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Biomolecules Jan 2024Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex...
Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO -glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO -glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique -glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric -glycans, i.e., -acetyllactosamine repeats and -acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX.
Topics: Humans; Glycosylation; Erythropoietin; Protein Processing, Post-Translational; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Acetylation
PubMed: 38254725
DOI: 10.3390/biom14010125 -
Comparative Biochemistry and... Mar 2024The toxicity of copper nanoparticles (CuNPs) to aquatic animals, particularly their effects on the cardiovascular system, has not been thoroughly investigated. In the...
The toxicity of copper nanoparticles (CuNPs) to aquatic animals, particularly their effects on the cardiovascular system, has not been thoroughly investigated. In the present study, zebrafish embryos were used as a model to address this issue. After exposure to different concentrations (0.01, 0.1, 1, and 3 mg/L) of CuNPs for 96 h (4 to 100 h post-fertilization), cardiac parameters of the heart rate (HR), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), ejection fraction (EF), and cardiac output (CO), and vascular parameters of the aortic blood flow velocity (ABFV) and aortic diameter (AD) were examined by a video-microscopic method. Morphologically, CuNPs induced concentration-dependent pericardial edema. Although CuNPs did not alter the HR, they significantly reduced the EDV, SV, and CO at ≥0.1 mg/L, the ESV and EF at 3 mg/L, the ABFV at ≥0.1 mg/L, and the AD at ≥1 mg/L. Transcript levels of several cardiac genes, nppa, nppb, vmhc, and gata4, were also examined. CuNPs significantly suppressed nppa and nppb at ≥0.1 mg/L, gata4 at ≥0.01 mg/L, and vmhc at 1 mg/L. This study demonstrated that CuNPs can induce cardiovascular toxicity at environmentally relevant concentrations during fish embryonic development and highlight the potential ecotoxicity of CuNPs to aquatic animals.
Topics: Animals; Zebrafish; Copper; Nanoparticles; Cardiovascular System; Nitrobenzoates; Procainamide
PubMed: 38220071
DOI: 10.1016/j.cbpc.2024.109838 -
Analytical Methods : Advancing Methods... Jan 2024Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling...
Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.
Topics: Female; Pregnancy; Humans; Chondroitin Sulfates; Procainamide; Pre-Eclampsia; Disaccharides; Placenta; Glycosaminoglycans
PubMed: 38189556
DOI: 10.1039/d3ay01578e -
Talanta Apr 2024N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide...
N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide (ProcA) labelling and online solid phase extraction (online SPE). N-glycans were enzymatically released, then labeled with ProcA and cleaned up via the online SPE using HILIC chemistry (online HILIC SPE). Two preparation protocols were optimized: a short one (1 h 30) and a long one (18 h). Furthermore, the developed approach was compared to RapiFluor-MS (RFMS) kit (from Waters) and to InstantPC kit (from Agilent) which both include a classical HILIC μElution plate SPE purification. Samples were analyzed using HILIC separation coupled to fluorescence and MS detection (HILIC-FLD-MS) with or without the online HILIC SPE. During the validation, repeatability, intermediate precision, stability, response function and injection volume were tested. Human IgG mix (Multigam®) and NIST mAb standard were used as references as their glycoprofiles are well described. A comparison of three batches of a rituximab biosimilar (Truxima®) and one batch of its originator (MabThera®) was also performed. Online HILIC SPE sample cleanup shows a higher sensitivity and repeatability compared to the classical HILIC μElution SPE. Our online HILIC SPE approach also offers the highest MS signal compared to both commercial kits. However, InstantPC shows the highest FLD signal. The analyses of rituximab samples were in line with the literature showing the efficiency of the method for N-glycan monitoring of biotherapeutics. In conclusion, the results demonstrated the usefulness and ease of application of the developed protocol with the online HILIC SPE purification.
Topics: Humans; Glycosylation; Rituximab; Glycoproteins; Polysaccharides; Solid Phase Extraction
PubMed: 38101031
DOI: 10.1016/j.talanta.2023.125541 -
Small (Weinheim An Der Bergstrasse,... Apr 2024Green synthesis of stable metal-organic frameworks (MOFs) with permanent and highly ordered porosity at room temperature without needing toxic and harmful solvents and...
Green synthesis of stable metal-organic frameworks (MOFs) with permanent and highly ordered porosity at room temperature without needing toxic and harmful solvents and long-term high-temperature reactions is crucial for sustainable production. Herein, a rapid and environmentally friendly synthesis strategy is reported to synthesize the complex topological bismuth-based-MOFs (Bi-MOFs), [Bi(CHO)(HO)] (denoted CAU-17), in water under ambient conditions by surfactant-mediated sonochemical approach, which could also be applicable to other MOFs. This strategy explores using cetyltrimethylammonium bromide (CTAB) amphiphilic molecules as structure-inducing agents to control the removal of non-coordinated water (dehydration) and enhance the degree of deprotonation of the ligands, thereby regulating the coordination and crystallization in aqueous solutions. In addition, another two new strategies for synthesizing CAU-17 by crystal reconstruction and one-step synthesis in binary solvents are provided, and the solvent-induced synthesis mechanism of CAU-17 is studied. The as-prepared CAU-17 presents a competitive iodine capture capability and effective delivery of the antiarrhythmic drug procainamide (PA) for enteropatia due to the broad pH tolerance and the unique phosphate-responsive destruction in the intestine. The findings will provide valuable ideas for the follow-up study of surfactant-assisted aqueous synthesis of MOFs and their potential applications.
PubMed: 38050936
DOI: 10.1002/smll.202307484 -
Heart Rhythm Feb 2024More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved.
BACKGROUND
More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved.
OBJECTIVE
This study aimed to investigate the mechanism and potential treatment of NPPA mutation-associated AF.
METHODS
Nppa knock-in (KI, p.I137T) rats were generated, and cardiac function was evaluated. Blood pressure was recorded using a tail-cuff system. The expression levels were measured using real-time polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot analysis, and RNA-sequence analysis. Programmed electrical stimulation, patch clamp, and multielectrode array were used to record the electrophysical characteristics.
RESULTS
Mutant rats displayed downregulated expression of atrial natriuretic peptide but elevated blood pressure and enlarged left atrial end-diastolic diameter. Further, gene topology analysis suggested that the majority of differently expressed genes in Nppa KI rats were related to inflammation, electrical remodeling, and structural remodeling. The expression levels of C-C chemokine ligand 5 and galectin-3 involved in remodeling were higher, while there were declined levels of Na1.5, Ca1.2, and connexin 40. AF was more easily induced in KI rats. Electrical remodeling included abbreviated action potentials, effective refractory period, increased late sodium current, and reduced calcium current, giving rise to conduction abnormalities. These electrophysiological changes could be reversed by the late sodium current blocker ranolazine and the Na1.8 blocker A-803467.
CONCLUSION
Our findings suggest that structural remodeling related to inflammation and fibrosis and electrical remodeling involved in late sodium current underly the major effects of the Nppa (p.I137T) variant to induce AF, which can be attenuated by the late sodium current blocker and Na1.8 blocker.
Topics: Animals; Rats; Action Potentials; Atrial Fibrillation; Atrial Natriuretic Factor; Atrial Remodeling; Heart Atria; Inflammation; Mutation; Myocytes, Cardiac; Procainamide; Sodium
PubMed: 37924963
DOI: 10.1016/j.hrthm.2023.10.025 -
Bioorganic & Medicinal Chemistry Letters Nov 2023Malignant melanoma has an aggressive nature and a high metastatic propensity resulting in the highest mortality rate of any skin cancer. In this study, we synthesized...
Malignant melanoma has an aggressive nature and a high metastatic propensity resulting in the highest mortality rate of any skin cancer. In this study, we synthesized F-labeled procainamide (PCA) for detection of melanoma using positron emission tomography (PET), and evaluated its biological characteristics. The non-decay-corrected radiochemical yield of F-PCA was 10-15% and its in vitro stability was over 98% for 2 h. At 1 h, cellular uptake of F-PCA was 3.8-fold higher in a group with the presence of l-tyrosine than in a non-l-tyrosine-treated group. Furthermore, F-PCA permitted visualization of B16F10 (mouse melanoma) xenografts on microPET after intravenous injection, and was retained in the tumor for 60 min, with a high tumor-to-liver uptake ratio. F-PCA showed specific melanoma uptake in primary lesions with a high melanin targeting ability in small animal models. F-PCA may have potential as a PET imaging agent for direct melanoma detection.
Topics: Animals; Mice; Humans; Procainamide; Melanoma; Skin Neoplasms; Positron-Emission Tomography; Radiopharmaceuticals; Cell Line, Tumor; Fluorine Radioisotopes; Melanoma, Cutaneous Malignant
PubMed: 37852422
DOI: 10.1016/j.bmcl.2023.129528 -
Annals of Medicine and Surgery (2012) Oct 2023Aluminum phosphide (ALP) is a commonly used suicidal agent in an agrarian country like Nepal. The unmasking of the Brugada pattern in the electrocardiogram (ECG)...
INTRODUCTION AND IMPORTANCE
Aluminum phosphide (ALP) is a commonly used suicidal agent in an agrarian country like Nepal. The unmasking of the Brugada pattern in the electrocardiogram (ECG) associated with ALP poisoning is a rare phenomenon, and studies pertaining to it are scarce in the medical literature.
CASE PRESENTATION
An 18-year-old female presented to the emergency department with multiple episodes of vomiting, headache, blurring of vision, and abdominal pain after 4 h of consumption of ALP with suicidal intent. A 12-lead ECG revealed a coved ST-segment elevation and T-wave inversion in leads V1-V3 with right bundle branch block suggestive of a type 1 Brugada pattern. Her past medical and family history was not significant. The patient made an uneventful recovery with the required supportive treatments.
CLINICAL DISCUSSION
Cardiac arrhythmias are the major cause of death in ALP poisoning. Unmasking of the Brugada ECG pattern is a rare but potentially fatal complication implicated in various pharmacological toxicities, including tricyclic antidepressants, cocaine, procainamide, disopyramide, flecainide, and rarely with ALP.
CONCLUSIONS
ALP poisoning can unmask the Brugada ECG pattern, which can lead to ventricular fibrillation and/or sudden cardiac death.
PubMed: 37811028
DOI: 10.1097/MS9.0000000000001129 -
Chemico-biological Interactions Sep 2023Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of...
Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of organophosphorus compounds (OPs). Domestic animals such as cows, pigs, sheep, and goats have been used for the transgenic expression of a variety of valuable therapeutic proteins. Indeed, rHu BChE was successfully expressed in the milk of transgenic goats, but the presence of any endogenous cholinesterases (ChE) in milk would interfere with the isolation of expressed rHu BChE. The aim of this study was to determine the presence of endogenous ChEs in bovine, ovine, caprine, and porcine milk to determine the suitability of these species for the production of rHu BChE. Using acetyl- and butyryl- thiocholine as substrates, ChE activity (2-4 U/mL) was detected in pig milk only. ChE activities in milk from other animals were <0.01 U/mL and could only be detected following enrichment on procainamide-Sepharose gel. Two different methods based on measuring activity in the presence of acetylcholinesterase (AChE)- or BChE- specific inhibitors were used to estimate the proportions of AChE and BChE activities in enriched milk. Monoclonal antibodies (MAbs), against fetal bovine serum AChE that recognize AChEs from ruminants only, were also used to confirm the identity of AChEs. While bovine and ovine milk contain both AChE and BChE activities, caprine and porcine milk contain predominantly BChE activity. The presence of very low ChE activity supports the choice of cows, sheep, and goats for the transgenic expression of rHu BChE in milk.
Topics: Female; Animals; Humans; Sheep; Cattle; Swine; Butyrylcholinesterase; Goats; Acetylcholinesterase; Milk; Animals, Genetically Modified; Pain
PubMed: 37659623
DOI: 10.1016/j.cbi.2023.110691