Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as a tag on the protein of interest and the second fragment as a supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells

Nonheme Iron–Thiolate Complexes as Structural Models of Sulfoxide Synthase Active Sites

Two mononuclear iron(II)–thiolate complexes have been prepared that represent structural models of the nonheme iron enzymes EgtB and OvoA, which catalyze the O2-dependent formation of carbon–sulfur bonds in the biosynthesis of thiohistidine compounds. The series of Fe(II) complexes reported here feature tripodal N4 chelates (LA and LB) that contain both pyridyl and imidazolyl donors (LA = (1H-imidazol-4-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine; LB = N,N-bis((1-methylimidazol-2-yl)methyl)-2-pyridylmethylamine). Further coordination with monodentate aromatic or aliphatic thiolate ligands yielded the five-coordinate, high-spin Fe(II) complexes [FeII(LA)(SMes)]BPh4 (1) and [FeII(LB)(SCy)]BPh4 (2), where SMes = 2,4,6-trimethylthiophenolate and SCy = cyclohexanethiolate. X-ray crystal structures revealed that 1 and 2 possess trigonal bipyramidal geometries formed by the N4S ligand set. In each case, the thiolate ligand is positioned cis to an imidazole donor, replicating the arrangement of Cys- and His-based substrates in the active site of EgtB. The geometric and electronic structures of 1 and 2 were analyzed with UV-vis absorption and Mössbauer spectroscopies in tandem with density functional theory (DFT) calculations. Exposure of 1 and 2 to nitric oxide (NO) yielded six-coordinate FeNO adducts that were characterized with infrared and electron paramagnetic resonance (EPR) spectroscopies, confirming that these complexes are capable of binding diatomic molecules. Reaction of 1 and 2 with O2 causes oxidation of the thiolate ligands to disulfide products. The implications of these results for the development of functional models of EgtB and OvoA are discussed

Synchronized dynamics of cortical neurons with time-delay feedback

The dynamics of three mutually coupled cortical neurons with time delays in the coupling are explored numerically and analytically. The neurons are coupled in a line, with the middle neuron sending a somewhat stronger projection to the outer neurons than the feedback it receives, to model for instance the relay of a signal from primary to higher cortical areas. For a given coupling architecture, the delays introduce correlations in the time series at the time-scale of the delay. It was found that the middle neuron leads the outer ones by the delay time, while the outer neurons are synchronized with zero lag times. Synchronization is found to be highly dependent on the synaptic time constant, with faster synapses increasing both the degree of synchronization and the firing rate. Analysis shows that presynaptic input during the interspike interval stabilizes the synchronous state, even for arbitrarily weak coupling, and independent of the initial phase. The finding may be of significance to synchronization of large groups of cells in the cortex that are spatially distanced from each other.Comment: 21 pages, 11 figure

Designing forest biodiversity experiments : general considerations illustrated by a new large experiment in subtropical China

Funded by German Research Foundation. Grant Number: DFG FOR 891/1 and 2 National Natural Science Foundation of China. Grant Numbers: NSFC 30710103907, 30930005, 31170457 , 31210103910 Swiss National Science Foundation (SNSF) Sino-German Centre for Research Promotion in BeijingPeer reviewedPublisher PD

Profound Depletion of HIV-1 Transcription in Patients Initiating Antiretroviral Therapy during Acute Infection

Early intervention resulted in profound depletion of PBMC expressing HIV-1 RNA. This is contrary to chronically infected patients who predominantly showed continuous UsRNA expression on cART. Thus, antiretroviral treatment initiated during the acute phase of infection prevented establishment or expansion of long-lived transcriptionally active viral cellular reservoirs in peripheral blood

Validation of 2006 WHO Prediction Scores for True HIV Infection in Children Less than 18 Months with a Positive Serological HIV Test

All infants born to HIV-positive mothers have maternal HIV antibodies, sometimes persistent for 18 months. When Polymerase Chain Reaction (PCR) is not available, August 2006 World Health Organization (WHO) recommendations suggest that clinical criteria may be used for starting antiretroviral treatment (ART) in HIV seropositive children <18 months. Predictors are at least two out of sepsis, severe pneumonia and thrush, or any stage 4 defining clinical finding according to the WHO staging system.From January 2005 to October 2006, we conducted a prospective study on 236 hospitalized children <18 months old with a positive HIV serological test at the national reference hospital in Kigali. The following data were collected: PCR, clinical signs and CD4 cell count. Current proposed clinical criteria were present in 148 of 236 children (62.7%) and in 95 of 124 infected children, resulting in 76.6% sensitivity and 52.7% specificity. For 87 children (59.0%), clinical diagnosis was made based on severe unexplained malnutrition (stage 4 clinical WHO classification), of whom only 44 (50.5%) were PCR positive. Low CD4 count had a sensitivity of 55.6% and a specificity of 78.5%.As PCR is not yet widely available, clinical diagnosis is often necessary, but these criteria have poor specificity and therefore have limited use for HIV diagnosis. Unexplained malnutrition is not clearly enough defined in WHO recommendations. Extra pulmonary tuberculosis (TB), almost impossible to prove in young children, may often be the cause of malnutrition, especially in HIV-affected families more often exposed to TB. Food supplementation and TB treatment should be initiated before starting ART in children who are staged based only on severe malnutrition

Ageing with HIV: medication use and risk for potential drug-drug interactions

Objectives To compare the use of co-medication, the potential drug-drug interactions (PDDIs) and the effect on antiretroviral therapy (ART) tolerability and efficacy in HIV-infected individuals according to age, ≥50 years or <50 years. Methods All ART-treated participants were prospectively included once during a follow-up visit of the Swiss HIV Cohort Study. Information on any current medication was obtained by participant self-report and medical prescription history. The complete treatment was subsequently screened for PDDIs using a customized version of the Liverpool drug interaction database. Results Drug prescriptions were analysed for 1497 HIV-infected individuals: 477 age ≥50 and 1020 age <50. Older patients were more likely to receive one or more co-medications compared with younger patients (82% versus 61%; P < 0.001) and thus had more frequent PDDIs (51% versus 35%; P < 0.001). Furthermore, older patients tended to use a higher number of co-medications and certain therapeutic drug classes more often, such as cardiovascular drugs (53% versus 19%; P < 0.001), gastrointestinal medications (10% versus 6%; P = 0.004) and hormonal agents (6% versus 3%; P = 0.04). PDDIs with ART occurred mainly with cardiovascular drugs (27%), CNS agents (22%) and methadone (6%) in older patients and with CNS agents (27%), methadone (15%) and cardiovascular drugs (11%) in younger patients. The response to ART did not differ between the two groups. Conclusions The risk for PDDIs with ART increased in older patients who take more drugs than their younger HIV-infected counterparts. However, medication use in older and younger patients did not differ in terms of effect on antiretroviral tolerability and respons