Plant Cellulose as a Substrate for 3D Neural Stem Cell Culture.

Neural stem cell (NSC)-based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury, and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D), in vitro culture of NSCs. Plant cellulose scaffolds were shown to support the attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance the differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct the differentiation of NSCs.

The Influence of Protein Secretomes of Enterococcus durans on ex vivo Human Gut Microbiome.

The gut microbiome plays a critical role to all animals and humans health. Methods based on ex vivo cultures are time and cost-effective solutions for rapid evaluation of probiotic effects on microbiomes. In this study, we assessed whether the protein secretome from the potential probiotic Enterococcus durans LAB18S grown on fructoligosaccharides (FOS) and galactoligosaccharides (GOS) had specific effects on ex vivo cultured intestinal microbiome obtained from a healthy individual. Metaproteomics was used to evaluate changes in microbial communities of the human intestinal microbiome. Hierarchical clustering analysis revealed 654 differentially abundant proteins from the metaproteome samples, showing that gut microbial protein expression varied on the presence of different E. durans secretomes. Increased amount of Bacteroidetes phylum was observed in treatments with secretomes from E. durans cultures on FOS, GOS and albumin, resulting in a decrease of the Firmicutes to Bacteroidetes (F/B) ratio. The most functionally abundant bacterial taxa were Roseburia, Bacteroides, Alistipes and Faecalibacterium. The results suggest that the secretome of E. durans may have favorable effects on the intestinal microbial composition, stimulating growth and different protein expression of beneficial bacteria. These findings suggest that proteins secreted by E. durans growing on FOS and GOS have different effects on the modulation of gut microbiota functional activities during cultivation.

Early postnatal defects in neurogenesis in the 3xTg mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia. The hippocampus, which is one of the sites where neural stem cells reside and new neurons are born, exhibits the most significant neuronal loss in AD. A decline in adult neurogenesis has been described in several animal models of AD. However, the age at which this defect first appears remains unknown. To determine at which stage, from birth to adulthood, the neurogenic deficits are found in AD, we used the triple transgenic mouse model of AD (3xTg). We show that defects in neurogenesis are present as early as postnatal stages, well before the onset of any neuropathology or behavioral deficits. We also show that 3xTg mice have significantly fewer neural stem/progenitor cells, with reduced proliferation and decreased numbers of newborn neurons at postnatal stages, consistent with reduced volumes of hippocampal structures. To determine whether there are early changes in the molecular signatures of neural stem/progenitor cells, we perform bulk RNA-seq on cells sorted directly from the hippocampus. We show significant changes in the gene expression profiles at one month of age, including genes of the Notch and Wnt pathways. These findings reveal impairments in neurogenesis very early in the 3xTg AD model, which provides new opportunities for early diagnosis and therapeutic interventions to prevent neurodegeneration in AD.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

Comprehensive Assessment of Functional Effects of Commonly Used Sugar Substitute Sweeteners on Ex Vivo Human Gut Microbiome.

The composition and function of the human gut microbiome are often associated with health and disease status. Sugar substitute sweeteners are widely used food additives, although many studies using animal models have linked sweetener consumption to gut microbial changes and health issues. Whether sugar substitute sweeteners directly change the human gut microbiome functionality remains largely unknown. In this study, we systematically investigated the responses of five human gut microbiomes to 21 common sugar substitute sweeteners, using an approach combining high-throughput in vitro microbiome culturing and metaproteomic analyses to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The noncaloric artificial sweetener (NAS) cluster was composed of NASs and two sugar alcohols with shorter carbon backbones (4 or 5 carbon atoms), and the carbohydrate (CHO) cluster was composed of the remaining sugar alcohols. The metaproteomic functional responses of the CHO cluster were clustered with those of the prebiotics fructooligosaccharides and kestose. The sugar substitute sweeteners in the CHO cluster showed the ability to modulate the metabolism of Clostridia. This study provides a comprehensive evaluation of the direct effects of commonly used sugar substitute sweeteners on the functions of the human gut microbiome using a functional metaproteomic approach, improving our understanding of the roles of sugar substitute sweeteners on microbiome-associated human health and disease issues. IMPORTANCE The human gut microbiome is closely related to human health. Sugar substitute sweeteners as commonly used food additives are increasingly consumed and have potential impacts on microbiome functionality. Although many studies have evaluated the effects of a few sweeteners on gut microbiomes using animal models, the direct effect of sugar substitute sweeteners on the human gut microbiome remains largely unknown. Our results revealed that the sweetener-induced metaproteomic responses of individual microbiomes had two major patterns, which were associated with the chemical properties of the sweeteners. This study provided a comprehensive evaluation of the effects of commonly used sugar substitute sweeteners on the human gut microbiome.

Women's and Nurses' Perceptions of Visitor Restrictions After Childbirth During the COVID-19 Pandemic.

OBJECTIVE: To examine the perceptions of labor and delivery (L&D) nurses and childbearing women in the postpartum period regarding a restricted visitor policy during the COVID-19 pandemic. DESIGN: Descriptive mixed-methods survey and open-ended questions. SETTING/LOCAL PROBLEM: One hospital in the southwestern United States. There is limited evidence regarding recently imposed visitor restrictions related to COVID-19. PARTICIPANTS: Individuals who were pregnant and self-identified as women who gave birth during October 2020 through March 2021 (n = 674) and L&D nurses (n = 47). INTERVENTION/MEASUREMENTS: Participants who had given birth with visitor restrictions completed an online survey, and L&D nurses completed a paper survey. RESULTS: Childbearing women had positive and negative views; they valued a more intimate familial bonding and recovery without visitors and appreciated decreased pressure to accommodate family/friends. They were also disappointed with sibling restrictions and were sad and frustrated with visitor limitations, especially in special circumstances (e.g., NICU admission or extended stays). Nurses expressed that visitor restrictions allowed more time for higher-quality nursing care/patient teaching and decreased distractions in emergencies, leading to safer care. Women and nurses reported that visitor restrictions allowed women more rest and relaxation as well as less worry and strain from juggling family and friends who wanted to visit, but they also identified that there was decreased family support when it was needed. CONCLUSION: Women's responses were mixed, with some preferring support from many visitors, while others appreciated the intimate focus of just their partner. Most nurses preferred fewer visitors but could empathize with women.

Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics.

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

Examining the Effects of an Anti-Salmonella Bacteriophage Preparation, BAFASAL®, on Ex-Vivo Human Gut Microbiome Composition and Function Using a Multi-Omics Approach.

Salmonella infections (salmonellosis) pose serious health risks to humans, usually via food-chain contamination. This foodborne pathogen causes major food losses and human illnesses, with significant economic impacts. Overuse of antibiotics in the food industry has led to multidrug-resistant strains of bacteria, and governments are now restricting their use, leading the food industry to search for alternatives to secure food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics, as they are natural components of the ecosystem. However, when specifically used in the industry, they can also make their way into humans through our food chain or exposure, as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented to animal feeds. To our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in-vitro assay called RapidAIM to investigate the effect of a bacteriophage mixture, BAFASAL®, used in poultry farming on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex-vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment.

Exploring the Microbiome-Wide Lysine Acetylation, Succinylation, and Propionylation in Human Gut Microbiota.

Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples. A new bioinformatic workflow consisting of an unrestricted database search confidently identified >60,000 acetylated, and ∼20,000 propionylated and succinylated gut microbial peptides. The characterization of these identified modification-specific metaproteomes, i.e., meta-PTMomes, demonstrates that lysine acylations are differentially distributed in microbial species with different metabolic capabilities. This study provides an analytical framework for the study of lysine acylations in the microbiome, which enables functional microbiome studies at the post-translational level.

Studying the Temporal Dynamics of the Gut Microbiota Using Metabolic Stable Isotope Labeling and Metaproteomics.

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here, we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15N hydrolysate from Ralstonia eutropha, we identified 12 326 nonredundant unlabeled peptides, of which 8256 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse microbiome. MetaProfiler and the bioinformatic pipeline are available at https://github.com/northomics/MetaProfiler.git.

Widespread protein lysine acetylation in gut microbiome and its alterations in patients with Crohn's disease.

Lysine acetylation (Kac), an abundant post-translational modification (PTM) in prokaryotes, regulates various microbial metabolic pathways. However, no studies have examined protein Kac at the microbiome level, and it remains unknown whether Kac level is altered in patient microbiomes. Herein, we use a peptide immuno-affinity enrichment strategy coupled with mass spectrometry to characterize protein Kac in the microbiome, which successfully identifies 35,200 Kac peptides from microbial or human proteins in gut microbiome samples. We demonstrate that Kac is widely distributed in gut microbial metabolic pathways, including anaerobic fermentation to generate short-chain fatty acids. Applying to the analyses of microbiomes of patients with Crohn's disease identifies 52 host and 136 microbial protein Kac sites that are differentially abundant in disease versus controls. This microbiome-wide acetylomic approach aids in advancing functional microbiome research.

pepFunk: a tool for peptide-centric functional analysis of metaproteomic human gut microbiome studies.

MOTIVATION: Enzymatic digestion of proteins before mass spectrometry analysis is a key process in metaproteomic workflows. Canonical metaproteomic data processing pipelines typically involve matching spectra produced by the mass spectrometer to a theoretical spectra database, followed by matching the identified peptides back to parent-proteins. However, the nature of enzymatic digestion produces peptides that can be found in multiple proteins due to conservation or chance, presenting difficulties with protein and functional assignment. RESULTS: To combat this challenge, we developed pepFunk, a peptide-centric metaproteomic workflow focused on the analysis of human gut microbiome samples. Our workflow includes a curated peptide database annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG) terms and a gene set variation analysis-inspired pathway enrichment adapted for peptide-level data. Analysis using our peptide-centric workflow is fast and highly correlated to a protein-centric analysis, and can identify more enriched KEGG pathways than analysis using protein-level data. Our workflow is open source and available as a web application or source code to be run locally. AVAILABILITY AND IMPLEMENTATION: pepFunk is available online as a web application at https://shiny.imetalab.ca/pepFunk/ with open-source code available from https://github.com/northomics/pepFunk. CONTACT: dfigeys@uottawa.ca. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparative proteomic analysis reveals metabolic variability of probiotic Enterococcus durans during aerobic and anaerobic cultivation.

The variation in the bioavailability of oxygen constitutes the environmental conditions found by bacteria in their passage through the host gastro-intestinal tract. Given the importance of oxygen in the defense mechanism of bacteria, it is important to understand how bacteria respond to this stress at a metabolic level. The probiotic strain Enterococcus durans LAB18S was cultivated under aerobic and anaerobic conditions using prebiotic oligosaccharides as carbon source. The whole cell proteome and secretome were analyzed through label-free quantitative proteomics approach. The results showed that the LAB18S isolate when grown with fructo-oligosacchrides (FOS) showed a higher number of differentially expressed proteins compared to samples with galacto-oligosaccharides (GOS) or glucose. Clinically important enzymes for the treatment of cancer, L-asparaginase and arginine deiminase, were overexpressed when the isolate was cultured in FOS. In addition, the absence of oxygen induced the strain to produce proteins related to cell multiplication, cell wall integrity and resistance, and H2O2 detoxification. This study showed that E. durans LAB18S growing on FOS was stimulated to produce clinically important biomolecules, including proteins that have been investigated as potential antineoplastic agents. Significance: The probiotic strain E. durans LAB18S produce clinically relevant enzymes for the treatment of cancer when cultivated in symbiosis with fructo-oligosacchrides (FOS). In addition, proteins associated with cellular multiplication, cell wall integrity and resistance, and H2O2 detoxification were induced under anaerobic growth. These characteristics could be relevant to support maintenance of intestinal health.

Metaproteomics Reveals Growth Phase-Dependent Responses of an In Vitro Gut Microbiota to Metformin.

Metaproteomics has been used in combination with in vitro gut microbiota models to study drug-microbiome interactions. However, it remains unexplored whether the metaproteomics profile of in vitro gut microbiota responds differently to a same stimulus added at different growth phases. In this study, we cultured a human gut microbiota in 96-deep well plates using a previously validated model. Metformin was added during the lag, log, and stationary phases. Microbiome samples, collected at different time points, were analyzed by optical density and function by metaproteomic. The in vitro gut microbiota growth curves, taxonomy, and functional responses were different depending whether metformin was added during the lag, log, or stationary phases. The addition of drugs at the log phase may lead to the greatest decline of bacterial growth. Metaproteomic analysis suggests that the strength of the metformin effect on the gut microbiome functional profile may be ranked as lag phase > log phase > stationary phase. Metformin added at the lag phase may result in a significantly reduced level of the Clostridiales order and an increased level of the Bacteroides genus, which is different from stimulations during the rest of the growth phases. Metformin may also result in alterations of several pathways, including energy production and conversion, lipid transport and metabolism, translation, ribosomal structure, and biogenesis. Our results indicate that the timing for drug stimulation should be considered when studying drug-microbiome interactions in vitro.

Differential Lysis Approach Enables Selective Extraction of Taxon-Specific Proteins for Gut Metaproteomics.

Changes in microbiome composition and function have been linked to human health and diseases. Metaproteomics provides invaluable functional information on the state of a microbiome. However, lower-abundance bacteria in complex microbiomes are difficult to observe by metaproteomics. In this study, stepwise differential lysis protocols were developed for human stool microbiomes to separate different microbial species and to increase the depth of metaproteomic measurements. We achieved differential lysis of Gram-positive (G+) and Gram-negative (G-) bacteria, selective enrichment of specific bacteria, and functional enrichment by our stepwise differential lysis protocols. Therefore, differential lysis can serve as a fractionation method to reduce sample complexity and selectively extract proteins from specific taxa for deep metaproteomic studies.

Chemoenzymatic Method for Glycoproteomic N-Glycan Type Quantitation.

Glycosylation is one of the most important post-translational modifications in biological systems. Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-mannose/hybrid, biantennary, and triantennary with/without core fucose, were quantified. As a proof of principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine the N-glycan type composition of the immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method to analyze the glycan type profile of proteins from the breast cancer cell line MCF7, and we quantitatively revealed the N-glycan type microheterogeneity at the glycopeptide and glycoprotein level. The novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type glycosylation on each site provides a new avenue to investigate the function of glycoproteins in broad areas, such as cancer biomarker research, pharmaceuticals characterization, and antiglycan vaccine development.

An in vitro model maintaining taxon-specific functional activities of the gut microbiome.

In vitro gut microbiome models could provide timely and cost-efficient solutions to study microbiome responses to drugs. For this purpose, in vitro models that maintain the functional and compositional profiles of in vivo gut microbiomes would be extremely valuable. Here, we present a 96-deep well plate-based culturing model (MiPro) that maintains the functional and compositional profiles of individual gut microbiomes, as assessed by metaproteomics, while allowing a four-fold increase in viable bacteria counts. Comparison of taxon-specific functions between pre- and post-culture microbiomes shows a Pearson's correlation coefficient r of 0.83 ± 0.03. In addition, we show a high degree of correlation between gut microbiome responses to metformin in the MiPro model and those in mice fed a high-fat diet. We propose MiPro as an in vitro gut microbiome model for scalable investigation of drug-microbiome interactions such as during high-throughput drug screening.

Therapeutic Targeting of Casein Kinase 1δ/ε in an Alzheimer's Disease Mouse Model.

Sleep disturbances and memory impairment are common symptoms of Alzheimer's disease (AD). Given that the circadian clock regulates sleep, hippocampal function, and neurodegeneration, it represents a therapeutic target against AD. Casein kinase 1δ/ε (CK1δ/ε) are clock regulators and overexpressed in AD brains, making them viable targets to improve sleep and cognition. In this study, we evaluated the therapeutic potential of a small molecule CK1δ/ε inhibitor (PF-670462) in a triple transgenic mouse model of AD (3xTg-AD). Mass spectrometry-based proteomic analyses revealed that PF-670462 administration in 3xTg-AD mice reversed hippocampal proteomic alterations in several AD-related and clock-regulated pathways, including synaptic plasticity and amyloid precursor protein processing. Furthermore, PF-670462 administration rescued working memory deficits and normalized behavioral circadian rhythm disturbances in 3xTg-AD mice. Our study provides in vivo proof of concept for CK1δ/ε inhibition against AD-associated hippocampal proteomic changes, memory impairment, and circadian disturbances.

Associations Between Soluble LDLR and Lipoproteins in a White Cohort and the Effect of PCSK9 Loss-of-Function.

Context: Elevated circulating cholesterol-rich low-density lipoprotein (LDL) particles increase coronary artery disease risk. Cell-surface hepatic LDL receptors (LDLRs) clear 70% of these particles from circulation. The ectodomain of LDLR is shed into circulation, preventing it from removing LDL particles. The role that LDLR ectodomain shedding plays as a regulatory mechanism is unknown. Objective: We describe LDLR shedding via the relationships between circulating soluble LDLRs (sLDLRs) and serum lipoproteins, serum proprotein convertase subtilin/kexin type 9 (PCSK9; a negative regulator of LDLR), and clinical parameters in a white Canadian population. Design: Population-based, cross-sectional study. Settings: Clinical Research Center, The Ottawa Hospital, and Faculty of Medicine, University of Ottawa. Participants: Two hundred seventy-three white Canadians. Intervention: None. Main Outcome Measures: sLDLR measured by ELISA; serum lipids and PCSK9, PCSK9 genotypes, and clinical parameters from previous analyses. Results: sLDLRs correlated strongly with triglycerides (TG; r = 0.624, P < 0.0001) and moderately with LDL cholesterol (r = 0.384, P < 0.0001), and high-density lipoprotein cholesterol (r = -0.307, P = 0.0003). Only TG correlations were unaffected by PCSK9 variations. sLDLR levels were significantly elevated in those with TG >50th or LDL cholesterol >75th percentiles. Conclusions: Serum sLDLR levels correlate with several lipoprotein parameters, especially TG, and the presence of PCSK9 loss-of-function variants alters sLDLR levels and correlations, except for TG. Ectodomain LDLR shedding has a role in LDL metabolism, distinct from PCSK9, with interplay between these two pathways that regulate cell-surface LDLRs. Findings suggest alteration of LDLR shedding could emerge as a target to treat dyslipidemia.

Long-term performance of bicarbonate-form anion exchange: removal of dissolved organic matter and bromide from the St. Johns River, FL, USA.

The goal of this research was to evaluate the long-term performance of magnetic ion exchange (MIEX) treatment using bicarbonate as the mobile counter ion (i.e., MIEX-HCO(3)) and sodium bicarbonate for regeneration. This work is important because there are many unknowns concerning the affinity and regeneration efficiency of bicarbonate-form anion exchange, whereas chloride-form anion exchange (i.e., MIEX-Cl resin) is well-studied. Raw water samples were collected approximately two times per month for one year from a single location on the St. Johns River (SJR), FL, USA. The SJR is characterized by high concentrations of dissolved organic carbon (DOC; 12-26 mg C/L) and bromide (550-1100 µg/L), and is being considered as an alternative drinking water supply. Jar tests were conducted using MIEX-HCO(3) resin, and MIEX-Cl resin was used as a baseline for comparison. The same batch of MIEX-HCO(3) and MIEX-Cl resin was used for the entire study, which was accomplished by regenerating the resins after each jar test in concentrated solutions of sodium bicarbonate and sodium chloride, respectively, and resulted in 21 regeneration cycles. Maximum removal efficiency was achieved with fresh MIEX-HCO(3) resin and virgin MIEX-Cl resin. Both forms of fresh/virgin MIEX resin also had the same affinity sequence with sulfate ≈ UV-absorbing substance > DOC > bromide. The removal efficiency of both forms of MIEX resin decreased as the number of regeneration cycles increased, with MIEX-HCO(3) resin showing 7-18% lower removals than MIEX-Cl resin after 21 regeneration cycles. The affinity sequence of regenerated MIEX-HCO(3) and MIEX-Cl resins differed from fresh resin with UV-absorbing substances > DOC > sulfate > bromide. Scanning electron microscopy and simulated MIEX-HCO(3) treatment under rapidly changing water quality were also used to improve the understanding of bicarbonate-form anion exchange. The major contribution of this research is a systematic study of the extended use of bicarbonate-form anion exchange resin in the context of affinity, regeneration efficiency, and changing water quality.