Alternative host model to evaluate Aeromonas virulence

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.

Assay of β-hematin formation by malaria parasite

Novel leads are urgently required for designing antimalarials due to the reduced efficacy of presently available drugs. The malaria parasite has a unique reaction of heme polymerization, which has attracted much attention in the recent past as a target for the design of antimalarial drugs. The process is hampered by non-availability of a proper assay method. Currently available methods are cumbersome and require advanced instrumentation or radioactive substrates. Here, we are describing an assay for hemozoin formation that is simple and reproducible. This assay has routinely been used by us for the identification of potential compounds with antimalarial activity.

An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Simple Estimation of Incident HIV Infection Rates in Notification Cohorts Based on Window Periods of Algorithms for Evaluation of Line-Immunoassay Result Patterns

Background Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient's antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods. Methods We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm's window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship ‘Prevalence = Incidence x Duration’ in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship ‘incident = true incident + false incident’ and also to the IIR derived from the BED incidence assay. Results Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms' diagnostic sensitivity (R2 = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods. Conclusions IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.