Due to the large number of basic therapeutic and illicit drugs, systematic toxicological analysis has widely been performed with liquid chromatography coupled to mass spectrometry using positive electrospray ionization. However, there exist a smaller number of drugs, typically acidic drugs, which require the use of negative electrospray ionization either via a separate analysis or polarity switching. Here, targets relating to salicylic acid and ibuprofen in positive electrospray ionization were determined through a metabolomics-driven retrospective investigation of forensic casework. Samples were previously screened using liquid chromatography coupled with high-resolution mass spectrometry with quantification of target analytes performed using liquid chromatography with tandem mass spectrometry. Of the 1,717 whole-blood samples submitted between 2014 and 2019, 48 were positive for salicylic acid (1.1-1,400 mg/kg) and 78 for ibuprofen (1-46 mg/kg). Based on the retrospective analysis, 19 and 90 targets were identified for salicylic acid and ibuprofen, respectively. For targets of salicylic acid, the protonated adduct of salicyluric acid ([M + H]+ , m/z 196.0605) was present in 89.6% (n = 32) of the salicylic acid positive cases, while the [M + HCOOH + CH3 CN + Ca - H]+ adduct (m/z 264.0179) of salicylic acid was present in all positive samples with concentrations above 66 mg/kg salicylic acid. Similarly, the [M + 2Na - H]+ adduct (m/z 251.1018) of ibuprofen was present in 98.7% (n = 77) of positive cases and was present in all samples with concentrations above 3 mg/kg ibuprofen.
In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges. Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL-15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). Secretion of TNFR:Fc (a disulfide-rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum. PDI overexpression did not result in intracellular retention of IL15. The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein.
A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.
Dynamic combinatorial chemistry has emerged as a promising tool for the discovery of complex receptors in supramolecular chemistry. At the heart of dynamic combinatorial chemistry are the reversible reactions that enable the exchange of building blocks between library members in dynamic combinatorial libraries (DCLs) ensuring thermodynamic control over the system. If more than one reversible reaction operates in a single dynamic combinatorial library, the complexity of the system increases dramatically, and so does its possible applications. One can imagine two reversible reactions that operate simultaneously or two reversible reactions that operate independently. Both these scenarios have advantages and disadvantages. In this contribution, we show how disulfide exchange and boronic ester transesterification can function simultaneous in dynamic combinatorial libraries under appropriate conditions. We describe the detailed studies necessary to establish suitable reaction conditions and highlight the analytical techniques appropriate to study this type of system.
Abstract Background and purpose The endocannabinoid system (ECS) has been found altered in patients with multiple sclerosis (MS). However, whether the ECS alteration is present in the early stage of MS remains unknown. First, we aimed to compare the ECS profile between newly diagnosed MS patients and healthy controls (HCs). Next, we explored the association of the ECS, biomarkers of inflammation, and clinical parameters in newly diagnosed MS patients. Methods Whole blood gene expression of ECS components and levels of endocannabinoids in plasma were measured by real‐time quantitative polymerase chain reaction and ultra‐high‐pressure liquid chromatography–mass spectrometry, respectively, in 66 untreated MS patients and 46 HCs. Results No differences were found in the gene expression or plasma levels of the selected ECS components between newly diagnosed MS patients and HCs. Interferon‐γ, encoded by the gene IFNG, correlated positively ( ρ = 0.60) with the expression of G protein‐coupled receptor 55 ( GPR55 ), and interleukin1β ( IL1B ) correlated negatively ( ρ = −0.50) with cannabinoid receptor 2 ( CNR2 ) in HCs. Conclusions We found no alteration in the peripheral ECS between untreated patients with MS and HC. Furthermore, our results indicate that the ECS has a minor overall involvement in the early stage of MS on inflammatory markers and clinical parameters when compared with HCs.
Alternative specimens collected during autopsies can be valuable in postmortem toxicology in cases where peripheral blood is not available. The applicability of brain tissue as an alternative matrix for drug screening by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was investigated in this study. Results of the 50 most frequently detected drugs and metabolites of toxicological interest in blood and brain tissue samples from 1,719 autopsy cases were compared. Examination of the results in paired blood and brain tissue samples revealed that the two matrices were in general comparable, as the majority of the 50 analytes were observed in a high number of the examined cases in both blood and brain tissue. This demonstrates the potential of brain tissue as an alternative matrix for drug screening in postmortem toxicology or as a secondary matrix for confirmation.
Abstract There is limited knowledge on the global prescription and consumption patterns of therapeutic (TD) and illicit drugs (ID). Pooled urine analysis and wastewater-based epidemiology (WBE) has been used for local-based drug screening. It is, however, difficult to study the global epidemiology due to difficulties in obtaining samples. The aims of the study were to test the detectability of TD and ID in airplane wastewater samples categorized according to their geographical origin. Wastewater samples (n= 17) were collected from long-distance flights and prepared with enzymatic conjugate cleaving followed by either precipitation or solid phase extraction. Aliquots were analysed on various liquid chromatography – mass spectrometers. TDs were grouped according to their Anatomical Therapeutic Chemical (ATC) codes. Identification confidence was assigned to three levels based on variables including detection on multiple instruments and number of targets per compound. A total of 424 compounds were identified across all samples, distributed on 87 unique TD and 2 ID. Two principal components in a principal component analysis separated three clusters of wastewater samples corresponding to geographical origin of the airplanes with therapeutic subgroup ATC codes as variables. Airplane wastewater analysis is useful for identifying targets for WBE and toxicological analysis and explore drug use and abuse patterns.
Small molecule additives to cell culture media (e.g., sodium butyrate) that are capable of enhancing the expression of recombinant proteins have significant utility in the production and manufacture of therapeutic polypeptides. To identify novel small molecule enhancers (SMEs) of recombinant protein expression in Chinese Hamster Ovary (CHO) cells, we screened two separate small molecule libraries for compounds capable of enhancing the expression of either a fluorescent reporter protein or a monoclonal antibody. Several compounds that increased recombinant protein expression were identified, and these compounds fell into three broad classes: (1) aromatic carboxylic acids, (2) hydroxamic acids, and (3) acetamides. We examined the impact of SME addition to CHO cell cultures expressing different classes of recombinant proteins including monoclonal antibodies (MAbs). For CHO cell pools or clones grown in production shake-flasks or bioreactors, recombinant protein titers up to 60% higher than control cultures were observed. Analysis of mRNA levels suggest that transcriptional activation plays a role in the expression enhancement seen for some SMEs, but other mechanisms may be involved for at least one compound. Finally, we tested many of the identified SMEs for their ability to increase MAb production by a hybridoma cell line. Hexanohydroxamic acid increased shake-flask MAb production by 40% relative to a control. Taken together, these data demonstrate the potential utility of the compounds in the production of therapeutically relevant proteins from diverse cell-based production systems.
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