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Analytical and Bioanalytical Chemistry Jun 2024Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns...
Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, K ~ 4.4 nM, was consistent with literature values.
PubMed: 38942955
DOI: 10.1007/s00216-024-05413-7 -
Methods in Enzymology 2024Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene...
Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.
Topics: Alkyl and Aryl Transferases; Dimethylallyltranstransferase; Diterpenes; Enzyme Assays; Polyisoprenyl Phosphates; Cyclization
PubMed: 38942517
DOI: 10.1016/bs.mie.2023.11.003 -
Microbial Pathogenesis Jun 2024Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of...
Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 10 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.
PubMed: 38942248
DOI: 10.1016/j.micpath.2024.106766 -
Journal of Ethnopharmacology Jun 2024The traditional medicinal formulation, Qifu-yin (QFY), has been widely prescribed for Alzheimer's disease (AD) treatment in China, yet the comprehensive mechanisms...
ETHNOPHARMACOLOGICAL RELEVANCE
The traditional medicinal formulation, Qifu-yin (QFY), has been widely prescribed for Alzheimer's disease (AD) treatment in China, yet the comprehensive mechanisms through which QFY mitigates AD pathology remain to be fully delineated.
AIM OF THE STUDY
This study aimed to explore the therapeutic implications of QFY on the synaptic injury and oxidative stress in the hippocampus of APPswe/PS1dE9 (APP/PS1) mice, with a concerted effort to elucidate the molecular mechanisms related to synaptic preservation and memory improvement.
MATERIALS AND METHODS
The components of QFY were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The neuroprotective effects of QFY was evaluated using six-month-old male APP/PS1 mice. Subsequent to a 15 days of QFY regimen, spatial memory was assessed utilizing the Morris water maze (MWM) test. Amyloid-beta (Aβ) aggregation was detected via immunostaining, while the quantification of Aβ and Aβ was achieved through enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was used to investigate the synaptic structure and mitochondrial morphology. Golgi staining was applied to examine dendritic spine density. Reactive oxygen species (ROS), 3-nitrotyrosine (3-NT) and 4-hydroxy-nonenal (4-HNE) assays were employed to assess oxidative stress. The expression profiles of Aβ metabolism-associated enzymes and the Keap1/Nrf2/ARE signaling pathway were determined by Western blot.
RESULTS
A total of 20 principal compounds in QFY were identified. QFY mitigated memory deficits of APP/PS1 mice, including reducing escape latency and search distance and increasing the time and distance spent in the target quadrant. In addition, QFY increased platform crossings of APP/PS1 mice in the probe trial of MWM tests. TEM analysis showed that QFY increased synapse number in the CA1 region of APP/PS1 mice. Further studies indicated that QFY elevated the expression levels of Post synaptic density protein 95 (PSD95) and synaptophysin, and mitigated the loss of dendritic spine density in the hippocampus of APP/PS1 mice. QFY has been shown to ameliorated the structural abnormalities of mitochondria, including mitochondrial dissolution and degradation, up-regulate ATP synthesis and membrane potential in the hippocampus of APP/PS1 mice. Moreover, QFY activated the Keap1/Nrf2/ARE signaling pathway in the hippocampus of APP/PS1 mice, which might contribute to the neuroprotective effects of QFY.
CONCLUSION
QFY activates the Keap1/Nrf2/ARE signaling, and protects against synaptic and mitochondrial dysfunction in APP/PS1 mice, proposing a potential alternative therapeutic strategy for AD management.
PubMed: 38942156
DOI: 10.1016/j.jep.2024.118497 -
Journal of the American Chemical Society Jun 2024Identifying the active phase with the highest activity, which is long-believed to be a steady state of the catalyst, is the basis of rational design of heterogeneous...
Identifying the active phase with the highest activity, which is long-believed to be a steady state of the catalyst, is the basis of rational design of heterogeneous catalysis. In this work, we performed detailed investigations, successfully capturing the instantaneous structure-activity change in oscillating Pd nanocatalysts during methane oxidation, which reveals an unprecedented oscillatory active state. Combining quantitative environmental transmission electron microscopy and highly sensitive online mass spectrometry, we identified two distinct phases for the reaction: one where the Pd nanoparticles refill with oxygen, and the other, a period of abrupt pumping of oxygen and boosted methane oxidation within about 1 s. It is the rapid reduction process that shows the highest activity for total oxidation of methane, not a PdO or Pd steady state under the conditions applied here (methane:oxygen = 5:1). This observation challenges the traditional understanding of the active phase and requires a completely different strategy for catalyst optimization.
PubMed: 38942067
DOI: 10.1021/jacs.4c02830 -
American Journal of Veterinary Research Jun 2024To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing...
OBJECTIVE
To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (≥ 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age.
ANIMALS
16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses ≥ 25 years old).
METHODS
Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 °C until assayed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic analyses were performed using Phoenix WinNonlin, version 8.0 (Certara). Both clinical and pharmacokinetic data were compared between age groups using independent samples t tests, with P < .05 considered significant.
RESULTS
Baseline characteristics did not differ between groups, with the exception of age, weight, and plasma total solids. Plasma concentrations of PBZ were best described by a two-compartment model. The maximum plasma concentration of OPBZ was reached at 5 hours for both age groups, and the metabolite-to-parent-drug area-under-the-curve ratios were approximately 20% for both groups. None of the pharmacokinetic parameters of PBZ or its metabolite, OPBZ, differed significantly between age groups.
CLINICAL RELEVANCE
The hypothesis was rejected as there was no significant difference in PBZ disposition in young-adult horses compared to geriatric horses. Our data do not support the need for dose adjustments of PBZ in clinically healthy geriatric horses.
PubMed: 38942059
DOI: 10.2460/ajvr.24.01.0012 -
Environment International Jun 2024The National Academies of Sciences, Engineering, and Medicine recommends per- and polyfluoroalkyl substance (PFAS) blood testing for patients with risk of elevated...
The National Academies of Sciences, Engineering, and Medicine recommends per- and polyfluoroalkyl substance (PFAS) blood testing for patients with risk of elevated exposure, and the Agency for Toxic Substances and Disease Registry (ATSDR) suggests PFAS blood testing based on exposure. Barriers to PFAS blood testing include cost, access to labs, and evolving laboratory methods. We quantify water and serum PFAS levels among a highly-exposed cohort in an area with groundwater contaminated by historical agricultural biosolid application. We compare the gold standard PFAS serum test with a commercial test and results from a one-compartment toxicokinetic model. Participants were adults (n = 30) whose household (n = 19) water had levels of the sum of six PFAS > 500 ng/L. Serum PFAS were measured using liquid chromatography-tandem mass spectrometry. Demographic and water consumption data were collected via telephone. Serum PFAS results from the commercial test were accessed via medical record. Statistical analysis included descriptive statistics and bivariate plots of serum levels. Perfluorohexanoic acid, perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorobutanesulfonic acid, perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS) were detected in 19 wells, and PFHpA, PFOA, PFNA, perfluorodecanoic acid, perfluoroundecanoic acid, PFHxS, and PFOS were detected in at least 19 participants' serum. In well water, PFOA and PFOS levels had geometric means (GMs) of 1749 ng/L (geometric standard deviation [GSD] 2.4) and 887 ng/L (GSD 19.7), respectively. In serum, PFOA and PFOS had GMs of 116.2 µg/L (GSD 13.5) and 58.3 µg/L (GSD 13.8), respectively. Our results are comparable with and had a wider mix of PFAS than other high-exposure cohorts. There was good agreement between the commercial and gold standard tests for PFOA, PFNA, and PFHxS, and mixed agreement between the gold standard test and modeled predictions, suggesting water-based toxicokinetic models of serum PFAS may be inadequate for assessing exposure in this population.
PubMed: 38941944
DOI: 10.1016/j.envint.2024.108850 -
Biophysical Chemistry Jun 2024Human islet amyloid polypeptide (hIAPP) forms amyloid deposits that contribute to β-cell death in pancreatic islets and are considered a hallmark of Type II diabetes...
Human islet amyloid polypeptide (hIAPP) forms amyloid deposits that contribute to β-cell death in pancreatic islets and are considered a hallmark of Type II diabetes Mellitus (T2DM). Evidence suggests that the early oligomers of hIAPP formed during the aggregation process are the primary pathological agent in islet amyloid induced β-cell death. The self-assembly mechanism of hIAPP, however, remains elusive, largely due to limitations in conventional biophysical techniques for probing the distribution or capturing detailed structures of the early, structurally dynamic oligomers. The advent of Ion-mobility Mass Spectrometry (IM-MS) has enabled the characterisation of hIAPP early oligomers in the gas phase, paving the way towards a deeper understanding of the oligomerisation mechanism and the correlation of structural information with the cytotoxicity of the oligomers. The sensitivity and the rapid structural characterisation provided by IM-MS also show promise in screening hIAPP inhibitors, categorising their modes of inhibition through "spectral fingerprints". This review delves into the application of IM-MS to the dissection of the complex steps of hIAPP oligomerisation, examining the inhibitory influence of metal ions, and exploring the characterisation of hetero-oligomerisation with different hIAPP variants. We highlight the potential of IM-MS as a tool for the high-throughput screening of hIAPP inhibitors, and for providing insights into their modes of action. Finally, we discuss advances afforded by recent advancements in tandem IM-MS and the combination of gas phase spectroscopy with IM-MS, which promise to deliver a more sensitive and higher-resolution structural portrait of hIAPP oligomers. Such information may help facilitate a new era of targeted therapeutic strategies for islet amyloidosis in T2DM.
PubMed: 38941872
DOI: 10.1016/j.bpc.2024.107285 -
Water Research Jun 2024Dissolved black carbon (DBC) released from biochar, is an essential group in the dissolved organic matter (DOM) pool and is widely distributed in aquatic environments....
Dissolved black carbon (DBC) released from biochar, is an essential group in the dissolved organic matter (DOM) pool and is widely distributed in aquatic environments. In various advanced oxidation processes (AOPs), DBC exhibits enhanced free radical scavenging compared to typical DOM, attributed to its smaller molecular weight and more compacted aromatic structure; however, the molecular-level transformations of DBC in different AOPs, such as UV/HO, UV/PDS, and UV/Chlorine, remain unclear. This study employed a DBC derived from wheat biochar for experimentation. Characterization involved ultraviolet-visible (UV-Vis) spectroscopy and fluorescence excitation-emission-matrix (EEM) spectroscopy, revealing the transformation of DBC through diminished SUVA values and reduced intensity of three-dimensional fluorescence peaks. Further insights into the transformation were gained through Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). After each UV-AOP treatment, a conspicuous augmentation in the oxygen content of DBC was observed. The detailed oxygenation processes were elucidated through mass difference analysis, based on 23 types of typical reactions. Results indicated that oxygenation reactions were most frequently detected in all three UV-AOP treatments. Specifically, the hydroxylation (+O) predominated in UV/HO, while the di-hydroxylation (+2O) prevailed in UV/PDS. UV/Chlorine treatments commonly exhibited tri-hydroxylation (+3O), with the identification of 1194 Cl-BPs of unknown structures. This study contributes to a comprehensive understanding of the molecular transformations of DBC induced by various free radicals in different UV-AOP processes, leading to a better understanding of the different fates of DBC in UV-AOP processes. In addition, the identification of DBC as a precursor of by-products will also contribute to the understanding of how to inhibit the generation of by-products.
PubMed: 38941867
DOI: 10.1016/j.watres.2024.121962 -
Journal of Hazardous Materials Jun 2024Nanoplastics (NPs, size <1000 nm) are ubiquitous plastic particles, potentially more abundant than microplastics in the environment; however, studies highlighting their...
Environmental nanoplastics quantification by pyrolysis-gas chromatography-mass spectrometry in the Pearl River, China: First insights into spatiotemporal distributions, compositions, sources and risks.
Nanoplastics (NPs, size <1000 nm) are ubiquitous plastic particles, potentially more abundant than microplastics in the environment; however, studies highlighting their distribution dynamics in freshwater are rare due to analytical limitations. Here, we investigated spatiotemporal levels of nine polymers of NPs in surface water samples (n = 30) from the full stretch of the Pearl River (sites, n = 15) using pyrolysis gas chromatography-mass spectrometry (Py-GC/MS). Six polymers were detected, including polystyrene (PS), polyvinyl chloride (PVC), nylon/polyamide 66 (PA66), polyester (PES), poly(methyl methacrylate) (PMMA) and polyethylene (PE), where three polymers showed high detection frequencies; PS (100 % in winter and summer), followed by PVC (73 % in winter and 87 % in summer) and PA66 (53 % in winter and 67 % in summer). The spatiotemporal distribution revealed the sites related to aquaculture (AQ) and shipping (SHP) showed higher NP levels than those of human settlement (HS) and wastewater treatment plants (WWTPs) (p = 0.004), and relatively high average levels of NPs in the urban sites compared to rural sites (p = 0.04), albeit showed no obvious seasonal differences (p = 0.78). For instance, the average PS levels in the Pearl River were in the following order: AQ 411.55 µg/L > SHP 81.75 µg/L > WWTP 56.66 µg/L > HS 47.75 µg/L in summer and HS 188.1 µg/L > SHP 103.55 µg/L > AQ 74.7 µg/L > WWTP 62.1 µg/L in winter. Source apportionment showed a higher contribution through domestic plastic waste emissions among urban sites, while rural sites showed an elevated contribution via aquaculture, agriculture, and surface run-off to the NP pollution. Risk assessment revealed that NPs at SHP and AQ sites posed a higher integrated risk in terms of pollution load index (PLI) than those at WWTP and HS sites. Regarding polymer hazard index (HI), 80 % of sampling sites in summer and 60 % of sampling sites in winter posed level III polymer risk, with PVC posing the highest risk. This study provides novel insights into the seasonal contamination and polymer risks of NP in the Pearl River, which will help to regulate the production and consumption of plastics in the region. ENVIRONMENTAL IMPLICATIONS: The contamination dynamics of field nanoplastics (NPs) in freshwater resources remain little understood, mainly attributed to analytical constraints. This study aims to highlight the spatiotemporal distribution of NPs in the Pearl River among various land use types, urban-rural comparison, seasonal comparison, their compositional profiles, potential sources, interaction with environmental factors, and ecological and polymer hazard assessments of investigated polymers in the full stretch of the Pearl River from Liuxi Reservoir to the Pearl River Delta (PRD) region. This study, with a comparatively large number of samples and NP polymers, will offer novel insights into the contamination profiles of nano-sized plastic particles in one of the important freshwater riverine systems in China.
PubMed: 38941826
DOI: 10.1016/j.jhazmat.2024.135055