Enrichment Strategies Research Articles

138 An examination of cow behavior with an automated brush system in an intensively managed dairy system

Abstract In recent years, the dairy industry has seen a significant shift in animal welfare practices, with growing emphasis on providing optimal living conditions for indoor-housed animals. Intensive management of dairy cows has become increasingly common, offering a controlled setting that ensures the well-being and productivity of the cow. Enrichment strategies such as automated brushes are proposed as a means for intensively managed dairy cows to improve their physical, mental, and emotional well-being. Automated brushes have been shown to promote natural behaviors such as grooming and scratching, reducing boredom and promoting social interactions among cattle. These behavioral benefits can contribute to an enriched living environment. With this known, the objective of this study was to evaluate the usage of an automated brush system for grooming behaviors by lactating dairy cows housed in a freestall setting. A total of 29 cows (Holstein-Friesian and Jersey) over three trials (21 d each) were analyzed for duration of automated brush use using scan sampling of video recordings (0500h-0900h and 1700h-2100h); post milking) totaling eight hours per day throughout each trial. Animals were housed in a single pen with sand bedding at the Joe Bearden Dairy Research Center and managed in accordance with standard operating procedures of the unit. All data were passively collected without influencing behavioral changes. Video capture was in 4k resolution using a four camera closed-circuit media recording system. Cameras were placed in the corners of the pen, allowing full view of animals interacting with the brush system. Video images were scored using the open-source behavioral analysis software BORIS. Temperature and humidity were recorded daily using wall mounted monitors. Results indicate that cows increased brush use in the evenings (277.7±18.1s vs 221.7± 18.1s; P < 0.01). An analysis of heat index and brush usage was performed for relationships towards assessing thermal effects of automated brush usage. However, heat index was not related to brush usage (R2 = 0.003; P = 0.69), suggesting animal motivation for brush interaction was not affected by temperature or humidity during the trial periods. Lastly, an examination of continued interest in the automated brush system was performed by partitioning each trial into two equal fractions representing the first and second halves. Results provide evidence of animals utilizing the automated brush similarly throughout the trial (P = 0.18), suggesting there was continued interest throughout the evaluated period. In conclusion, this study sheds light on the behavior of lactating dairy cows in response to an automated brush system in a freestall setting. These findings have implications for animal welfare and farm management strategies, suggesting that automated brush systems can be effectively utilized to enhance the well-being of dairy cows, providing them with opportunities for comfortable grooming experiences, regardless of environmental variations.

The role of mir96 in predicting CTC status and prognostic evaluation in gastric cancer patients.

In this study, 20 patients clinically diagnosed with advanced gastric cancer were selected as subjects. Circulating tumor cells (CTCs) in the peripheral blood of gastric cancer patients were detected and counted by collecting peripheral blood samples at Ningbo No. 2 Hospital and using the Cell Rich TM system combined with a negative enrichment strategy. In addition, routine pathological examination and immunohistochemical staining were performed on surgical specimens, including HER2, EBER, E-cadherin, and vimentin indicators. These indicators were correlated and analyzed with CTC counts and routine clinical tests. At the same time, miRNA groups were performed to explore the miRNAs with or without correlation with CTC and to try to construct a predictive model for CTC status. Tumor tissue from patients whose CTC counts did not match the results of the miRNA prediction model was subjected to second-generation gene sequencing to analyze indicators such as tumor heterogeneity and immune microenvironment. The results of the study showed that CTCs were detected in the peripheral blood of 50% (10/20) of gastric cancer patients using the Cell Rich TM system, serum fibrinogen was negatively correlated with CTC counts, whereas TNM staging elements, HER2 receptor and p53 were not correlated with CTC counts. miRNA assays showed that the expression levels of miR218, miR1207, miR96, miR409, miR149, miR148a, miR155, miR370 and miR223 were significantly different between the two groups of CTC≥2 and CTC<2. Among them, miR-96 showed good efficacy as an indicator for predicting CTC status and assisting in determining the prognosis of gastric cancer. In conclusion, the present study demonstrated that the Cell Rich TM system combined with the negative enrichment strategy can effectively detect CTCs in the peripheral blood of gastric cancer patients and that the expression of miR-96 can be used as an effective indicator to predict CTC status and assist in determining the prognosis of gastric cancer. These findings have important implications for the diagnosis and treatment of gastric cancer and provide new clues for the further study of the tumor immune microenvironment and tumor heterogeneity.

Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche

Immunotherapies with antibody–drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.

In situ membrane separation drives nitrous oxide enrichment from nitrate denitrification for energy recovery

Efficiently enriching nitrous oxide (N2O) can provide a foundation for energy recovery during wastewater treatment. In this study, gas-liquid separation membranes were placed in a sequencing batch reactor fed with acidic nitrate-containing wastewater to determine the feasibility of enriching N2O by in situ separation of dissolved gases. Gas separation across the membrane (GSM) had no obvious effect on chemical oxygen demand and nitrate removal, but N2O production efficiency (NPE) increased from 11.0 % to 82.6 %. Denitrifying bacteria with low N2O yield such as Thauera, Diaphorobacter, and Flavobacterium were eliminated, and Propioniclava (46.79 %) and Candidatus_Competibacter (22.53 %) became dominant. Metagenomic sequencing showed that the abundance of the nosZ gene encoding N2O reductase decreased from 17.3 % to 6.1 %. In conclusion, GSM prevented the further reduction of N2O, eliminating denitrifying bacteria carrying the nosZ gene, which the enriched Propioniclava likely lacked. This demonstrates that GSM is an alternative strategy for N2O enrichment through nitrate denitrification.

Mass Spectrometry Strategies for O-Glycoproteomics.

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in O-glycoproteomics, a field that lags behind N-glycoproteomics due to several unique challenges associated with the complexity of O-glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of O-glycoproteins.

Effective enrichment of trace exosomes for the label-free SERS detection via low-cost thermophoretic profiling

Exosome-based liquid biopsies possess great potential in monitoring cancer development However, current exosome detection biosensors require large exosome volumes, showing the weak detection sensitivity. Besides, these methods pay little attention to in situ analysis of exosomes, hence limiting the provision of more accurate clinically-relevant information. Herein, we develop an innovative label-free biosensor combining the low-cost thermophoretic enrichment method with the surface-enhanced Raman spectroscopy (SERS) detection. Based on the thermophoretic enrichment strategy, exosomes and gold nanoparticles can be enriched together into a small area with a scale of 500 μm within 10 min. The Raman signals of various exosomes derived from normal, cancerous cell lines and human serum are dynamically monitored in situ, with the limit of detection of 102–103 particles per microliter, presenting higher sensitivity compared with the similar label-free SERS detection. The spectral data set of different exosomes is applied to train for multivariate classification of cell types and to estimate how the normal exosome data resemble cancer cell exosome. The reliable classification and identification of different exosomes can be realized. The current biosensor is convenient, low-cost and requires small exosome volumes (∼3 μL), and if validated in larger cohorts may contribute to the tumor prediction and diagnosis.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

MRNA-based precision targeting of neoantigens and tumor-associated antigens in malignant brain tumors

BackgroundDespite advancements in the successful use of immunotherapy in treating a variety of solid tumors, applications in treating brain tumors have lagged considerably. This is due, at least in part, to the lack of well-characterized antigens expressed within brain tumors that can mediate tumor rejection; the low mutational burden of these tumors that limits the abundance of targetable neoantigens; and the immunologically “cold” tumor microenvironment that hampers the generation of sustained and productive immunologic responses. The field of mRNA-based therapeutics has experienced a boon following the universal approval of COVID-19 mRNA vaccines. mRNA-based immunotherapeutics have also garnered widespread interest for their potential to revolutionize cancer treatment. In this study, we developed a novel and scalable approach for the production of personalized mRNA-based therapeutics that target multiple tumor rejection antigens in a single therapy for the treatment of refractory brain tumors.MethodsTumor-specific neoantigens and aberrantly overexpressed tumor-associated antigens were identified for glioblastoma and medulloblastoma tumors using our cancer immunogenomics pipeline called Open Reading Frame Antigen Network (O.R.A.N). Personalized tumor antigen-specific mRNA vaccine was developed for each individual tumor model using selective gene capture and enrichment strategy. The immunogenicity and efficacy of the personalized mRNA vaccines was evaluated in combination with anti-PD-1 immune checkpoint blockade therapy or adoptive cellular therapy with ex vivo expanded tumor antigen-specific lymphocytes in highly aggressive murine GBM models.ResultsOur results demonstrate the effectiveness of the antigen-specific mRNA vaccines in eliciting robust anti-tumor immune responses in GBM hosts. Our findings substantiate an increase in tumor-infiltrating lymphocytes characterized by enhanced effector function, both intratumorally and systemically, after antigen-specific mRNA-directed immunotherapy, resulting in a favorable shift in the tumor microenvironment from immunologically cold to hot. Capacity to generate personalized mRNA vaccines targeting human GBM antigens was also demonstrated.ConclusionsWe have established a personalized and customizable mRNA-therapeutic approach that effectively targets a plurality of tumor antigens and demonstrated potent anti-tumor response in preclinical brain tumor models. This platform mRNA technology uniquely addresses the challenge of tumor heterogeneity and low antigen burden, two key deficiencies in targeting the classically immunotherapy-resistant CNS malignancies, and possibly other cold tumor types.

Sequencing of Physically Interacting Cells in Human Kidney Allograft Rejection to Infer Contact-dependent Immune Cell Transcription.

Rejection requires cell-cell contact involving immune cells. Inferring the transcriptional programs of cell-cell interactions from single-cell RNA-sequencing (scRNA-seq) data is challenging as spatial information is lost. We combined a CD45 pos enrichment strategy with Cellular Indexing of Transcriptomes and Epitopes by sequencing based quantification of leukocyte surface proteins to analyze cell-cell interactions in 11 human kidney transplant biopsies encompassing a spectrum of rejection diagnoses. scRNA-seq was performed using the 10X Genomics platform. We applied the sequencing physically interacting cells computational method to deconvolute the transcriptional profiles of heterotypic physically interacting cells. The 11 human allograft biopsies generated 31 203 high-quality single-cell libraries. Clustering was further refined by combining Cellular Indexing of Transcriptomes and Epitopes by sequencing data from 6 different leukocyte-specific surface proteins. Three of 6 doublet clusters were identified as physically interacting cell complexes; macrophages or dendritic cells bound to B cells or plasma cells; natural killer (NK) or T cells bound to macrophages or dendritic cells and NK or T cells bound to endothelial cells. Myeloid-lymphocyte physically interacting cell complexes expressed activated and proinflammatory genes. Lymphocytes physically interacting with endothelial cells were enriched for NK and CD4 T cells. NK cell-endothelial cell contact caused increased expression of endothelial proinflammatory genes CXCL9 and CXCL10 and NK cell proinflammatory genes CCL3 , CCL4 , and GNLY . The transcriptional profiles of physically interacting cells from human kidney transplant biopsies can be inferred from scRNA-seq data using the sequencing physically interacting cells method. This approach complements previous methods that estimate cell-cell physical contact from scRNA-seq data.

Feeding predictability as a cognitive enrichment protects brain function and physiological status in rainbow trout: a multidisciplinary approach to assess fish welfare

Cognitive enrichment is a promising but understudied type of environmental enrichment that aims to stimulate the cognitive abilities of animals by providing them with more opportunities to interact with (namely, to predict events than can occur) and to control their environment. In a previous study, we highlighted that farmed rainbow trout can predict daily feedings after two weeks of conditioning, the highest conditioned response being elicited by the combination of both temporal and signalled predictability. In the present study, we tested the feeding predictability that elicited the highest conditioned response in rainbow trout (both temporal and signalled by bubbles, BUBBLE + TIME treatment) as a cognitive enrichment strategy to improve their welfare. We thus analysed the long-term effects of this feeding predictability condition as compared with an unpredictable feeding condition (RANDOM treatment) on the welfare of rainbow trout, including the markers in the modulation of brain function, through a multidisciplinary approach. To reveal the brain regulatory pathways and networks involved in the long-term effects of feeding predictability, we measured gene markers of cerebral activity and plasticity, neurotransmitter pathways and physiological status of fish (oxidative stress, inflammatory status, cell type and stress status). After almost three months under these predictability conditions of feeding, we found clear evidence of improved welfare in fish from BUBBLE + TIME treatment. Feeding predictability allowed for a food anticipatory activity and resulted in fewer aggressive behaviours, burst of accelerations, and jumps before mealtime. BUBBLE + TIME fish were also less active between meals, which is in line with the observed decreased expression of transcripts related to the dopaminergic system. BUBBLE + TIME fish tented to present fewer eroded dorsal fin and infections to the pathogen Flavobacterium psychrophilum. Decreased expression of most of the studied mRNA involved in oxidative stress and immune responses confirm these tendencies else suggesting a strong role of feeding predictability on fish health status and that RANDOM fish may have undergone chronic stress. Fish emotional reactivity while isolated in a novel-tank as measured by fear behaviour and plasma cortisol levels were similar between the two treatments, as well as fish weight and size. To conclude, signalled combined with temporal predictability of feeding appears to be a promising approach of cognitive enrichment to protect brain function via the physiological status of farmed rainbow trout in the long term.

Refined parallel adaptive Bayesian quadrature for estimating small failure probabilities

Bayesian active learning methods have emerged for structural reliability analysis, showcasing more attractive features compared to existing active learning methods. The parallel adaptive Bayesian quadrature (PABQ) method, as a representative of them, allows to efficiently assessing small failure probabilities but faces the problem of empirically specifying several important parameters. The unreasonable parameter settings could lead to the inaccurate estimates of failure probability or the non-convergence of active learning. This study proposes a refined PABQ (R-PABQ) method by presenting three novel refinements to overcome the drawbacks of PABQ. Firstly, a sequential population enrichment strategy is presented and embedded into the importance ball sampling technique to solve the computer memory problem when involving large sample population. Secondly, an adaptive determination strategy for radius is developed to automatically adjust the sampling region during the active learning procedure. Lastly, an adaptive multi-point selection method is proposed to identify a batch of points to enable parallel computing. The effectiveness of the proposed R-PABQ method is demonstrated by four numerical examples. Results show that the proposed method can estimate small failure probabilities (e.g., 10−7∼10−9) with superior accuracy and efficiency over several existing active learning reliability methods.

Single-Atom Site SERS Chip for Rapid, Ultrasensitive, and Reproducible Direct-Monitoring of RNA Binding.

Ribonucleic acids (RNA) play active roles within cells or viruses by catalyzing biological reactions, controlling gene expression, and communicating responses to cellular signals. Rapid monitoring RNA variation has become extremely important for appropriate clinical decisions and frontier biological research. However, the most widely used method for RNA detection, nucleic acid amplification, is restricted by a mandatory temperature cycling period of ≈1 h required to reach target detection criteria. Herein, a direct detection approach via single-atom site integrated surface-enhanced Raman scattering (SERS) monitoring nucleic acid pairing reaction, can be completed within 3 min and reaches high sensitivity and extreme reproducibility for COVID-19 and two other influenza viruses' detection. The mechanism is that a single-atom site on SERS chip, enabled by positioning a single-atom oxide coordinated with a specific complementary RNA probe on chip nanostructure hotspots, can effectively bind target RNA analytes to enrich them at designed sites so that the binding reaction can be detected through Raman signal variation. This ultrafast, sensitive, and reproducible single-atom site SERS chip approach paves the route for an alternative technique of immediate RNA detection. Moreover, single-atom site SERS is a novel surface enrichment strategy for SERS active sites for other analytes at ultralow concentrations.

Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.

The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Running toward Sustainability: Exploring Off-Peak Destination Resilience through a Mixed-Methods Approach—The Case of Sporting Events

The sustainability of tourism activities faces many challenges. Furthermore, in the context of the Sustainable Development Goals (SDGs) and their overall slow progress, tourism-related goals and targets are underachieved. To address these challenges, this study has recognized sporting events as an opportunity to enhance the socio-economic activities in tourism destinations during off-season periods. More specifically, the aim of this study is to explore the dynamics of off-season destination resilience through small-scale running events, focusing on three key aspects: the strategies employed by organizers, the synergy created within the community, and the sustainable outcomes. Drawing on insights from qualitative interviews with twenty-five running event organizers across Greece, supported by the Delphi method to confirm and validate the results, their perceptions of the economic and socio-cultural dimensions are explored. This study identifies a number of strategies that enrich running events and can contribute to the sustainability of off-peak destinations. Additionally, the concept of synergy is identified and explored, emphasizing the importance of local engagement, participant encouragement, and community collaborations. These findings provide a comprehensive understanding of how these factors can influence the sustainability of off-peak destinations. To further validate and extend these findings, the second part of this study performs a quantitative analysis using PLS-SEM, involving eighty local authorities in Greece. The results highlight the mediating role of small-scale event enrichment strategies on the relationship between synergy and destination sustainability. This multifaceted approach indicates the dynamics of sustainable tourism, by examining the case of small-scale running events in the off-peak season. The impact of this balanced approach on the broader achievement of relevant SDGs is also supported. The implications of this research, in terms of its strategic and stakeholder orientation for tourism professionals and government agencies, are also discussed.

Importance of Elemental Chemical Speciation Studies in Enriched Food: Nutritional Quality, Toxicity, and Economic Improvement

For several reasons, mainly cost and local productivity, the world population does not have access to a balanced diet that contains all the macro and micronutrients necessary to maintain physiological functions for a healthy life. Nutritional education, supplementation, and consuming enriched (or fortified) foods appear as alternatives to supply daily demands and minimize malnutrition. Adding essential elements as salts (e.g., iron, calcium, and zinc) to ready-to-eat processed foods, such as milk, flour, and juices is already adopted in several countries. The choice of the compound to be added, as well as the transport vehicle (foods), must be very well evaluated since the cost, long-term consumption, and bioavailability of the added chemical species are imperative to ensure the nutritional quality of enriched food.1 Another alternative to produce enriched foods is cultivating an enriched medium (Figure 1), adding essential elements to soil or in nutritive solution (hydroponic procedure), irrigating leaves, or immersing seeds.2 In this case, the chemical species used to the enrich food must be absorbed, translocated, and accumulated in the edible part.2 Studies have shown that the iron enrichment of adzuki beans using iron nitrate or iron chloride was unsuccessful since iron inorganic species interact strongly with the antinutrients (tannins or phytates) present in the roots, forming insoluble complexes and preventing their translocation.3 Alternatives found to overcome this obstacle were enrichment by applying iron complexes with EDTA (ethylenediaminetetra-acetic acid)3 or iron nanoparticles, mainly encapsulated.4 The nanoparticle application has been gaining prominence in agriculture, aiming to carry fertilizer, pesticides, and nutrients to stimulate plant growth and increase macro and micronutrient availability and absorption efficiency.5,6 Besides the interaction between essential elements with antinutrients, evaluating the competition between elemental species is important, because synergistic or antagonistic effects can be observed. In both cases, chemical species must interact with other components present in food or cultivation medium, altering its chemical composition when compared to food cultivated in conventional conditions. The antagonistic effect between selenium and mercury was observed in edible mushrooms, while the synergistic effect was observed with lead and selenium.7,8 Finally, it must be evaluated if the enrichment promotes the production of non-bioavailable or toxic species. Regardless of the food enrichment strategy, it is important to highlight the need to identify and quantify the elemental chemical species in the enriched foods by chemical speciation analysis. In the Figure 1 is shown examples of elemental chemical species; they can differ according to their oxidation states, inorganic forms, and organometallic or isotopic composition.9 For chemical speciation studies, initial fractionation steps (e.g., extraction procedures) are carried out; subsequently, chromatographic, or non-chromatographic methods can be used to identify/determine the chemical species. The hyphenation (Figure 1) between separation techniques, mainly chromatography, with high sensitivity detectors, such as inductively coupled plasma mass spectrometry (ICP-MS), is commonly applied.9-11 Nonetheless, extremely creative procedures used in non-chromatographic chemical speciation, working only with chemical reactions and solubility differences, can also be applied to determine chemical species. Non-chromatographic strategies were applied for iron (reaction with hydroxylamine and precipitation with trichloroacetic acid and HCl) and selenium (cloud point extraction) speciation in enriched adzuki sprouts.3 In summary, food enrichment success is closely associated with chemical speciation studies since there are chemical species that will be absorbed in a cultivation medium, as well as the chemical species that are formed during translocation and accumulation, which must be in bioavailable forms, in order to act on the different metabolic systems of the human body, including remedying prevalences. In this scenario, it is imperative to go beyond determining the total concentration of essential elements, since quantifying their species will provide information regarding essentiality and toxicity. Finally, the formation of bioavailable chemical species will add nutritional quality and, consequently, economic benefits that are so essential for countries with specific prevalences to combat in a predominantly agricultural economy.

Individualized and Biomarker-Based Prognosis of Longitudinal Cognitive Decline in Early Symptomatic Alzheimer's Disease.

Although individualized models using demographic, MRI, and biological markers have recently been applied in mild cognitive impairment (MCI), a similar study is lacking for patients with early Alzheimer's disease (AD) with biomarker evidence of abnormal amyloid in the brain. We aimed to develop prognostic models for individualized prediction of cognitive change in early AD. A total of 421 individuals with early AD (MCI or mild dementia due to AD) having biomarker evidence of abnormal amyloid in the brain were included in the current study. The primary cognitive outcome was the slope of change in Alzheimer's Disease Assessment Scale-cognitive subscale-13 (ADAS-Cog-13) over a period of up to 5 years. A model combining demographics, baseline cognition, neurodegenerative markers, and CSF AD biomarkers provided the best predictive performance, achieving an overfitting-corrected R2 of 0.59 (bootstrapping validation). A nomogram was created to enable clinicians or trialists to easily and visually estimate the individualized magnitude of cognitive change in the context of patient characteristics. Simulated clinical trials suggested that the inclusion of our nomogram into the enrichment strategy would lead to a substantial reduction of sample size in a trial of early AD. Our findings may be of great clinical relevance to identify individuals with early AD who are likely to experience fast cognitive deterioration in clinical practice and in clinical trials.

An efficient strategy with a synergistic effect of hydrophilic and electrostatic interactions for simultaneous enrichment of N- and O-glycopeptides.

N- and O-glycosylation modifications of proteins are closely linked to the onset and development of many diseases and have gained widespread attention as potential targets for therapy and diagnosis. However, the low abundance and low ionization efficiency of glycopeptides as well as the high heterogeneity make glycosylation analysis challenging. Here, an enrichment strategy, using Knoevenagel copolymers modified with polydopamine-adenosine (denoted as PDA-ADE@KCP), was firstly proposed for simultaneous enrichment of N- and O-glycopeptides through the synergistic effects of hydrophilic and electrostatic interactions. The adjustable charged surface and hydrophilic properties endow the material with the capability to achieve effective enrichment of intact N- and O-glycopeptides. The experimental results exhibited excellent selectivity (1 : 5000) and sensitivity (0.1 fmol μL-1) of the prepared material for N-glycopeptides from standard protein digest samples. Moreover, it was further applied to simultaneous capturing of N- and O-glycopeptides from mouse liver protein digests. Compared to the commercially available zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) material, the number of glycoproteins corresponding to all N- and O-glycopeptides enriched with PDA-ADE@KCP was much more than that with ZIC-HILIC. Furthermore, PDA-ADE@KCP captured more O-glycopeptides than ZIC-HILIC, revealing its superior performance in O-glycopeptide enrichment. All these results indicated that the strategy holds immense potential in characterizing N- and O-intact glycopeptides in the field of proteomics.

Chemical reagents for the enrichment of modified peptides in MS-based identification.

Chemical reagents with special groups as enrichable handles have empowered the ability to label and enrich modified peptides. Here is an overview of different chemical reagents with affinity tags to isolate labeled peptides and the latest developments of enrichment strategies. Biotin is the most used affinity tag due to its high interaction with avidin. To decrease the unfavorable influence of biotin for its poor efficiency in ionization and fragmentation in downstream MS analysis, cleavable moieties were installed between the reactive groups and biotin to release labeled peptides from the biotin. To minimize the steric hindrance of biotin, a two-step method was developed, for which alkyne- or azide-tagged linkers were firstly used to label peptides and then biotin was installed through click chemistry. Recently, new linkers using a small phosphonic acid as the affinity tag for IMAC or TiO2 enrichment have been developed and successfully used to isolate chemically labeled peptides in XL-MS. A stable P-C instead of P-O bond was introduced to linkers to differentiate labeled and endogenous phosphopeptides. Furthermore, a membrane-permeable phosphonate-containing reagent was reported, which facilitated the study of living systems. Taking a cue from classic chemical reactions, stable metal-complex intermediates, including cobalt and palladium complexes, have been developed as peptide purification systems. Advanced enrichment strategies have also been proposed, such as the two-stage IMAC enrichment method and biotin-based two-step reaction strategy, allowing the reduction of unwanted peptides and improvements for the analysis of specific labeled peptides. Finally, future trends in the area are briefly discussed.

Single-Cell Histone Modification Profiling with Cell Enrichment Using sortChIC.

Histone post-translational modifications (PTMs) influence the overall structure of the chromatin and gene expression. Over the course of cell differentiation, the distribution of histone modifications is remodeled, resulting in cell type-specific patterns. In the past, their study was limited to abundant cell types that could be purified in necessary numbers. However, studying these cell type-specific dynamic changes in heterogeneous in vivo settings requires sensitive single-cell methods. Current advances in single-cell sequencing methods remove these limitations, allowing the study of nonpurifiable cell types. One complicating factor is that some of the most biologically interesting cell types, including stem and progenitor cells that undergo differentiation, only make up a small fraction of cells in a tissue. This makes whole-tissue analysis rather inefficient. In this chapter, we present a sort-assisted single-cell Chromatin ImmunoCleavage sequencing technique (sortChIC) to map histone PTMs in single cells. This technique combines the mapping of histone PTM location in combination with surface staining-based enrichment, to allow the integration of established strategies for rare cell type enrichment. In general terms, this will enable researchers to quantify local and global chromatin changes in dynamic complex biological systems and can provide additional information on their contribution to lineage and cell-type specification in physiological conditions and disease.