizer genes but resulted in embryos with morphologically defective heart and eyes and suppressed cardiac markers. These markers were restored by overexpressed XDbf4, or an XDbf4 mutant that inhibits Wnt signaling but lacks the ability to regulate Cdc7. This indicates that the function of XDbf4 in heart development is independent of its role in the cell cycle. Moreover, our data suggest that XDbf4 acts through the physical and functional interaction with Frodo, a context-dependent regulator of Wnt signaling. These findings establish an unexpected function for a vertebrate Dbf4 homolog and demonstrate the requirement for Wnt inhibition in early cardiac specification.     

Sequence microheterogeneity of parvalbumin pI 5.0 of pike: a mass spectrometric study

Parvalbumin (PA) is a muscle and neuronal calcium-binding protein, the major fish and frog allergen. Its characteristic feature is the presence of multiple isoforms with significantly different amino acid sequences. Here we show that the major isoform of northern pike muscle PA (pI 5.0, alpha-PA) exhibits microheterogeneity of amino acid sequence. ESI Q-TOF mass-spectrometry (MS) analysis of alpha-PA sample showed the presence of two components with mass difference of 71 Da. Analysis of tryptic and endoproteinase Asp-N digests of alpha-PA by MALDI-TOF MS revealed peptides, corresponding to two different amino acid sequences. The sequence differences between variant proteins are limited to AB-domain and include substitutions K27A and L31K, and an extra Leu residue between K11 and K12. Since the affected residues comprise a cluster on the surface of PA, an involvement of the identified region into target recognition is suggested. The substitutions at positions 27 and 31 are located in the region of previously identified epitopes of parvalbumin relevant for PA-specific IgE and IgG binding, which suggests different immunoactivities of the variants. The found microheterogeneity of PA is suggested to be of importance for physiological adaptation of the propulsive musculature to developmental and/or environmental requirements and may contribute to PA allergenicity.