BRCT domains: Easy as one,two, three

BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero-domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.Key words: BRCT domain, DNA repair, phosphorylation, phospho-peptide interaction, protein interaction, DNA binding, DNA damage response     

Cytokine response patterns in severe pandemic 2009 H1N1 and seasonal influenza among hospitalized adults

Background

Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis.

Methods

Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed.

Results

63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2–15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2–27 times lower than seasonal influenza; P−values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3–4, 55% vs 85%; day 6–7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6–61.5, per log₁₀unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman''s rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their ‘responsiveness’ to stimuli was shown to change dynamically during the illness course.

Conclusions

A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.     

Role of IGF signaling in olfactory sensory map formation and axon guidance

Olfactory neurons project their axons to spatially invariant glomeruli in the olfactory bulb, forming an ordered pattern of innervation comprising the olfactory sensory map. A mirror symmetry exists within this map, such that neurons expressing a given receptor typically project to one glomerulus on the medial face and one glomerulus on the lateral face of the bulb. The mechanisms underlying an olfactory neuron's choice to project medially versus laterally remain largely unknown, however. Here we demonstrate that insulin-like growth factor (IGF) signaling is required for sensory innervation of the lateral olfactory bulb. Mutations that eliminate IGF signaling cause axons destined for targets in the lateral bulb to shift to ectopic sites on the ventral-medial surface. Using primary cultures of olfactory and cerebellar neurons, we further show that IGF is a chemoattractant for axon growth cones. Together these observations reveal a role of IGF signaling in sensory map formation and axon guidance.     

Receptor subtypes mediating adenosine-induced dilation of cerebral arterioles

The purpose of this study was to investigate the receptor subtypes that mediate the dilation of rat intracerebral arterioles elicited by adenosine. Penetrating arterioles were isolated from the rat brain, cannulated with the use of a micropipette system, and luminally pressurized to 60 mmHg. Both adenosine and the A2A receptor-selective agonist CGS-21680 induced dose-dependent vasodilation (-logEC(50): 6.5 +/- 0.2 and 8.6 +/- 0.3, respectively). However, adenosine, which is capable of activating both A2A and A2B receptors, caused a greater maximal dilation than CGS-21680. The A2A receptor-selective antagonist ZM-241385 (0.1 microM) only partially inhibited the dilation induced by adenosine but almost completely blocked CGS-21680-induced dilation. Neither 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), an A1 receptor-selective antagonist, nor MRS-1191 (0.1 microM), an A3 receptor-selective antagonist, attenuated adenosine dose responses. Moreover, ZM-241385 had no effect on the dilation induced by ATP (10 microM) or acidic (pH 6.8) buffer. We concluded that the A2A receptor subtype mediates adenosine-induced dilation of intracerebral arterioles in the rat brain. Furthermore, our results suggest that A2B receptors may also participate in the dilation response to adenosine.     

Onset of odorant receptor gene expression during olfactory sensory neuron regeneration

Individual olfactory sensory neurons are thought to express only one odorant receptor gene from a repertoire of hundreds to thousands of genes. How do these sensory neurons choose just one specific odorant receptor to express during their differentiation? As an initial attempt toward understanding the process of odorant receptor gene regulation, we studied when odorant receptor expression is activated during sensory neuron regeneration. We find that receptor gene expression is activated in postmitotic neurons and can occur in the absence of the olfactory bulb. These results suggest that receptor expression is restricted to the terminal stages of olfactory neuron differentiation, and sensory neurons do not simply inherit the odorant receptor that is already expressed in mitotic precursor cells. Our results also support a model in which odorant receptor gene expression occurs independent of the olfactory bulb.     

Development of the vertebrate main olfactory system

Olfactory receptor neurons project from the sensory epithelium to stereotyped targets within the olfactory bulb. Recent studies suggest that the generation of this precise spatial map probably involves a hierarchy of guidance events, as receptor neurons integrate information present in the epithelium and bulb to reach their target.