Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry

In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and poor spot-to-spot reproducibilities. We found that the quantitative precisions improved significantly when DMSO (dimethylsulfoxide) was added to the matrix solution. For lopinavir and ritonavir, currently the most frequently prescribed HIV-1 protease inhibitors, the signal-to-noise ratios improved significantly when potassium iodide was added to the matrix solution. The mean quantitative precisions, expressed as % relative standard deviation, were 6.4% for saquinavir, 7.3% for lopinavir, 8.5% for ritonavir, 11.1% for indinavir, and 7.2% for nelfinavir. The mean quantitative accuracies, expressed as % deviation, were 4.5% for saquinavir, 6.0% for lopinavir, 5.9% for ritonavir, 6.6% for indinavir, and 8.0% for nelfinavir. The concentrations measured for the individual quality control samples were all within 85-117% of the theoretical concentrations. The lower limits of quantification in cell lysates were 4 fmol/microL for saquinavir, 16 fmol/microL for lopinavir, 31 fmol/microL for ritonavir, and 100 fmol/microL for indinavir and nelfinavir. The mean mass accuracies for the protease inhibitors were 0.28 ppm using external calibration. Our results show that MALDI-FTICR mass spectrometry can be successfully used for precise, accurate, and selective quantitative analyses of HIV-1 protease inhibitors in cell lysates. In addition, the lower limits of quantification obtained allow clinical applications of the technique.     

Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.     

Ramping down a clinical 3 T scanner: a journey into MRI and MRS at 0.75 T

Object

Lower-field MR is reemerging as a viable, potentially cost-effective alternative to high-field MR, thanks to advances in hardware, sequence design, and reconstruction over the past decades. Evaluation of lower field strengths, however, is limited by the availability of lower-field systems on the market and their considerable procurement costs. In this work, we demonstrate a low-cost, temporary alternative to purchasing a dedicated lower-field MR system.

Materials and Methods

By ramping down an existing clinical 3 T MRI system to 0.75 T, proton signals can be acquired using repurposed ¹³C transmit/receive hardware and the multi-nuclei spectrometer interface. We describe the ramp-down procedure and necessary software and hardware changes to the system.

Results

Apart from presenting system characterization results, we show in vivo examples of cardiac cine imaging, abdominal two- and three-point Dixon-type water/fat separation, water/fat-separated MR Fingerprinting, and point-resolved spectroscopy. In addition, the ramp-down approach allows unique comparisons of, e.g., gradient fidelity of the same MR system operated at different field strengths using the same receive chain, gradient coils, and amplifiers.

Discussion

Ramping down an existing MR system may be seen as a viable alternative for lower-field MR research in groups that already own multi-nuclei hardware and can also serve as a testing platform for custom-made multi-nuclei transmit/receive coils.

     

Access to High-Purity 7mG-cap RNA in Substantial Quantities by a Convenient All-Chemical Solid-Phase Method

Given the importance of mRNA with 5’-cap, easy access to RNA substrates with different ^7mG caps, of high quality and in large quantities is essential to elucidate the roles of RNA and the regulation of underlying processes. In addition to existing synthetic routes to 5’-cap RNA based on enzymatic, chemical or chemo-enzymatic methods, we present here an all-chemical method for synthetic RNA capping. The novelty of this study lies in the fact that the capping reaction is performed on solid-support after automated RNA assembly using commercial 2’-O-propionyloxymethyl ribonucleoside phosphoramidites, which enable final RNA deprotection under mild conditions while preserving both ^7mG-cap and RNA integrity. The capping reaction is efficiently carried out between a 5’-phosphoroimidazolide RNA anchored on the support and ^7mGDP in DMF in the presence of zinc chloride. Substantial amounts of ^7mG-cap RNA (from 1 to 28 nucleotides in length and of any sequence with or without internal methylations) containing various cap structures (^7mGpppA, ^7mGpppA_m, ^7mGppp^m6A, ^7mGppp^m6A_m, ^7mGpppG, ^7mGpppG_m) were obtained with high purity after IEX-HPLC purification. This capping method using solid-phase chemistry is convenient to perform and provides access to valuable RNA substrates as useful research tools to unravel specific issues regarding cap-related processes.     

Self-Targeted Co-Delivery of an Antibiotic and a Cancer-Chemotherapeutic from Synthetic Liposomes for the Treatment of Infected Tumors

Intra-tumor bacteria promote tumor growth and inactivate cancer-chemotherapeutics, causing poor treatment prognoses. Combined administration of cancer-chemotherapeutics and antibiotics may disturb the oral and intestinal microflora in critically-ill patients. To establish intra-tumor co-delivery of cancer-chemotherapeutics and antibiotics, gemcitabine and ciprofloxacin are loaded in so-called “self-targeting”, highly blood-compatible, synthetic DCPA-H₂O liposomes equipped with complexed water for pH-responsiveness. Liposomal pH-responsiveness can be maintained by in-shell loading of gemcitabine and in-core loading of ciprofloxacin. These dual-loaded liposomes are stealthily transported in the blood circulation to accumulate in the acidic environment of an infected tumor. Upon tumor self-targeting, liposomes are fused with tumor cells and infecting bacteria and are disassembled to simultaneously release gemcitabine and ciprofloxacin. Treatment of mice with these self-targeting liposomes yields significantly higher responses of Escherichia coli infected tumors with respect to both infection and tumor volume than gemcitabine and ciprofloxacin co-delivered from non-self-targeting liposomes or free gemcitabine with or without ciprofloxacin in solution.     

A structural equation model to analyze the use of a new multi media platform for increasing awareness of cultural heritage

Dedicated multi media platforms have the potential to enhance experiences and increase people’s awareness of cultural heritage. It is, however, not clear to what extent and by which functions these platforms accomplish this. Therefore, the aim of this research is to investigate the influence of functionalities offered by a well-designed multi-media web-platform on the awareness and experience of cultural heritage. The multi media platform used is focused on Strijp-S, an industrial cultural heritage in Eindhoven, the Netherlands. The platform was created as a prototype based on a study of users’ preferences for contents and media types offered. To collect data, a sample of 302 respondents used the platform to explore and view Strijp-S. To test assumed relationships between different functionalities, awareness and experience in an integrated fashion, the data are analyzed using Structural Equation Modelling (SEM). The results show that information-collection funtionality plays a significant role in improving awareness of cultural heritage, whereas auxiliary functions are particularly helpful for enhancing experiences. The perceived helpfulness of these two sets of functions differ however between users depending on theitr level of prior knowledge. Policymakers and urban planners can use these results to create their own platform based on local heritage to increase the citizens’ awareness of cultural heritage.