Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype

Computed tomography of solitary fibrous tumor of the pleura abutting the mediastinum: A diagnostic challenge

Background: Solitary fibrous tumor of the pleura (SFTP) arising from the mediastinal pleura may be confused with primary mediastinal tumors. We studied the computerized tomographic (CT) findings of patients with SFTP that could suggest a diagnosis of SFTP. Materials and Methods: At our hospital from January 1995 to June 2012, 39 patients with histologically confirmed SFTP were surgically treated; seven of them abutting the mediastinal pleura. The study group included seven patients aged between 53 and 81 years. Baseline CT scans were retrospectively reviewed to identify radiological findings suggestive of SFTP including: (1) smooth and sharply delineated contours; (2) obtuse, acute, or tapering angles between the lesion and the mediastinum depending on the size; (3) homogeneous soft-tissue attenuation; (4) “geographic pattern” due to the contemporary presence of large vessels, necrosis, and calcifications; (5) displacement of the lung parenchyma; (6) presence of a cleavage plane; and (7) absence of lymphadenopathy or pleural methastasis. Results: All tumors formed acute angles with the pleura. Six out of the seven presented smoothly tapering margins, three had a “geographic pattern” of attenuation and displaced the anterior junction line; one showed an outside junction line development. Four cases had a clear pleural origin. Conclusions: The possibility of SFTP should be taken into account when a mass abuts the mediastinum projecting inside the thoracic cavity in the presence of an intense and “geographical pattern” of enhancement without lymphoadenopathy or pleural metastasis. These findings assume greater significance in the presence of discrepancy between the size of the lesion and the clinical presentation

Anthracycline-Induced Cardiotoxicity: Cardiac Monitoring by Continuous Wave-Doppler Ultrasound Cardiac Output Monitoring and Correlation to Echocardiography

Background: Anthracyclines are agents with a well-known cardiotoxicity. The study sought to evaluate the hemodynamic response to an anthracycline using real-time continuous-wave (CW)-Doppler ultrasound cardiac output monitoring (USCOM) and echocardiography in combination with serum biomarkers. Methods: 50 patients (26 male, 24 female, median age 59 years) suffering from various types of cancer received an anthracycline-based regimen. Patients' responses were measured at different time points (T0 prior to infusion, T1 6 h post infusion, T2 after 1 day, T3 after 7 days, and T4 after 3 months) with CW-Doppler ultrasound (T0-T4) and echocardiography (T1, T4) for hemodynamic parameters such as stroke volume (SV; SVUSCOM ml) and ejection fraction (EF; EFechocardiography%) and with NT-pro-BNP and hs-Troponin T (T0-T4). Results: During the 3-month observation period, the relative decrease in the EF determined by echocardiography was -2.1% (Delta T0-T4, T0 71 +/- 7.8%, T4 69.5 +/- 7%, p = 0.04), whereas the decrease in SV observed using CW-Doppler was -6.5% (Delta T0-T4, T0 54 +/- 19.2 ml, T4 50.5 +/- 20.6 ml, p = 0.14). The kinetics for serum biomarkers were inversely correlated. Conclusions: Combining real-time CW-Doppler USCOM and serum biomarkers is feasible for monitoring the immediate and chronic hemodynamic changes during an anthracycline-based regimen; the results obtained were comparable to those from echocardiography

Prevalence and risk factors susceptibility of Salmonella spp., Campylobacter spp. and Listeria spp. isolated from pork and poultry sausages, in Reunion Island, France

Zoonoses are a public health burden in France. The most predominant pathogens incriminated in TIAC are Salmonella spp. Campylobacter spp., and Listeria spp. The population of Reunion Island eats a lot of pork and poultry meats. One of the local cooking characteristic is the sausage 100% poultry or 100% pig

Strigolactones affect phosphorus acquisition strategies in tomato plants

Strigolactones (SLs) are plant hormones that modulate morphological, physiological and biochemical changes as part of the acclimation strategies to phosphorus (P) deficiency, but an in‐depth description of their effects on tomato P‐acquisition strategies under P shortage is missing. Therefore, in this study, we investigate how SLs impact on root exudation and P uptake, in qualitative and quantitative terms over time, in wild‐type and SL‐depleted tomato plants grown with or without P. Under P shortage, SL‐depleted plants were unable to efficiently activate most mechanisms associated with the P starvation response (PSR), except for the up‐regulation of P transporters and increased activity of P‐solubilizing enzymes. The reduced SL biosynthesis had negative effects also under normal P provision, because plants over‐activated high‐affinity transporters and enzymatic activities (phytase, acidic phosphatase) to sustain elevated P uptake, at great carbon and nitrogen costs. A shift in the onset of PSR was also highlighted in these plants. We conclude that SLs are master kinetic regulators of the PSR in tomato and that their defective synthesis might lead both to suboptimal nutritional outcomes under P depletion and an unbalanced control of P uptake when P is available

mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with K_d = 1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication

Estimating specific surface area of fine stream bed sediments from geochemistry

Specific surface area (SSA) of headwater stream bed sediments is a fundamental property which determines the nature of sediment surface reactions and influences ecosystem-level, biological processes. Measurements of SSA – commonly undertaken by BET nitrogen adsorption – are relatively costly in terms of instrumentation and operator time. A novel approach is presented for estimating fine (2.5 mg kg−1), four elements were identified as significant predictors of SSA (ordered by decreasing predictive power): V > Ca > Al > Rb. The optimum model from these four elements accounted for 73% of the variation in bed sediment SSA (range 6–46 m2 g−1) with a root mean squared error of prediction – based on leave-one-out cross-validation – of 6.3 m2 g−1. It is believed that V is the most significant predictor because its concentration is strongly correlated both with the quantity of Fe-oxides and clay minerals in the stream bed sediments, which dominate sediment SSA. Sample heterogeneity in SSA – based on triplicate measurements of sub-samples – was a substantial source of variation (standard error = 2.2 m2 g−1) which cannot be accounted for in the regression model. The model was used to estimate bed sediment SSA at the other 1792 sites and at 30 duplicate sites where an extra sediment sample had been collected, 25 m from the original site. By delineating sub-catchments for the headwater sediment sites only those sub-catchments were selected with a dominant (>50% of the sub-catchment area) bedrock formation and land use type; the bedrock and land use classes accounted for 39% and 7% of the variation in bed sediment SSA, respectively. Variation in estimated, fine bed sediment SSA from the paired, duplicate sediment sites was small (2.7 m2 g−1), showing that local variation in SSA at stream sites is modest when compared to that between catchments. How the approach might be applied in other environments and its potential limitations are discussed

Intracerebral electrical stimulations of the temporal lobe: a stereo-electroencephalography study

The functional anatomy of the anteromesial portion of the temporal lobe and its involvement in epilepsy can be explored by means of intracerebral electrical stimulations. Here, we aimed to expand the knowledge of its physiological and pathophysiological symptoms by conducting the first large-sample systematic analysis of 1529 electrical stimulations of this anatomical region. We retrospectively analysed all clinical manifestations induced by intracerebral electrical stimulations in 173 patients with drug-resistant focal epilepsy with at least one electrode implanted in this area. We found that high-frequency stimulations were more likely to evoke electroclinical manifestations (p < .0001) and also provoked ‘false positive’ seizures. Multimodal symptoms were associated with EEG electrical modification (after discharge) (p < .0001). Visual symptoms were not associated with after discharge (p = .0002) and were mainly evoked by stimulation of the hippocampus (p = .009) and of the parahippocampal gyrus (p = .0212). ‘False positive seizures’ can be evoked by stimulation of the hippocampus, parahippocampal gyrus and amygdala, likely due to their intrinsic low epileptogenic threshold. Visual symptoms evoked in the hippocampus and parahippocampal gyrus, without EEG changes, are physiological symptoms and suggest involvement of these areas in the visual ventral stream. Our findings provide meaningful guidance in the interpretation of intracranial EEG studies of the temporal lobe