Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.     

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p_meta-Euro = 2.08 × 10⁻¹⁰), transmembrane protein 39A (TMEM39A; rs1132200; p_meta-all = 8.62 × 10⁻⁹), and 17q21 (rs1453560; p_meta-all = 3.48 × 10⁻¹⁰) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10⁻⁸ < p_meta-Euro < 9.99 × 10⁻⁵) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.     

Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

Glutamic acid decarboxylase activity in striatal slices: Persistent increase following depolarization

When slices prepared from rat corpus striatum were preincubated for 15 min in potassium-enriched Krebs Ringer-Phosphate medium (K⁺-KRP), the activity of glutamic acid decarboxylase measured upon reincubation in normal Krebs-Ringer-Phosphate (KRP) was doubled as compared to GAD activity in slices preincubated in normal KRP. Similarly, when striatal slices were preincubated in KRP containing 100 μM veratridine, GAD activity upon reincubation in normal KRP was increased 66% as compared to activity in slices preincubated in normal KRP. The observed increase in GAD activity was not a function of alterations in glutamate uptake by the slices. These results suggest that GABAergic neurons may regulate transmitter synthesis during the process of depolarization by increasing GAD activity.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.     

A computational framework to investigate retinal haemodynamics and tissue stress

Biomechanics and Modeling in Mechanobiology - The process of vision begins in the retina, yet the role of biomechanical forces in the retina is relatively unknown and only recently being explored....     

The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria

The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.     

Model based decoding of spike trains

Reliably decoding neuronal responses requires knowing what aspects of neuronal responses are stimulus related, and which aspects act as noise. Recent work shows that spike trains can be viewed as stochastic samples from the rate variation function, as estimated by the time dependent spike density function (or normalized peristimulus time histogram). Such spike trains are exactly described by order statistics, and can be decoded millisecond-by-millisecond by iterative application of order statistics.