Depletion Step Research Articles

Development of a Fit-For-Purpose Multi-Marker Panel for Early Diagnosis of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) suffers from a lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for the discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating the analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, the throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from the early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In the discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In the verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including a short gradient (<10min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.

Effects of irradiation on the insolubility of sodium fast reactor (U,Pu)O2 mixed oxide with a very high amount of plutonium

Sodium fast nuclear reactors might operate with MOX fuels containing up to 45% of plutonium, nevertheless, the reprocessability of spent nuclear fuel is sparsely investigated in the literature for plutonium contents higher than 30%. The dissolution step is a critical stage for MOX fuels since plutonium oxide exhibits a much slower dissolution kinetics as compared to uranium oxide. In particular, many authors suggested a significant change in the dissolution behavior of mixed oxides for plutonium contents between 30% and 50%. In this paper, the dissolution tests of unirradiated and irradiated CAPRA pellets with an initial plutonium content of 45% is described. Up to our knowledge, this is the first dissolution test reported in the literature with such high plutonium content in irradiated nuclear fuel. The fresh unirradiated pellets appears almost insoluble due to a very slow dissolution kinetics. Although the insolubility remains high after irradiation, a beneficial effect of irradiation on the dissolution behavior is observed with a depletion factor of five. The dissolution rates observed are very consistent with previous studies reported in the literature. The results show that complementary steps such as fuel pre-treatment or depletion steps should be considered in order to recycle spent nuclear fuel highly enriched in plutonium.

P14 Optimization of real-time sequencing for rapid detection of bacterial species and antimicrobial resistance in blood and urine infections

Abstract Objectives To optimize the process to obtain bacterial DNA directly from human urine and blood samples to produce high-quality genomic data using long-read Nanopore sequencer. Methods A total of 22 blood cultures and 24 urine samples positive for Escherichia coli or Klebsiella pneumoniae and corresponding isolates were collected from the microbiology diagnostics laboratory. DNA isolation optimization was performed testing three different sample volumes and seven DNA extraction kits. DNA concentration was quantified with Qubit fluorometer. Nanopore sequencing was performed on a MinION Mk1C device in R9.4.1 flow cells using the Rapid Barcoding Kit. Bacterial species and antimicrobial resistance (AMR) genes were determined with EPI2ME ARMA pipeline. Genomes were assembled with Flye, polished with Medaka and remaining human contigs removed with Krakentools. Bacterial isolates from the same samples were also sequenced using Illumina technology. Illumina and Nanopore assemblies were quality checked with Quast, Kraken was used to identify bacterial species, MLST for multilocus sequence typing and ABRicate with ResFinder database to detect AMR genes. Results A total of 72 DNA extractions from 24 urine and 22 blood samples with different kits were performed to find the optimal extraction protocol for each sample type. DNA from urine samples was isolated from 5 mL, while 1 mL was best for blood cultures. We found that the host depletion step prior to DNA extraction using MolYsis basic 5 Kit (VH-Bio) was essential. DNA from urine samples was extracted with GenElute Bacterial Genomic DNA Kit (Merck) and QIAamp Biostic bacteremia Kit (Qiagen) was best to allow successful bacterial genome sequencing for blood samples. Bacterial isolates from 11 urine and 1 blood sample (out of 54 sequenced) were successfully sequenced. In all 12 cases, the highest percentage of bacterial reads matched with the causative organism found during routine microbiology diagnostics (E. coli). A variety of clinically relevant AMR genes were detected, such as dfrA and bla CTX-M genes, which matched the phenotypic antimicrobial susceptibility testing results. Comparison of genomic assemblies obtained through Nanopore sequencing of 10 urine samples and Illumina sequencing of the corresponding clinical isolates showed a high degree of matching, validating Nanopore sequencing as an alternative option to support diagnosis and treatment choice. Conclusions We have successfully optimized the protocols to extract and sequence pathogenic bacterial DNA directly from blood and urine samples. Short-read sequencing confirmed results acquired by Nanopore sequencer, endorsing long-read sequencing in clinical settings to support timely diagnosis and treatment.

Abstract 2020: Nascent proteome analysis of drug response in precision-cut tumor slices

Abstract The complex and dynamic microenvironment of tumors influences their development, progression, and response to therapy. In addition to cancer cells, solid tumors consist of a tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. A wide variety of secreted proteins maintain the heterotypic interactions between the cell types in the TME. However, it is not well understood how the TME responds to treatment at the proteome level. Here, we developed a unique nascent proteomic approach for precision-cut tumor slices to address this issue. Precision-cut tumor slices (PCTS) are a technology in which tumor tissues are cut to a defined thickness of 150-300 µm and cultured ex vivo for a certain time. PCTS maintain both the three-dimensional architecture and tumor heterogeneity and preserve their TME with respect to different cell types and the extracellular matrix. Our approach for PCTS nascent proteome analysis combines pulsed-SILAC (stable isotope labeling with amino acids in cell culture) with click chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon drug treatment. PCTS were generated from patient-derived xenografts and primary human ovarian tumors. After a depletion step, the PCTS were cultured in AHA-SILAC medium and treated with cisplatin. PCTS and culture media containing secreted proteins were harvested separately. Newly synthesized proteins were enriched via click chemistry and analyzed using mass spectrometry. A maximum labelling efficiency of &amp;gt;60% was achieved. Human PCTS showed a higher labelling efficiency than mouse xenografts. The PCTS of different tumors showed varying labeling efficiencies, indicating patient heterogeneity. Nascent proteome analysis enables the investigation of drug resistance and response in different patients. Cisplatin treatment resulted in the downregulation of &amp;gt;200 proteins in responsive tumors. A PCTS resistant to cisplatin did not show this response. Moreover, the corresponding patient had a worse clinical outcome than patients whose tumors were sensitive ex vivo. GSEA revealed the involvement of components such as cadherin binding and DNA translation. Protein-protein interactions can be predicted via STRING analysis. Tumor response or resistance was validated using viability assays and immunohistochemical staining for biomarkers of DNA damage and cell death. In conclusion, we established an ex vivo nascent proteome analysis method to study drug response within the complex TME, which can predict the tumor in vivo drug response. By combining the PCTS culture system with pulsed SILAC-AHA treatment, this approach allows the tracking of compositional and dynamic changes within the proteome and monitoring of the direct proteome response on a rapid timescale. It can be used to study cellular communication, predict therapeutic outcomes, and identify new therapeutic targets. Citation Format: Julia Thiel, Lina-Marie Wagner, Karim Aljakouch, Julia Schüler, Bernd Winkler, Kathrin Böpple, Thomas E. Mürdter, Georg Sauer, German Ott, Walter E. Aulitzky, Matthias Schwab, Jeroen Krijgsveld, Meng Dong. Nascent proteome analysis of drug response in precision-cut tumor slices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2020.

A simple, time- and cost-effective, high-throughput depletion strategy for deep plasma proteomics.

We introduce a cost-effective, robust high-throughput-compatible plasma depletion method enabling in-depth profiling of plasma that detects >1300 proteins per run with a throughput of 60 samples per day. The method has been fully validated by processing >3000 samples with no apparent batch effect at a cost for the depletion step of ~$2.5 per sample.

An antibody-free LC-MS/MS method for the quantification of sex hormone binding globulin in human serum and plasma.

Sex hormone binding globulin (SHBG) is a hormone binding protein which plays an important role in regulating the transport and availability of biologically active androgens and estradiol to target cells and used to calculate free testosterone concentrations. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, featuring an albumin removal step followed by a tryptic digestion. After a reduction step with dithiothreitol and alkylation with iodoacetamide three signature peptides were used for the quantification of SHBG. The method enables the quantification of serum and plasma SHBG over the clinically relevant range of 200-20,000ng/mL and was validated according to the mostrecent guidelines. The LC-MS/MS method correlates wellwith the Abbott Alinity immunoassay (R2>0.95), but theLC-MS/MS results are on average 16-17% lower than the immunoassay results, which is consistent for all three signature peptides. The LC-MS/MS method which includes an albumin depletion step allows quantification of SHBG in serum and plasma without an immunocapture step at clinically relevant SHBG levels, thus contributing to better lab-to-lab consistency of results.

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method.

Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5′–3′ backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.

Novel isoelectric target depletion (ITaD) protocol reduces the need for specific reagents for immunogenicity testing.

Background: The development of immunogenicity assays for clinical drug candidates targeting soluble proteins is challenging when the soluble target might produce either false-positive or false-negative signals in bridging anti-drug antibody screening assays. A generic soluble target removal protocol that uses a pH-dependent depletion was evaluated. Results: An anti-drug antibody bridging assay with a pH-dependent soluble target depletion step was successfully developed. Endogenous target levels of ∼600nM could be depleted below 8pM. The assay was highly drug tolerant and met regulatory requirements. Conclusion: A reagent-independent target depletion protocol can be used for immunogenicity testing in the presence of a soluble target. The generic protocol circumvents common depletion or masking protocols.

REX: An analytical tool for reactor operating envelope expansion through fuel-clad thermo-mechanics

REX, an analytical reactor design tool for the identification and expansion of reactor operating envelopes, is introduced and detailed. Written in MATLAB, REX takes core geometries and axially discretized detector and depletion output from the Serpent neutron transport code and performs full-core, pin-specific thermal-hydraulic and thermo-mechanical calculations. REX identifies the operating envelopes of advanced, solid-fuel nuclear reactors on the basis of their fuel-cladding, fuel-gas, and coolant-cladding interfacial thermo-mechanics. It iteratively pushes coolant inlet temperatures and modifies fuel pin geometries to induce mechanical failure in their original cladding materials at the fullest extent of their fuel cycle lengths and permissible coolant inlet temperatures. It then attempts to expand their operating envelopes by determining the mechanical responses of alternative cladding materials under the same geometric conditions. The result of the REX calculation sequence is a set of 5-D variables describing the temperatures, pressures, geometries, and mechanics of the core as functions of assembly, fuel pin, axial zone, depletion step, and coolant inlet temperature for each candidate cladding material with the limiting fuel pin geometry for the original material.

Diagnosis of Viral Diseases Using Deep Sequencing and Metagenomics Analyses.

Viruses are ubiquitous in nature and exist in a variety of habitats. The advancement in sequencing technologies has revolutionized the understanding of viral biodiversity associated with plant diseases. Deep sequencing combined with metagenomics is a powerful approach that has proven to be revolutionary in the last decade and involves the direct analysis of viral genomes present in a diseased tissue sample. This protocol describes the details of RNA extraction and purification from wild rice plant and their yield, RNA purity, and integrity assessment. As a final step, bioinformatics data analysis including demultiplexing, quality control, de novo transcriptome assembly, taxonomic allocation and read mapping following Illumina HiSeq small and total RNA sequencing are described. Furthermore, the total RNAs extraction protocol and an additional ribosomal rRNAs depletion step which are significantly important for viral genomes construction are provided.

Parallel evaluation of cell‑based phage display panning strategies: Optimized selection and depletion steps result in AML blast‑binding consensus antibodies.

Phage display technology (PD) is a powerful technique for the generation of tumor-targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell-based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell-based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast-binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor-targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML-binding phages during panning. Furthermore, suggesting a common design-related mechanism using a ‘single-pot’ PD library, such as the well-known Tomlinson single-chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast-binding phage particles were isolated and soluble scFv-Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv-Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Grain boundary segregation induced precipitation in a non equiatomic nanocrystalline CoCuFeMnNi compositionally complex alloy

Compositionally complex alloys (CCAs) in a nanocrystalline state often involve complex and poorly understood phase transformations which can consequently result in grain growth even at low temperatures. A detailed study of the microstructure and phase stability in CCAs is challenging due to the presence of multiple principal components. In view of these challenges the objective of the present study is to establish a systematic understanding of the phase evolution in a face centered cubic non equiatomic nanocrystalline CCA (CoCuFeMnNi). To accomplish this objective, we employed in-situ transmission electron microscope heating in combination with automated crystal orientation mapping (ACOM) and energy filtered transmission electron microscopy (EFTEM) to elucidate the sequence of phase decomposition of the high-pressure torsion (HPT) processed CoCuFeMnNi. Our analysis reveals a complex succession of grain boundary segregation and depletion steps leading to the formation of a FeCo-rich secondary phase. Our results show that prior to the formation of the secondary phase, Cu, Ni and Co segregate and Fe and Mn deplete at the grain boundaries. After the FeCo precipitation is triggered, Mn segregates to the grain boundaries along with Ni and Cu, whereas Fe and Co are depleted. The FeCo precipitates have a B2 crystal structure and typically exhibit a Kurdjumov-Sachs (K-S) and/or Nishyama-Wasserman (N-W) orientation relationships with adjacent fcc grains. Ex-situ heat treated CoCuFeMnNi analyzed by atom probe tomography (APT) revealed a highly heterogeneous segregation of the different elements to different grain boundaries. The FeCo-rich precipitates contain trace amounts of Ni, whereas Cu is rejected leading to the formation of a separate Cu rich phase. This complex segregation phenomenon is assisted by the high fraction of grain boundaries and triple junctions in the nanocrystalline material, which are critical for the phase evolution in this alloy, which is not frequently observed in the corresponding coarse-grained material.

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography – mass spectrometry

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Affinity Proteomics Assays for Cardiovascular and Atherosclerotic Disease Biomarkers.

Systematic exploration of the dynamic human plasma proteome enables the discovery of novel protein biomarkers. Using state-of-the-art technologies holds the promise to facilitate a better diagnosis and risk prediction of diseases. Cardiovascular disease (CVD) pathophysiology is characterized for unbalancing of processes such as vascular inflammation, endothelial dysfunction, or lipid profiles among others. Such processes have a direct impact on the dynamic and complex composition of blood and hence the plasma proteome. Therefore, the study of the plasma proteome comprises an excellent exploratory source of biomarker research particularly for CVD. We describe the protocol for performing the discovery of protein biomarker candidates using the suspension bead array technology. The process does not require depletion steps to remove abundant proteins and consumes only a few microliters of sample from the body fluid of interest. The approach is scalable to measure many analytes as well as large numbers of samples. Moreover, we describe a bead-assisted antibody-labeling process that helps to develop quantitative assays for validation purposes and facilitate the translation of the identified candidates into clinical studies.

Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs

BackgroundThere are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 μg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation.ResultsProbe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3′ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85).ConclusionIn this study, our results showed that 1 μg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.

Study on β-Casein Depleted Casein Micelles: Micellar Stability, Enzymatic Cross-Linking, and Suitability as Nanocarriers.

β-Casein is an amphiphilic protein and thus considered as multilaterally bound in casein micelles. Its polar molecule part, in particular the phosphoserine residues, can interact electrostatically with colloidal calcium phosphate (CCP) to form nanoclusters and its nonpolar molecule part enhances micellar stability by forming hydrophobic bonds to other caseins. Because cooling weakens hydrophobic interactions, a substantial portion of β-casein can be irreversibly removed from the casein micelle by repeated depletion steps, including cooling and subsequent ultracentrifugation. Although this effect of cooling on the micellar β-casein concentration has been well known for decades, the influence of depletion on the main characteristics of casein micelles has been less investigated yet. Therefore, we aimed to analyze the consequences of β-casein depletion on the stability as well as the functionality of casein micelles to evaluate the suitability of depleted compared to native casein micelles as nanocarriers. Up to 43.2% of the total β-casein was irreversibly sequestered from native casein micelles by repeated cooling and ultracentrifugation steps. Depletion showed no effect on size distribution as well as polydispersity and particle concentration of micelle suspensions as measured via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), respectively. Furthermore, the stability of the micelles against ethanol or the chelating agent ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was not influenced by β-casein depletion. Notwithstanding, depleted micelles were less susceptible to enzymatic cross-linking by microbial transglutaminase (mTG), indicating narrowed water channels due to depletion. Additionally, loading experiments showed that depleted micelles could be loaded with linoleic acid (LA) as intensively as native micelles, whereupon LA displaces up to 81.3% of β-casein from native micelles. Our results confirm that depletion does not enhance the ability of the casein micelle to act as a nanocarrier for hydrophobic substances but could support the understanding of the casein micelle structure. Based on the observed unchanged stability against EGTA, the hindered enzymatical cross-linking, and the efficient displacing of β-casein by LA, we suggest that the major portion of micellar β-casein is hydrophobically incorporated into the micelle structure without impact on the formation of calcium phosphate nanoclusters. The main role of β-casein for the casein micelle structure, therefore, might be to facilitate the high hydration of the interior and thus the high permeability of casein micelles.

Gas Pressure Cycling (GPC) and Solvent-Assisted Gas Pressure Cycling (SA-GPC) Enhanced Oil Recovery Processes in a Thin Heavy Oil Reservoir

In this paper, gas pressure cycling (GPC) and solvent-assisted gas pressure cycling (SA-GPC) were developed as two new and effective enhanced oil recovery (EOR) processes. Eight coreflood tests were conducted by using a 2-D rectangular sandpacked physical model with a one or two-well configuration. More specifically, two cyclic solvent injection (CSI), three GPC, and three SA-GPC tests were conducted after the primary production, whose pressure was declined in steps from Pi = 3.0 MPa to Pf = 0.2 MPa. It was found that the CSI tests had poor performances because of the known CSI technical shortcomings and an additional technical issue of solvent trapping found in this study. Quick heavy oil viscosity regainment resulted in the solvent-trapping zone. In contrast, C3H8-GPC test at a pressure depletion step size of ∆PEOR = 0.5 MPa and C3H8-SA-CO2-GPC test at ∆PEOR = 1.0 MPa had the highest total heavy oil recovery factors (RFs) of 41.9% and 36.6% of the original oil-in-place (OOIP) among the two respective series of GPC and SA-GPC tests. The better performances of these two tests than C3H8- or CO2-CSI test were attributed to the effective displacement of the foamy oil toward the producer in the two-well configuration. Thus the back-and-forth movements of the foamy oil in CSI test in the one-well configuration were eliminated in these GPC and SA-GPC tests. Furthermore, C3H8-GPC test outperformed C3H8-SA-CO2-GPC test in terms of the heavy oil RF and cumulative gas-oil ratio (cGOR) because of the formation of stronger foamy-oil flow and the absence of CO2, which reduced the solubility of C3H8 in the heavy oil in the latter test. In summary, different solvent-based EOR processes were ranked based on the heavy oil RFs as follows: C3H8-GPC &gt; C3H8-SA-CO2-GPC &gt; CO2-GPC &gt; C3H8-CSI &gt; CO2-CSI.

Solvent-assisted water pressure cycling (SA-WPC) process in a thin heavy oil reservoir

In this paper, solvent-assisted water pressure cycling (SA-WPC) was developed as a new and effective enhanced heavy oil recovery process. Two respective series of WPC and SA-WPC tests were conducted by using a rectangular sandpacked physical model with a two-well configuration and a height-to-length ratio of 1:20 to represent a thin heavy oil reservoir. Three WPC tests were initiated after the primary production pressure declined from Pi = 3.0 MPa to Pf = 2.0, 1.0, and 0.2 MPa, respectively. Moreover, three C3H8-SA-WPC tests and one CO2-SA-WPC test were commenced after the final primary production pressure of Pf = 0.2 MPa was reached. It was found that the three WPC tests had the total heavy oil recovery factors (RFs) of 23.0%, 29.4%, and 22.0%, respectively. In comparison, the four SA-WPC tests had the total heavy oil RFs of 62.0% for C3H8-SA-WPC with the pressure depletion step size of ΔPEOR = 0.1 MPa, 68.4% for C3H8-SA-WPC at ΔPEOR = 0.5 MPa, 42.3% for C3H8-SA-WPC at ΔPEOR = 1.0 MPa, and 29.2% for CO2-SA-WPC at ΔPEOR = 0.5 MPa, respectively. Overall, SA-WPC performed much better than WPC because of effective solvent dissolution to reduce the heavy oil viscosity as well as combined enhanced oil recovery (EOR) and improved oil recovery (IOR) mechanisms. The experimental data showed that C3H8 was a far more effective solvent than CO2 when the same solvent injection pressure was used. It was also found that the intermediate pressure depletion step size of ΔPEOR = 0.5 MPa resulted in the highest heavy oil RF among the three C3H8-SA-WPC tests.

Polymeric nanoassemblies for enrichment and detection of peptides and proteins in human breast milk.

Human breast milk is an understudied biological fluid that may be useful for early detection of breast cancer. Methods for enriching and detecting biomarkers in human breast milk, however, are not as well-developed as compared with other biological fluids. In this work, we demonstrate a new enrichment method based on polymeric nanoassemblies that is capable of enhancing the mass spectrometry-based detection of peptides and proteins in human breast milk. In this method, positively charged nanoassemblies are used to selectively deplete abundant proteins in milk based on electrostatic interactions, which simplifies the mixture and enhances detection of positively charged peptides and proteins. Negatively charged nanoassemblies are used in a subsequent enrichment step to further enhance the detection and quantification of trace-level peptides and proteins. Together the depletion and enrichment steps allow model biomarkers to be detected at low nM levels, which are close to instrumental limits of detection. This new method not only demonstrates the ability to detect proteins in human breast milk but also provides an alternative approach for targeted protein detection in complex biological matrices. Graphical abstract.