Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES + S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).     

New bradykinin analogues substituted in positions 7 and 8 with sterically restricted 1-aminocyclopentane-1-carboxylic acid.

A sterically constrained non-coded amino acid, 1-aminocyclopentane-1-carboxylic acid (Apc), was introduced in position 7 or 8 of the bradykinin (BK) B(2) receptor antagonist, [D-Arg(0), Hyp(3), Thi(5, 8), D-Phe(7)]BK, previously synthesized by Stewart's group. This modification is believed to reduce the flexibility of the peptides, thereby forcing the peptide backbone and side chains to adopt specific orientations. Apc substitution was combined with acylation of the N-terminus with 1-adamantaneacetic acid (Aaa). The activity of four new analogues was assayed in isolated rat uterus and in rat blood pressure tests. The results clearly demonstrated that the Apc residue inserted in position 7 led to a reduction of antagonistic properties in the rat uterus assay or even restored the agonism in the blood pressure test, whereas Apc at position 8 enhanced antagonistic potency in both the tests. In both cases, acylation of the N-terminus led to the enhancement of the antagonistic potency. On the basis of these findings, new potent and selective B(2) blockers might be designed.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) and 2 (mTORC2) Control the Dendritic Arbor Morphology of Hippocampal Neurons

Dendrites are the main site of information input into neurons. Their development is a multistep process controlled by mammalian target of rapamycin (mTOR) among other proteins. mTOR is a serine/threonine protein kinase that forms two functionally distinct complexes in mammalian cells: mTORC1 and mTORC2. However, the one that contributes to mammalian neuron development remains unknown. This work used short hairpin RNA against Raptor and Rictor, unique components of mTORC1 and mTORC2, respectively, to dissect mTORC involvement in this process. We provide evidence that both mTOR complexes are crucial for the proper dendritic arbor morphology of hippocampal neurons. These two complexes are required for dendritic development both under basal conditions and upon the induction of mTOR-dependent dendritic growth. We also identified Akt as a downstream effector of mTORC2 needed for proper dendritic arbor morphology, the action of which required mTORC1 and p70S6K1.     

The bulge region of HIV-1 TAR RNA binds metal ions in solution

Binding of Mg²⁺, Ca²⁺ and Co(NH₃)₆³⁺ ions to the HIV-1 TAR RNA in solution was analysed by ¹⁹F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for ¹⁹F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg²⁺ and Ca²⁺ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K_d) of 0.9 ± 0.6 and 2.7 ± 1.7 mM, respectively. Argininamide, inducing largest ¹⁹F chemical shifts changes at FU-23, was used as a reference ligand (K_d = 0.3 ± 0.1 mM). In the Pb²⁺-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg²⁺ and Ca²⁺ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb²⁺-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH₃)₆³⁺ > Mg²⁺ > Ca²⁺] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.     

Enhanced formation of aerenchyma and induction of a barrier to radial oxygen loss in adventitious roots of Zea nicaraguensis contribute to its waterlogging tolerance as compared with maize (Zea mays ssp. mays)

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging‐tolerant plants. Zea nicaraguensis (teosinte), a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in comparison with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epidermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solution were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis.     

PROPER: Performance visualization for optimizing and comparing ranking classifiers in MATLAB

Background

One of the recent challenges of computational biology is development of new algorithms, tools and software to facilitate predictive modeling of big data generated by high-throughput technologies in biomedical research.

Results

To meet these demands we developed PROPER - a package for visual evaluation of ranking classifiers for biological big data mining studies in the MATLAB environment.

Conclusion

PROPER is an efficient tool for optimization and comparison of ranking classifiers, providing over 20 different two- and three-dimensional performance curves.

     

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

Insertion reactions of organic anhydrides into the M-O bond of CuOBu and Sb(OMe)3

The synthesis and structural characterization of the copper salts [Cu₂(2-Boc-benzoate)₄(dme)₂] (1), [Cu(2-Boc-benzoate)₂(tmeda)] (2), [Cu₂(2-Boc-benzoate)₂(dppm)] (3), [Cu(2-Boc-nicotinate)(PPh₃)₂] (4), [Cu₂{2-Boc-5,6-anhydride-naphthylcarboxylate}₂(dppm)₂] (5) [dme = 1,2-dimethoxyethane, dppm = bis(diphenylphosphino)methane, tmeda = N,N′-tetramethylethylenediamine, Boc = tert-butoxycarbonyl] prove that cyclic organic anhydrides and dianhydrides readily insert into the Cu-O bond of [CuO^tBu] forming carboxylate ligands with ester functionalities in the ligand periphery. [Sb(CO₂Ph-o-CO₂Me)₂(OMe)(tmeda)] (6) was synthesised by insertion reaction of Sb(OMe)₃ with phthalic anhydride.     

Biophotonic markers of malignancy: Discriminating cancers using wavelength-specific biophotons

Early detection is a critically important factor when successfully diagnosing and treating cancer. Whereas contemporary molecular techniques are capable of identifying biomarkers associated with cancer, surgical interventions are required to biopsy tissue. The common imaging alternative, positron-emission tomography (PET), involves the use of nuclear material which poses some risks. Novel, non-invasive techniques to assess the degree to which tissues express malignant properties are now needed. Recent developments in biophoton research have made it possible to discriminate cancerous cells from normal cells both in vitro and in vivo. The current study expands upon a growing body of literature where we classified and characterized malignant and non-malignant cell types according to their biophotonic activity. Using wavelength-exclusion filters, we demonstrate that ratios between infrared and ultraviolet photon emissions differentiate cancer and non-cancer cell types. Further, we identified photon sources associated with three filters (420-nm, 620-nm., and 950-nm) which classified cancer and non-cancer cell types. The temporal increases in biophoton emission within these wavelength bandwidths is shown to be coupled with intrisitic biomolecular events using Cosic's resonant recognition model. Together, the findings suggest that the use of wavelength-exclusion filters in biophotonic measurement can be employed to detect cancer in vitro.