Complex Mixtures Research Articles

Generating pooled quality control samples of volatile organic compounds

Untargeted analysis of volatile organic compounds (VOCs) from exhaled breath and culture headspace are influenced by several confounding factors not represented in reference standards. In this study, we propose a method of generating pooled quality control (QC) samples for untargeted VOC studies using a split-recollection workflow with thermal desorption tubes. Sample tubes were desorbed and split from each sample and recollected onto a single tube, generating a pooled QC sample. This QC sample was then repeatedly desorbed and recollected with a sequentially lower split ratio allowing injection of multiple QC samples. We found pooled QC samples to be representative of complex mixtures using principal component analysis and may be useful in future longitudinal, multi-centre, and validation studies to assess data quality and adjust for batch effects.

Molecular Aspects of the Interactions between Selected Benzodiazepines and Common Adulterants/Diluents: Forensic Application of Theoretical Chemistry Methods

Benzodiazepines are frequently encountered in crime scenes, often mixed with adulterants and diluents, complicating their analysis. This study investigates the interactions between two benzodiazepines, lorazepam (LOR) and alprazolam (ALP), with common adulterants/diluents (paracetamol, caffeine, glucose, and lactose) using infrared (IR) spectroscopy and quantum chemical methods. The crystallographic structures of LOR and ALP were optimized using several functionals (B3LYP, B3LYP-D3BJ, B3PW91, CAM-B3LYP, M05-2X, and M06-2X) combined with the 6-311++G(d,p) basis set. M05-2X was the most accurate when comparing experimental and theoretical bond lengths and angles. Vibrational and 13C NMR spectra were calculated to validate the functional’s applicability. The differences between LOR’s experimental and theoretical IR spectra were attributed to intramolecular interactions between LOR monomers, examined through density functional theory (DFT) optimization and quantum theory of atoms in molecules (QTAIM) analysis. Molecular dynamics simulations modeled benzodiazepine–adulterant/diluent systems, predicting the most stable structures, which were further analyzed using QTAIM. The strongest interactions and their effects on IR spectra were identified. Comparisons between experimental and theoretical spectra confirmed spectral changes due to interactions. This study demonstrates the potential of quantum chemical methods in analyzing complex mixtures, elucidating spectral changes, and assessing the structural stability of benzodiazepines in forensic samples.

Prenatal exposure to PM2.5 led to impaired respiratory function in adult mice

BackgroundPM2.5 is a complex mixture, with water-soluble inorganic ions (WSII), mainly NH4+, SO42−, and NO3−, constituting major components. Early-life PM2.5 exposure has been shown to induce adverse health consequence but it is difficult to determine whether such an effect occurs prenatally (preconception, gestational) or postnatally in human studies. MethodsFour groups of C57BL/6 J mice were assigned to four exposure conditions: PM2.5 NO3−, PM2.5 SO42−, PM2.5 NH4+ and clean air, and exposure started at 4 weeks old. At 8 weeks old, mice bred within group. The exposure continued during gestation. After delivery, both the maternal and F1 mice (offspring) were kept in clean air without exposure to PM2.5. Respiratory function and pulmonary pathology were assessed in offspring mice at 8 weeks of age. In parallel, placenta tissue was collected for transcriptome profiling and mechanistic investigation. ResultsF1 mice in PM2.5 NH4+, SO42- and NO3− groups had 32.2 % (p=6.0e-10), 30.3 % (p=3.8e-10) and 16.9 % (p=5.7e-8) lower peak expiratory flow (PEF) than the clean air group. Importantly, the exposure-induced lung function decline was greater in male than female offspring. Moreover, exposure to PM2.5 WSII before conception and during gestation was linked to increased airway wall thickness and elevated pulmonary neutrophil and macrophage counts in the offspring mice. At the molecular level, the exposure significantly disrupted gene expression in the placenta, affecting crucial functional pathways related to sex hormone response and inflammation. ConclusionsPM2.5 WSII exposure during preconception and gestational period alone without post-natal exposure substantially impacted offspring’s respiratory function as measured at adolescent age. Our results support the paradigm of fetal origin of environmentally associated chronic lung disease and highlight sex differences in susceptibility to air pollution exposure.

Plasma concentrations of multiple oxysterols and risk of colorectal adenomas.

Oxysterols are metabolites of cholesterol that regulate homeostasis of cholesterol, fatty acids, and glucose. These metabolites are generated throughout the body, either enzymatically or from oxidative stress, and are detectable in peripheral circulation. We previously reported that circulating 27-hydroxycholesterol (27-OHC), an endogenous selective estrogen receptor modulator, may be a risk factor for colorectal adenomas. Here, in addition to 27-OHC, we report on four other circulating oxysterols: 25-hydroxycholesterol (25-OHC), 24(S)-hydroxycholesterol (24(S)-OHC), 7ɑ-hydroxycholesterol (7ɑ-OHC), and 4β-hydroxycholesterol (4β-OHC). Oxysterol concentrations were measured using liquid chromatography-mass spectrometry from fasting plasma collected at baseline from 1,246 participants of the Vitamin D/Calcium Polyp Prevention Study, a multicenter adenoma chemoprevention trial. To evaluate multiple oxysterols simultaneously, we used both log-linear regression and Bayesian kernel machine regression (BKMR) models developed for analyses of complex mixtures adjusted for potential confounding factors. Higher circulating 7ɑ-OHC was associated with higher adenoma risk (BKMR-based multivariable-adjusted risk ratios, RR, for the 75th vs. 25th percentile, 1.22; 95% credible interval, CI, 1.04-1.42). In contrast, higher circulating 4β-OHC was associated with lower risk of these polyps (RR, 0.84; 95% CI, 0.71-0.99). The positive association with advanced adenoma risk that we previously reported for circulating 27-OHC persisted when controlling for other oxysterols (RR, 1.26; 95% CI, 0.98-1.62), including among those with advanced adenomas at baseline (RR, 1.75; 95% CI, 1.01-3.06).

Occupational Exposure of On-Shift Ottawa Firefighters to Flame Retardants and Polycyclic Aromatic Hydrocarbons

Firefighters can be exposed to complex mixtures of airborne substances, including hazardous substances released during structural fires. This study employed silicone wristbands (SWBs) as passive samplers to investigate potential exposure to polycyclic aromatic hydrocarbons (PAHs) and flame retardants (FRs). SWBs were deployed at different areas of four fire stations, in four truck cabins, and at an office control location; they were also donned outside the jackets of 18 firefighters who responded to fire calls. Overall, office areas had significantly lower PAHs than fire station areas. Vehicle bays and truck cabins had significantly higher concentrations of low molecular weight (LMW) PAHs than sleeping and living room areas. For organophosphate ester flame retardants (OPFRs), tri-n-butyl phosphate (TnBP) and tris(1-chloro-2-propyl) phosphate (TCPP) were detected in all the samples; 2-ethylhexyl diphenyl phosphate (EHDPP) was more frequently detected in the fire station areas. Triphenyl phosphate (TPP) concentrations were highest in the truck cabin and office areas, and tris(1,3-dichloro-2-propyl)phosphate (TDCPP) was highest in truck cabins. Thirteen of 16 PAHs and nine of 36 OPFRs were detected in all the SWBs worn by firefighters, and tris (2-butoxyethyl) phosphate (TBEP) was the predominant OPFR. Levels of LMW PAHs were significantly lower when firefighters did not enter the fire. LMW PAHs, HMW (high molecular weight) PAHs, and EHDPP were significantly elevated when heavy smoke was reported. This work highlights the potential for occupational exposure to PAHs and flame retardants in some fire station areas; moreover, factors that may influence exposure during fire suppression. Whilst firefighters’ occupational exposure to PAHs is likely related to fire suppression and exposure to contaminated gear and trucks, exposure to OPFRs may be more related to their presence in truck interiors and electronics.

A Review on Single-Cell analysis of Macrophages in Cancer

Cancer is characterized by uncontrolled cell growth and spread into surrounding tissue. According to the North American Association of Central Cancer Registries (NAACCR) and the National Center for Health Statistics, there were 1,918,030 cancer cases and a total of 609,360 deaths estimated in the US in 2022. Although the direct cause of cancer is not fully understood, it is recognized that the immune system plays a major role in the development and progression of tumors. Investigating tumor-associated macrophages (TAMs) is crucial for understanding the immunosuppressive role that they may play in the tumor microenvironment, which makes them a target for immunotherapies to combat cancer. However, angiogenesis, tumor initiation and invasion promoting signals prevent gaining deeper insight into the intricate molecular mechanisms involved in TAMs and the tumor microenvironment. While researchers in the past have used population samples to analyze cells, recent efforts have incorporated single-cell technologies to study the complex mixture of cells in a tumor, monitor the effects of immunotherapies and more. With the revolutionary advent of the first single-cell RNA sequencing (scRNA-seq), researchers have since been able to gain insight into single-cell data with unprecedented accuracy and develop new immunotherapies through different single-cell technologies, such as polymethylsiloxane (PDMS) and Multiplexed Ion Beam Imaging (MIBI) multiplexing. In this review, we underline the benefits of adopting single-cell technologies to identify distinct cell types in complex environments (i.e., tumors) and highlight the potential application of these technologies along with various multiplexing techniques to immunotherapies.

Exploitation of Pore Structure for Increased CO2 Selectivity in Type 3 Porous Liquids.

CO2 capture requires materials with high adsorption selectivity and an industrial ease of implementation. To address these needs, a new class of porous materials was recently developed that combines the fluidity of solvents with the porosity of solids. Type 3 porous liquids (PLs) composed of solvents and metal-organic frameworks (MOFs) offer a promising alternative to current liquid carbon capture methods due to the inherent tunability of the nanoporous MOFs. However, the effects of MOF structural features and solvent properties on CO2-MOF interactions within PLs are not well understood. Herein experimental and computational data of CO2 gas adsorption isotherms were used to elucidate both solvent and pore structure influences on ZIF-based PLs. The roles of the pore structure including solvent size exclusion, structural environment, and MOF porosity on PL CO2 uptake were examined. A comparison of the pore structure and pore aperture was performed using ZIF-8, ZIF-L, and amorphous-ZIF-8. Adsorption experiments here have verified our previously proposed solvent size design principle for ZIF-based PLs (1.8× ZIF pore aperture). Furthermore, the CO2 adsorption isotherms of the ZIF-based PLs indicated that judicious selection of the pore environment allows for an increase in CO2 selectivity greater than expected from the individual PL components or their combination. This nonlinear increase in the CO2 selectivity is an emergent behavior resulting from the complex mixture of components specific to the ZIF-L + 2'-hydroxyacetophenone-based PL.

507 Effects of simple and complex polycrop mixtures on forage yield and quality, beef cow performance, and methane emissions

Abstract Polycrop mixtures are of increasing interest to western Canadian beef producers as alternative forages for grazing as they may promote plant biodiversity and nutritive value. This 2-yr study evaluated the effects of swath grazing either simple or complex polycrop mixtures on forage yield and quality, beef cow performance, and methane emissions. Forage treatments included 1) CDC Haymaker oat (Avena sativa; Control); 2) CDC Haymaker oat, Goliath brassica (Brassica napus), hairy vetch (Vicia villosa), CDC Jasper pea (Pisum sativum; Simple); and 3) CDC Haymaker oat, Goliath brassica, hairy vetch, CDC Jasper pea, CDC Maverick barley (Hordeum vulgare), Red proso millet (Panicum miliaceum), Teff grass (Eragrostis tef) and Choice chicory (Cichorium intybus; Complex). Dry pregnant beef cows [n = 90; body weight (BW) = 607.2 ± 32.3 kg; body condition score (BCS) = 2.9 ± 0.06] blocked by BW were randomly allocated to 1 of 9 paddocks (10 cows/paddock). Paddocks were randomly assigned 1 of 3 replicated (n = 3) systems. Cow BCS was measured at start (SOT) and end (EOT) of trial. Cow BW was recorded on 2 consecutive days at the SOT and EOT, and every 21 d during trial. Cows grazed for an average of 52 d from October to December (Control 51 ± 9.07 d; Simple 54 ± 9.20 d; Complex 53 ± 12.41 d). Data were analyzed as a completely randomized block design using the mixed model of SAS (9.4). Year and paddock were included as a random effect. Forage dry matter yield (DMY) was similar (P = 0.44) among Control (4,369 kg/ha), Simple (4,053 kg/ha), and Complex (4,245 kg/ha) treatments. Start of trial forage crude protein (CP) and total digestible nutrients (TDN) did not differ (P = 0.18 and P = 0.14) among treatments (Control 13.3% CP and 74.4% TDN; Simple 14.76% CP and 71.51% TDN; Complex 14.95% CP and 74.46% TDN). End of trial forage CP and TDN did not differ (P = 0.57 and P = 0.20) among treatments (Control 13.9% CP and 69.5% TDN; Simple 13.9% CP and 69.9% TDN; Complex 13.2% CP and 72.4% TDN). Cow BCS and average daily gain did not differ among forage treatments (P = 0.60 and P = 0.45, respectively). Cow dry matter intake (DMI) was similar (P = 0.19) among treatments (Control 11.13 kg/d; Simple 9.74 kg/d; Complex 9.96 kg/d). Methane emissions were greatest (P = 0.04) for cows grazing Simple (28.7 CH4 g/kg DMI) and Complex treatments (28.2 CH4 g/kg DMI) compared with monoculture oat treatment (22.6 CH4 g/kg DMI). Study results suggest that simple or complex polycrop mixtures may be used as forage alternatives to a monoculture oat grazing system as they produce adequate biomass and had nutritive value suitable for gestating beef cows.

Analysis of the sweetening effects of lactone and ketone compounds in a cheddar cheese matrix using sensory analysis and molecular simulation

This study evaluated the effects of lactone and ketone compounds on sweetness perception in a Cheddar cheese matrix. According to the static sensory evaluation, a ternary mixture of γ-decanolactone/δ-decanolactone/acetoin at middle and high concentrations exhibited a significant sweetening effect closely related to olfactory input. Dynamic sensory evaluation showed that a γ-decanolactone/δ-decanolactone/acetoin ternary mixture at middle and high concentrations could enhance the sweetness perception of a Cheddar cheese matrix at different sucrose concentrations during oral processing. Molecular docking showed that the ternary mixture of γ-decanolactone/δ-decanolactone/acetoin with sucrose-T1R2 increased the number of hydrogen bond binding sites and increased the number of hydrophobic interaction sites. Molecular dynamics suggested that the molecular conformation of sucrose and T1R2 was more compact and stable in the ternary mixture. Additionally, in the presence of the ternary system, the binding free energy of the re-docking of sucrose and T1R2 was −7.28 kcal/mol, indicating that the docking structure is stable. This stability is maintained by the hydrogen bonding and hydrophobic interaction between the T1R2-sucrose complex and aroma mixtures. These findings provide a theoretical basis for using cross-modal perception to sweeten Cheddar cheese to cater to consumers’ pursuit of sweetness.

Effects of microplastic interaction with persistent organic pollutants on the activity of the aryl hydrocarbon and estrogen receptors

Environmental microplastics (MPs) are complex mixtures of plastic polymers and sorbed chemical pollutants with high degrees of heterogeneity, particularly in terms of particle size, morphology and degree of weathering. Currently, limitations exist in sampling sufficient amounts of environmental particles for laboratory studies to assess toxicity endpoints with statistical rigor and to examine chemical pollutant interactions. This study seeks to bridge this gap by investigating environmental plastic particle mimetics and pollutant-polymer interactions by mixing polymer particles with persistent organic pollutants (POPs) at set concentrations over time. Solutions containing combinations of polymers including polystyrene (PS), polypropylene (PP), polyethylene terephthalate (PET), and polyamide (PA) and POPs including 2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD), bisphenol A (BPA), and atrazine, were stirred for up to 19 weeks and monitored using assays to test for aryl hydrocarbon (AhR) and estrogen receptor (ER) activity which are cell signaling pathways impacted by environmental pollutants. TCDD induced AhR activity decreased over time in the presence of PS in a surface area dependent manner. BPA and atrazine also exhibited AhR antagonist activity in the presence of TCDD. The addition of BPA slowed the loss of activity but atrazine did not, suggesting that polymer chemistry impacts interactions with POPs. We also observed potential differences in TCDD sorption with different plastic polymers and that higher concentrations of PS particles may inhibit BPA-induced estrogen receptor activation. These results emphasize the need for additional understanding of how POPs and polymer chemistry impact their interaction and toxicity.

Enlightening the toxinological dark matter of spider venom enzymes

Spiders produce highly adapted venoms featuring a complex mixture of biomolecules used mainly for hunting and defense. The most prominent components are peptidic neurotoxins, a major focus of research and drug development, whereas venom enzymes have been largely neglected. Nevertheless, investigation of venom enzymes not only reveals insights into their biological functions, but also provides templates for future industrial applications. Here we compared spider venom enzymes validated at protein level contained in the VenomZone database and from all publicly available proteo-transcriptomic spider venom datasets. We assigned reported enzymes to cellular processes and known venom functions, including toxicity, prey pre-digestion, venom preservation, venom component activation, and spreading factors. Our study unveiled extensive discrepancy between public databases and publications with regard to enzyme coverage, which impedes the development of novel spider venom enzyme-based applications. Uncovering the previously unrecognized abundance and diversity of venom enzymes will open new avenues for spider venom biodiscovery.

Modeling the thermodynamic properties of cyclic alcohols with the SAFT-γ Mie approach: application to cyclohexanol and menthol systems

Cyclic alcohols are commonly found in natural and synthetic products and are involved in many biological processes. An ability to model their thermodynamic properties is of interest in food, flavouring, and pharmaceutical manufacturing, particularly for mixtures for which available experimental data are limited. Good examples are mixtures including menthol, a naturally occurring cyclic alcohol widely used in the food and pharmaceutical industries, well-known for its cooling-sensation properties and its role in the discovery of the TRPM8 receptor. Here, we extend the SAFT-γ Mie group-contribution method to model cyclic alcohols, by introducing a new cCHOH group (c for cyclic) composed of two identical Mie segments. Three association sites (two electronic sites of type e and one hydrogen site of type H) are also included to mediate hydrogen bonding. New parameters that characterise the group interactions (one new like interaction and 26 new unlike interactions) are developed and are employed to determine the thermophysical properties (vapour pressure, saturation density, vaporisation enthalpy, and second-order thermodynamic derivative properties) of pure cyclic alcohols, and mixture properties (vapour–liquid equilibria, liquid–liquid equilibria, solid–liquid equilibria, density, and excess enthalpy) of cyclic alcohols in several solvents. The quality of the model is evaluated by comparing predictive calculations with experimental data of 76 systems: six pure fluids and 70 binary mixtures, selected from a large range of solvent families: cyclic, linear, and branched alkanes, 1-alcohols, 2-alcohols, 2-ketones, esters, aromatic compounds, water, and carbon dioxide. Very good overall agreement is found, including for the prediction of solid–liquid solubility, which confirms the transferability of the new group parameters. Together with previously developed parameters, these open the way for the prediction of the thermodynamic properties of further complex mixtures.

Experimental characterization and prediction of Escherichia coli host cell proteome retention during preparative chromatography.

Purification of recombinantly produced biopharmaceuticals involves removal of host cell material, such as host cell proteins (HCPs). For lysates of the common expression host Escherichia coli (E. coli) over 1500 unique proteins can be identified. Currently, understanding the behavior of individual HCPs for purification operations, such as preparative chromatography, is limited. Therefore, we aim to elucidate the elution behavior of individual HCPs from E. coli strain BLR(DE3) during chromatography. Understanding this complex mixture and knowing the chromatographic behavior of each individual HCP improves the ability for rational purification process design. Specifically, linear gradient experiments were performed using ion exchange (IEX) and hydrophobic interaction chromatography, coupled with mass spectrometry-based proteomics to map the retention of individual HCPs. We combined knowledge of protein location, function, and interaction available in literature to identify trends in elution behavior. Additionally, quantitative structure-property relationship models were trained relating the protein 3D structure to elution behavior during IEX. For the complete data set a model with a cross-validated R2 of 0.55 was constructed, that could be improved to a R2 of 0.70 by considering only monomeric proteins. Ultimately this study is a significant step toward greater process understanding.

Revisiting Dissolved Organic Matter Analysis Using High-Resolution Trapped Ion Mobility and FT-ICR Mass Spectrometry.

The molecular level characterization of complex mixtures remains an analytical challenge. We have shown that the integration of complementary, high-resolution, gas-phase separations allows for chemical formula level isomeric content description. In the current work, we revisited the current challenges associated with the analysis of dissolved organic matter using high-resolution trapped ion mobility separation (TIMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). In particular, we evaluated the separation capabilities provided by TIMS-MS compared to MS alone, the use of ICR complementary data acquisition (DAQ) systems and transient processing strategies, ICR cell geometries (e.g., Infinity cell vs harmonized cell), and magnetic field strengths (7 T vs 9.4 T vs 21 T) for the case of a Harney River DOM sample. Results showed that the external high-performance DAQ enables direct representation of mass spectra in absorption mode FT (aFT), doubling the MS resolution compared to the default magnitude mode FT (mFT). Changes between half- vs full-apodization result in greater MS signal/noise vs superior MS resolving power (RP); in the case of DOM analysis, a 45% increase in assigned formulas is observed when employing the DAQ half (Kaiser-type)-apodization window and aFT when compared to the default instrument mFT. Results showed the advantages of reprocessing 2D-TIMS-FT-ICR MS data with higher RP and magnetic field chemical formulas generated list acquired (e.g., 21 T led to a 24% increase in isomers reported) or the implementation of alternative strategies.

Conductive Framework Materials for Chemiresistive Detection and Differentiation of Toxic Gases.

ConspectusSensing complex gaseous mixtures and identifying their composition and concentration have the potential to achieve unprecedented improvements in environmental monitoring, medical diagnostics, industrial safety, and the food/agriculture industry. Electronically transduced chemical sensors capable of recognizing and differentiating specific target gases and transducing these chemical stimuli in a portable electronic device offer an opportunity for impact by bridging the utility of chemical information with global wireless connectivity. Among electronically transduced chemical sensors, chemiresistors stand out as particularly promising due to combined features of low-power requirements, room temperature operation, non-line-of-sight detection, high portability, and exceptional modularity. Relying on changes in resistance of a functional material triggered by variations in the surrounding chemical environment, these devices have achieved part-per-billion sensitivities of analytes by employing conductive polymers, graphene, carbon nanotubes (CNTs), metal oxides, metal nanoparticles, metal dichalcogenides, or MXenes as sensing materials. Despite these tremendous developments, the need for stable, selective, and sensitive chemiresistors demands continued innovation in material design in order to operate in complex mixtures with interferents as well as variations in humidity and temperature.To fill existing gaps in sensing capabilities, conductive metal-organic frameworks (MOFs) and covalent organic frameworks (COFs) have recently emerged as a promising class of materials for chemiresistive sensing. In contrast to previously reported chemiresistors, these materials offer at least three unique features for gas sensing applications: (i) bottom-up synthesis from molecularly precise precursors that allows for strategic control of material-analyte interactions, (ii) intrinsic conductivity that simultaneously facilitates charge transport and signal transduction under low power requirements, and (iii) high surface area that enables the accessibility of abundant active sites and decontamination of gas streams by coordinating to and, sometimes, detoxifying harmful analytes. Through an emphasis on molecular engineering of structure-property relationships in conductive MOFs and COFs, combined with strategic innovations in device integration strategies and device form factor (i.e., the physical dimensions and design of device components), our group has paved the way to demonstrating the multifunctional utility of these materials in the chemiresistive detection of gases and vapors. Backed by spectroscopic assessment of material-analyte interactions, we illustrated how molecular-level features lead to device performance in detection, filtration, and detoxification of gaseous analytes. By merging the bottom-up synthesis of these materials with device integration, we show the versatility and scalability of using these materials in low-power electronic sensing devices. Taken together, our achievements, combined with the progress spearheaded on this class of materials by other researchers, establish conductive MOFs and COFs as promising multifunctional materials for applications in electronically transduced, portable, low-power sensing devices.

Hf Isotopes and Detrital Zircon Geochronology of the Silasia Formation, Midyan Terrane, Northwestern Arabian Shield: An Investigation of the Provenance History

ABSTRACTU–Pb ages and Hf isotopic data from detrital zircon of the Silasia Formation in the Midyan Terrane record evidence for the provenance and tectonic evolution of the northern Arabian Shield. Given that the youngest acknowledged age of these detritus sediments is 735 ± 13 Ma, it is likely that the Silasia Formation was deposited during the closure of the Mozambique Ocean. The U–Pb ages define a major Mesoproterozoic peak, with two minor peaks of Neoproterozoic and Archean age. Combined with zircon Hf isotopic compositions, the sedimentary detritus of the Silasia Formation was mainly derived from source rocks formed during the Grenville Orogeny during the assembly of Rodinia, with a minor contribution from Archean and Paleoproterozoic crustal material, in addition to a limited arc‐basement supply related to the early Mozambique Ocean. The youngest Concordia age of 735 ± 13 Ma with highly variable εHf(t) values (11 to −35) indicates a complex mixture of sources from juvenile to extremely ancient. The Concordia ages at 1113 ± 11 and 1046 ± 10 Ma have positive hafnium isotope signatures (up to +10.45) that are consistent with juvenile source rocks formed during the Grenville Orogeny. Several detrital zircons with ages of 2622 ± 22 Ma and 2690 ± 7 Ma are similar to those reported in Yemen, whereas 1818 ± 19 Ma, 2071 ± 8 Ma and 2001 ± 19 Ma Paleoproterozoic ages are similar to dated outcrops in the Khida terrane in the eastern Arabian Shield.

Temporal analysis of water chemistry and smallmouth bass (Micropterus dolomieu) health at two sites with divergent land use in the Susquehanna River watershed, Pennsylvania, USA

Monitoring wild fish health and exposure effects in impacted rivers and streams with differing land use has become a valuable research tool. Smallmouth bass (Micropterus dolomieu) are a sensitive, indicator species that exhibit signs of immunosuppression and endocrine disruption in response to water quality changes and contaminant exposure. To determine the impact of agriculture and development on smallmouth bass health, two sites (a developed/agriculture site and a forested site) in the Susquehanna River watershed, Pennsylvania were selected where bass and water chemistry were sampled from 2015 to 2019. Smallmouth bass were sampled for histopathology to assess parasite and macrophage aggregate density in the liver and spleen, condition factor (Ktl), hepatic gene transcript abundance, hepatosomatic index (HSI), and a health assessment index (HAI). Land use at the developed/agriculture site included greater pesticide application rates and phytoestrogen crop cover and more detections and higher concentrations of pesticides, wastewater-associated contaminants, hormones, phytoestrogens, and mycotoxins than at the forested site. Additionally, at the developed/agriculture site, indicators of molecular changes, including oxidative stress, immune/inflammation, and lipid metabolism-related hepatic gene transcripts, were associated with more contaminants and land use variables. At both sites, there were multiple associations of contaminants with liver and/or spleen macrophage aggregate density, indicating that changes at the molecular level seemed to be a better indicator of exposures unique to each site. The findings illustrate the importance of timing for land management practices, the complex mixtures aquatic animals are exposed to, and the temporal changes in contaminant concentration. Agricultural practices that affect hepatic gene transcripts associated with immune function and disease resistance were demonstrated which could negatively affect smallmouth bass populations.

Parallel reaction monitoring targeted mass spectrometry as a fast and sensitive alternative to antibody-based protein detection

The reliable, accurate and quantitative targeted detection of proteins is a key technology in molecular and cell biology and molecular diagnostics. The current golden standard for targeted protein detection in complex mixtures such as complete cell lysates or body fluids is immunoblotting, a technology that was developed in the late 1970s and has not undergone major changes since. Although widespread, this methodology suffers from several disadvantages, such as the inability to detect low-abundant proteins or specific posttranslational modifications, the requirement for highly specific antibodies, the lack of quantitative power and the often-tedious practical procedures. Mass spectrometry (MS) based targeted protein detection is an alternative technology that could circumvent these caveats. Here, we compare immunoblotting with targeted protein mass spectrometry using a parallel reaction monitoring (PRM) regime on the Orbitrap mass spectrometer. We show that PRM based MS has superior sensitivity and quantitative accuracy over immunoblotting. The limit of detection for proteolytic peptides of a purified target protein was found to be in the mid- to low-attomole range and approximately one order of magnitude higher when embedded in a complex biological matrix. The incorporation of synthetic heavy isotope labeled (AQUA) peptides as internal calibrants into the PRM workflow allows for even higher accuracy for both the relative and absolute quantitation of tryptic target peptides. In conclusion, PRM is a versatile and sensitive technology, which can overcome the shortcomings of immunoblotting. We argue that PRM based MS could become the method of choice for the targeted detection of proteins in complex cellular matrices or body fluids and may eventually replace standard methods such as Western blotting and ELISA in biomedical research and in the clinic.

Rapid Production of Native and Mirror-Image Tumor Necrosis Factor-α Enabled by Automated Flow Peptide Synthesis Technology.

Tumor necrosis factor-α (TNF-α) plays a central role in immune response regulation. Because elevated TNF-α production is correlated with a range of diseases, inhibiting the interaction of this protein with its native receptors has been thoroughly explored as a therapeutic avenue. Despite advancements in the development of TNF-α inhibitors, concerns remain regarding immunogenicity and loss of activity in vivo. To facilitate the discovery of stable and less immunogenic therapeutic modalities, we applied a single-shot automated fast-flow peptide synthesis (AFPS) strategy to produce full-length TNF-α, resulting in a complex reaction mixture. Leveraging the ability of AFPS to generate long peptides with high purity, we combined this technology with native chemical ligation (NCL). An NCL reaction using two fragments readily produced by AFPS afforded synthetic L- and D-TNF-α in milligram quantities (up to 5.5 mg, ∼28% yield). Following the oxidative folding of synthetic TNF-α using established conditions, higher molecular weight species were generated. Through high-throughput screening of refolding conditions, functional synthetic L- and mirror-image D-TNF-α were obtained, exhibiting characteristics analogous to those of the recombinant TNF-α. Overall, this approach can serve as a general protocol for accessing proteins that are intractable by modern protein synthesis methods, therefore, streamlining the development of novel therapeutics.

Periodic membrane fractionation of freshwater organic matter reveals various reactivity patterns during chlorine/chloramine disinfection

Although drinking water disinfection has significantly reduced mortality from waterborne diseases, the formation of potentially harmful disinfection by-products (DBPs) remains a concern today. These DBPs emerge from the reaction of dissolved organic matter (DOM) with chlor(am)ine during disinfection. Identifying these DBP precursors from heterogeneous and complex DOM mixtures is challenging and to address this, a novel membrane fractionation protocol was developed to isolate and identify the organic DBP precursors from fresh water sources. A tight ultrafiltration and nanofiltration membrane were carefully selected to partition DOM into three molecular weight (MW) fractions: high (>20 kDa), medium (0.3–20 kDa) and low (<0.3 kDa) as defined by high performance-size exclusion chromatography- total organic carbon analysis. A mathematical tool was developed to optimize the fractionation protocol. Therefore, ultrafiltration was executed before nanofiltration and the concentration factor was maximized without inducing fouling to obtain the purest fractions. The mathematical tool was also set in place to predict the necessary diafiltration factor for each individual fractionation experiment based on the initial organic matter composition of the surface water allowing the fractionation protocol to be effective at all times. The protocol was applied to surface water samples collected six times across three seasons yielding a fraction enriched in high MW compounds (up to 50 %), a fraction having more than 80 % of medium MW compounds and a fraction only containing low MW compounds. Although the medium MW fraction showed the highest reactivity for the majority of the investigated DBPs, the low MW fraction showed high reactivity for iodinated trihalomethanes during chlorination and for haloacetonitriles during chloramination. The high MW fraction had the lowest reactivity towards DBPs, which can have important implications for a drinking water treatment since this fraction is generally the most effectively removed by e.g. coagulation, while the more important fractions for DBP formation such as the medium and low MW compounds remain in the water until the disinfection step.