Control Reagents Research Articles

A-107 Sample Matrix Matters: A Precision Study of BioRad, TechnoPath, and Patient Pooled Samples Across 20 High Volume Chemistries and Immunoassays

Abstract Background BioRad is a major provider of QC materials in the US with robust peer data, and Technopath has emerged as a recent competitor, especially on cost. While many factors are considered when selecting QC materials, primary considerations include commutability, matrix effects, decision levels, and acceptability limits. A couple studies comparing BioRad and Technopath QC performance were published in recent years. However, none have compared performance to patient-pooled samples. We undertook a comparison of BioRad/Technopath QC performance, with the addition of patient-pooled samples in order to assess potential matrix effects. Methods We compared precision for 20 high-volume chemistry/immunoassay tests (Table 1) across two levels of BioRad and Technopath QC and a patient pooled sample over 7 days on 2 Abbott Alinity-i and 4 Alinity-C instruments. Each immunoassay control level was run 5 times/day across 10 tests and 7 days per instrument, (i.e., 700 results/level), and the same for chemistry controls (1,400 results/level). Lithium-heparin plasma patient-pool was prepared by obtaining sufficient volume and preparing daily frozen aliquots (i.e., 1400 immunoassay and 2800 chemistry results). Results BioRad and Technopath Chemistry QCs were highly comparable with all levels demonstrating <1% CV difference. Notably, CO2, B12, FT4 pooled-patient CVs were >2% lower than the best performing high control. Additionally, greater differences in performance were observed across immunoassay controls with high BioRad QC performing better for 7 tests. Conclusions Chemistry QC for Technopath and BioRad are largely comparable (i.e., CV differences <1%). Overall, BioRad immunoassay control performance was slightly to somewhat better (i.e., B12, CA125, and CA15-3). B12 and CO2 were particularly unstable (as reflected by patient-pool CVs). That said, differences in performance were not untenable and could be handled based on a tailored approach of extending QC limits if assay/instrument performance allows, and/or changing control material or reagents on a tighter schedule.

Variability of lead-zinc sulfide ore slurry rheological properties and its influence on flotation conditions

We investigated how the rheological characteristics of a flotation slurry change in response to variations in mineral species, slurry concentration, and particle size; slurry pH; and trap and rheological control reagent concentrations using slurries containing galena, sphalerite, quartz, and kaolinite. The results indicate that reducing particle size and increasing slurry concentration leads to varying degrees of increase in apparent viscosity and yield stress. At the same particle size, the slurry exhibits the following order of apparent viscosity and yield stress: kaolinite > galena > sphalerite > quartz. In addition, as the slurry’s apparent viscosity and yield stress increase, the rheology decreases, creating progressively unfavorable conditions for the flotation of lead, zinc, and other target minerals. Furthermore, changes in pH have no significant effect on the slurry’s rheology when the slurry is comprised of vein minerals. Moreover, galena and sphalerite depict particle agglomeration in the slurry. Ultimately, the addition of sodium silicate as a rheological control reagent substantially enhances the slurry’s rheological properties. This results in a system where problematic minerals like kaolinite are more effectively dispersed, thereby promoting efficient lead-zinc mineral flotation. Regarding the flotation of lead sulfide and zinc minerals, the addition of kaolinite raises the apparent viscosity of the mixed slurry, hindering the flotation of the target minerals. Conversely, quartz lowers the apparent viscosity aiding the flotation separation process. Understanding the relationship between flotation conditions and the pulp’s rheological properties can provide valuable guidance for subsequent flotation tests.

Identification and Functional Analysis of Adipokinetic Hormone Receptor in Ostrinia furnacalis Guenée Larvae Parasitized by Macrocentrus cingulum.

As a typical G protein-coupled receptor, the adipokinetic hormone receptor (AKHR) has seven transmembrane domains (TMDs), and its structure and function are similar to the gonadotropin-releasing hormone receptor (GnRHR) in vertebrates. However, there is a dearth of information on other components of the AKHR signaling pathway and how it functions in the interaction between insect hosts and parasitoids. In this study, we cloned and analyzed the multifunctional Ostrinia furnacalis AKHR (OfAKHR) cDNA (GenBank accession number MF797868). OfAKHR has a 2206 bp full-length cDNA, which includes an open reading frame containing 1194 bp. OfAKHR contains the typical seven TMDs, and a "DRY" motif. OfAKHR has the highest relative expression in the fat body and the fifth instar larvae. The results revealed that ApoLpⅢ, PPO2, GS, TPS, Cecropin, and Moricin decreased the transcription levels from 48 to 72 h after the knockdown of OfAKHR expression by dsOfAKHR injection in the fourth instar O. furnacalis larvae. The parasitization of Macrocentrus cingulum selectively upregulated the expression levels of nutrition metabolism and immune-related genes in parasitized O. furnacalis larvae, stimulated lysozyme activity, and obviously raised the concentrations of triglyceride and trehalose in the hemolymph of O. furnacalis larvae. However, they inhibited the activities of PO and trehalase. This study is conducive to a deeper cognition of the roles of OfAKHR in nutrition and immune homeostasis, coevolution, and coexistence between parasitic wasps and hosts. It also sheds light on the potential as the target of pest control reagents.

Balancing pH and yield: exploring itaconic acid production in Ustilago cynodontis from an economic perspective

BackgroundItaconic acid is a promising bio-based building block for the synthesis of polymers, plastics, fibers and other materials. In recent years, Ustilago cynodontis has emerged as an additional itaconate producing non-conventional yeast, mainly due to its high acid tolerance, which significantly reduces saline waste coproduction during fermentation and downstream processing. As a result, this could likely improve the economic viability of the itaconic acid production process with Ustilaginaceae.ResultsIn this study, we characterized a previously engineered itaconate hyper-producing Ustilago cynodontis strain in controlled fed-batch fermentations to determine the minimal and optimal pH for itaconate production. Under optimal fermentation conditions, the hyper-producing strain can achieve the theoretical maximal itaconate yield during the production phase in a fermentation at pH 3.6, but at the expense of considerable base addition. Base consumption is strongly reduced at the pH of 2.8, but at cost of production yield, titer, and rate. A techno-economic analysis based on the entire process demonstrated that savings due to an additional decrease in pH control reagents and saline waste costs cannot compensate the yield loss observed at the highly acidic pH value 2.8.ConclusionsOverall, this work provides novel data regarding the balancing of yield, titer, and rate in the context of pH, thereby contributing to a better understanding of the itaconic acid production process with Ustilago cynodontis, especially from an economic perspective.

Hexagonal MoO3 Anode with Extremely High Capacity and Cyclability for Lithium-Ion Battery: A Combined Theoretical and Experimental Study.

It is essential and still remains a big challenge to obtain fast-charge anodes with large capacities and long lifespans for Li-ion batteries (LIBs). Among all of the alternative materials, molybdenum trioxide shows the advantages of large theoretical specific capacity, distinct tunnel framework, and low cost. However, there are also some key shortcomings, such as fast capacity decaying due to structural instability during Li insertion and poor rate performance due to low intrinsic electron conductivity and ion diffusion capability, dying to be overcome. A unique strategy is proposed to prepare Ti-h-MoO3-x@TiO2 nanosheets by a one-step hydrothermal approach with NiTi alloy as a control reagent. The density functional theory (DFT) calculations indicate that the doping of Ti element can make the hexagonal h-MoO3-x material show the best electronic structure and it is favor to be synthesized. Furthermore, the hexagonal Ti-h-MoO3-x material has better lithium storage capacity and lithium diffusion capacity than the orthogonal α-MoO3 material, and its theoretical capacity is more than 50% higher than that of the orthogonal α-MoO3 material. Additionally, it is found that Ti-h-MoO3-x@TiO2 as an anode displays extremely high reversible discharge/charge capacities of 1326.8/1321.3 mAh g-1 at 1 A g-1 for 800 cycles and 611.2/606.6 mAh g-1 at 5 A g-1 for 2000 cycles. Thus, Ti-h-MoO3-x@TiO2 can be considered a high-power-density and high-energy-density anode material with excellent stability for LIBs.

Selection of positive controls and their impact on anti-drug antibody assay performance

Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format. Neither anti-id affinity for drug, nor sensitivity in direct immunoassays related to sensitivity in ADA assays. Anti-ids were differentially able to detect damage to drug conjugates used in bridging assays and were differentially drug tolerant. These parameters also failed to relate to assay sensitivity, further complicating selection of anti-ids for use in ADA assay development based on functional characteristics. Given this variability among anti-ids, alternative controls that could be employed across multiple antibody drugs were investigated as a more uniform means to define ADA detection sensitivity across drug products and assay protocols, which could help better relate assay results to clinical risks of ADA responses. Overall, this study highlights the importance of positive control selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses.

Radiolytic evaluation of a new technetium redox control reagent for advanced used nuclear fuel separations.

Technetium is a problematic radioisotope for used nuclear fuel (UNF) and subsequent waste management owing to its high environmental mobility and coextraction in reprocessing technologies as the pertechnetate anion (TcO4-). Consequently, several strategies are under development to control the transport of this radioisotope. A proposed approach is to use diaminoguanidine (DAG) for TcO4- and transuranic ion redox control. Although the initial DAG molecule is ultimately consumed in the redox process, its susceptibility to radiolysis is currently unknown under envisioned UNF reprocessing conditions, which is a critical knowledge gap for evaluating its overall suitability for this role. To this end, we report the impacts of steady-state gamma irradiation on the rate of DAG radiolysis in water, aqueous 2.0 M nitric acid (HNO3), and in a biphasic solvent system composed of aqueous 2.0 M HNO3 in contact with 1.5 M N,N-di-(2-ethylhexyl)isobutyramide (DEHiBA) dissolved in n-dodecane. Additionally, we report chemical kinetics for the reaction of DAG with key transients arising from electron pulse radiolysis, specifically the hydrated electron (eaq-), hydrogen atom (H˙), and hydroxyl (˙OH) and nitrate (NO3˙) radicals. The DAG molecule exhibited significant reactivity with the ˙OH and NO3˙ radicals, indicating that oxidation would be the predominant degradation pathway in radiation environments. This is consistent with its role as a reducing agent. Steady-state gamma irradiations demonstrated that DAG is readily degraded within a few hundred kilogray, the rate of which was found to increase upon going from water to HNO3 containing solutions and solvents systems. This was attributed to a thermal reaction between DAG and the predominant HNO3 radiolysis product, nitrous acid (HNO2), k(DAG + HNO2) = 5480 ± 85 M-1 s-1. Although no evidence was found for the radiolysis of DAG altering the radiation chemistry of the contacted DEHiBA/n-dodecane phase in the investigated biphasic system, the utility of DAG as a redox control reagent will likely be limited by significant competition with its degradation by HNO2.

B-089 Development of a Secondary Standard for Quantitation of IgG Antibodies to SARS-CoV-2 Proteins in a Multiplex Assay

Abstract Background Serological surveillance is essential for monitoring disease burden and vaccine uptake at an individual and population level. During the pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), several serological assays were developed and obtained emergency use authorization to detect antibodies after infection. At that time, the need was to differentiate the infected from the naïve population, and these qualitative assays played a critical role in disease management. In the wake of vaccinations, there is a need for the development of quantitative assays. The world health organization (WHO) developed an international standard for quantitation. We performed this study to develop and validate an in-house secondary standard for our multiplex assay based on this WHO standard. Methods We developed a novel multiplex assay for quantifying human IgG antibodies to four different SARS-CoV-2 antigens in human serum or plasma. We have used Luminex® xMAP® technology for this assay and immobilized four recombinant proteins, namely, the receptor-binding domain of S1(RBD), nucleocapsid protein (NP), S1 protein (S1), and trimeric Spike protein (trimer) on internally coded magnetic microspheres. We procured lyophilized WHO quantification standard 20/136 from the National Institute for Biological Standards and Control (NIBSC), which has the arbitrary assignment of 1000 BAU/mL after reconstitution in 0.25 mL. We tested this standard in the range of 10 to 0.01 BAU/mL for the standard curve. We tested NIBSC 20/162 control reagent using this assay for the linearity of dilution. Thirty-seven samples from the NIBSC verification panel were also tested for this evaluation. We also quantified IgG antibodies in 25 samples from vaccinated individuals, ten samples from vaccine breakthrough individuals, 25 samples from PCR-positive individuals, and 75 samples collected before 2019. Results The secondary standard was calibrated using the 20/136 standard. The NIBSC 20/136 and the secondary standard displayed a wide dynamic range of quantitation. The difference in the expected and actual concentrations was less than 20%. Multiple dilutions of the serum or plasma samples were used to get readings within the standard curve range. Excellent linearity was noted at various sample dilutions to get the correct quantification. We observed that vaccinated individuals had a higher concentration of binding antibodies to S1, Trimer, and RBD and were negative for NP antibodies. Conversely, the acute samples from infected individuals collected less than ten days post-positive PCR showed higher antibodies to NP than different spike proteins. The vaccine break-through infection samples showed lower NP antibodies than unvaccinated but infected individuals. Conclusion Our development of a secondary standard calibrated against WHO international standard is vital for quantitatively measuring different SARS-CoV-2 antibodies. A secondary standard can help manufacturers and users to normalize results from various assays and lots over a prolonged period.

A-248 Evaluation of NextGene™ MTB/NTM Detection Kit for Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Sputum and Culture Specimens

Abstract Background Tuberculosis, an infectious disease, is a major cause of deteriorating health, and, until SARS-CoV-2 (COVID-19) became a pandemic disease, a major cause of death worldwide. The recently developed NextGene™ MTB/NTM Detection Kit is designed for detection of Mycobacterium tuberculosis complex (MTB) using IS6110 genes and MPB64 genes, and for detection of nontuberculous mycobacteria (NTM) using 16S rRNA genes for resolution of false positive and false negative results, and for development of more sensitive and specific real-time PCR products. The purpose of this study is to examine the clinical sensitivity and specificity of the NextGene™ MTB/NTM Detection Kit in order to verify its clinical efficacy. Methods A total of 446 residual clinical specimens were obtained from 274 sputum and 172 culture samples collected from July to August 2021. Diagnostic tests were performed in order to confirm that these residual samples were positive and negative; 344 specimens were positive for MTB or NTM, and 102 specimens were confirmed as negative for both MTB and NTM. Measurement of positive/negative percent agreement values and kappa coefficients was performed for comparison of diagnostic performance between the NextGene™ and Advansure assays. Positive and negative controls were included in each run. Results Of the 446 samples (274 sputum and 172 culture samples), 344 specimens showed a positive result and 102 specimens showed a negative result. Results of the comparison tests were all consistent for detection of MTB and NTM. Both MTB and NTM met the criterion for evaluation of target validity of kappa value 0.8 or higher in sputum and culture samples. Results of the evaluation of clinical sensitivity and specificity were consistent with both clinical diagnoses. Conclusion This study was conducted in order to examine the clinical sensitivity and specificity of the NextGene™ MTB/NTM Detection Kit in order to verify its clinical validity. Residual samples from sputum and culture specimens from patients who were suspected being infected that were confirmed positive or negative for MTB and NTM were used in this study with the goal of establishing the already commercially available licensed product as a control reagent for comparative analysis of the clinical performance of the NextGene™ MTB/NTM Detection Kit. Compared to existing kits that are widely used, consistent results from comparison of the NextGene™ MTB/NTM Detection Kit have been reported with comparable clinical sensitivity and specificity, thus it is regarded as showing the same performance.

Bougainvillea Flower Extract Utilization as Eosin Substitute in Worm Eggs Microscopic Observation

Introduction: Flavonoids, glycosides, alkaloids, phlobatannins, saponins, steroids, tannins, and terpenoids are all known components of bougainvillea flowers. The betalain compounds betasianin and betaxanthin, which give bougainvillea flowers their color, are present. Betalains are water-soluble pigments classified into betacyanins (red) and betaxanthins (yellow). The eosin dye, which can stain worm eggs, is comparable to the pigment in bougainvillea flowers. Because reagents are expensive, raw materials from the local area must be used instead. Local raw materials can replace eosin reagents. When examining intestinal nematode worm eggs, the extract of bougainvillea flowers can be used as an eosin reagent. We are interested in learning whether Bougenvillea flower extract can be used to observe worm eggs at a microscopic level instead of the expensive and difficult-to-come-by eosin. Aim: This study aimed to ascertain whether Bougainvillea extract could be used instead of eosin to observe worm eggs at a microscopic level. Method: Complete Randomised Design (CRD) is used in this quasi-experimental research. This study also aims to compare the efficacy of eosin reagent and bougainvillea flower extract in the protracted drying process. The Microbiology Laboratory, Department of Medical Laboratory Technology, Aceh Health Polytechnic, Ministry of Health, was the site of this study. Gampong Jawa, Banda Aceh, is where feces were sampled. Preparation of bougainvillea flower extract with a concentration of 100% as a substitute for 2% eosin on microscopic observations of Ascaris lumbricoides, Trichuris trichiura, and Hookworm eggs to see the comparison of the drying time for reagents in worm egg preparations using red, pink and orange Bougainvillea flower extract with eosin control. Repeat three more times. T-test used for data analysis. Result: When compared to the eosin control reagent, which dried in only 27.63 minutes in preparation for Soil Transmitted Helminth worm eggs, the average drying time for the reagents using natural reagents from the extract of red bougainvillea flowers was 23.96 minutes, that of pink bougainvillea flowers was 23.30 minutes, and that of orange bougainvillea flowers was longer at 28.40 minutes. A variety of bougainvillea species have different drying requirements for regeneration. Conclusion: The time it took for red, pink, and orange Bougainvillea extract to dry, as well as the control eosin, indicates that there is a significant difference in the dry times between the treatments. Keywords: Bougainvillaea extract, eosin reagents, worm eggs

A human papillomavirus whole genome plasmid repository: A resource for HPV DNA quality control reagents.

Well characterized reference reagents are useful for assay validation, proficiency/competency assessment, daily run controls, and to improve inter-laboratory comparisons. Synthetic human papillomavirus (HPV) DNA fragments and plasmid clones are available, but synthetic fragments include limited segments of the HPV genome and many HPV plasmids have interrupted coding regions or contain partial genomes. As a result, they are not compatible with all HPV DNA detection and typing assays. To address this need, we are establishing an HPV plasmid repository of HPV clones containing the whole genome of each type with no interruptions in coding regions. To date, HPV plasmid clones for 16 HPV types, (including all vaccine types and 14 types in clinical assays: HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) have been constructed using a Gibson assembly method and validated by sequencing and the Novaplex HPV typing assay. The newly constructed HPV whole genome plasmids can serve as a quality control reagent resource for HPV DNA assays and are available for public health and research laboratories.

Use of Corrosion Control Device to Control Reagent Water Treatment of Heating Networks

Use of Corrosion Control Device to Control Reagent Water Treatment of Heating Networks

Validation of immunoassays for the Chlamydia trachomatis antigen Pgp3 using a chimeric monoclonal antibody

Seroepidemiology, or measuring antibodies to pathogens to estimate population-level exposure, can provide useful public health data. The tests used, however, often lack sufficient validation data due to absence of a gold standard. For many pathogens, serum antibodies can be detected long after resolution of infection, but infection status is often used as a gold standard for antibody positivity. To ensure that recently developed antibody tests for seroepidemiology of Chlamydia trachomatis (Ct), the causative agent of urogenital chlamydia and the blinding eye disease trachoma, have high performance, we generated a chimeric antibody to the immunodominant Ct antigen Pgp3. Two clones were selected to evaluate the test performance of three assays to measure antibodies to Pgp3: multiplex bead assay (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). Overall, each assay demonstrated high accuracy and precision when tested using either clone, and the clones were stable when stored at − 20 °C and 4 °C for almost 2 years. The limit of detection was similar for MBA and LFA, but almost a log-fold higher (i.e. less sensitive) using ELISA. Overall, the chimeric antibodies represent stable control reagents for tests with robust performance and will facilitate deployment of these tests to other laboratories.

Abstract 3386: Development of next generation extracellular vesicle based urine tests for prostate cancer

Abstract Introduction and Objective: Non-invasive urine and blood biomarkers are commercially available to assess the risk of clinically significant prostate cancer (csPCa) and the need for a biopsy. However, these tests are limited in their specificity resulting in a significant number of men still undergoing biopsy to avoid missing a csPCa. Exosomes and other extracellular vesicles (EVs) provide a platform for blood and urine biomarkers since they are released by all types of cells and preserve molecular constituents from their cells of origin within lipid membranes. The EPI urine test from Exosome Diagnostics (ExoDx) is currently the only commercially available EV based cancer diagnostic test; it uses three well established EV RNA markers to estimate the risk of csPCa. Studies by us and others have shown that an expanded urine EV PCa marker panel would improve disease stratification if these markers could be incorporated into a clinical grade assay. Our objective in this current study is to identify and characterize the EV RNA transcripts that are enriched by using antibodies for prostate specific membrane antigen (PSMA) to capture urinary EVs that originated from PCa cells. This is the first step in the development of a next generation urinary EV test with increased specificity for csPCa within a rigorous and reproducible assay format. Methods: Urine EVs from patients with csPCa and from healthy controls were prepared using the ExoDx clinical platform. These total EV samples were then compared with EVs enriched by immunocapture either with a PSMA specific antibody or with an isotype IgG control reagent. RNA was prepared from each of these three types of urinary EV samples and analyzed using RNAseq. Results: RNAseq analysis of urinary total EVs, PSMA-antibody captured EVs and isotype IgG control captured EVs showed excellent mapping to transcriptome regions. We detected over 600 genes differentially enriched by PSMA antibody vs. isotype IgG EV capture and identified 18 genes from a candidate list of prostate biomarkers that have been assembled based on previous studies. A principal component analysis of 196 differentially expressed transcripts obtained from EVs enriched by PSMA immunocapture vs IgG controls showed excellent discrimination between prostate cancer patients and healthy controls. By using a feature selection approach to optimize for discrimination between prostate cancer patients and healthy controls we identified a novel 6-gene signature that, when derived from a PSMA capture EV dataset, shows a superior AUC for the accuracy of cancer detection (greater than 0.95) than that obtained from applying the same 6-gene signature on total EVs or on isotype IgG controls. Conclusions: EV PSMA immuno-capture provides a robust approach to expanding the panel of PCa biomarkers that can be incorporated into clinical grade PCa urine EV assays, supporting the translation of pre-clinical discovery into clinical practice. Citation Format: Sandra M. Gaston, Douglas Roberts, Sudipto Chakrabortty, Emily Mitsock, Kailey Babcock, Rikky Xing, Benjamin Spieler, Radka Stoyanova, Mark L. Gonzalgo, Chad Ryan Ritch, Bruno Nahar, Alan Pollack, Dipen J. Parekh, Seth Yu, Johan Skog, Sanoj Punnen. Development of next generation extracellular vesicle based urine tests for prostate cancer [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 3386.

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity.

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are characterized by their remarkable tumor-initiating and self-renewal capacity. Depending on the origin of the tumor (e.g., ovaries), the CSC surface biomarker profile can vary dramatically, making the identification of such cells via immunohistochemical staining a challenging endeavor. On the contrary, aldehyde dehydrogenase 1A1 (ALDH1A1) has emerged as an excellent marker to identify CSCs, owing to its conserved expression profile in nearly all progenitor cells including CSCs. The ALDH1A1 isoform belongs to a superfamily of 19 enzymes that are responsible for the oxidation of various endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products. Chan et al. recently developed AlDeSense, an isoform-selective "turn-on" probe for the detection of ALDH1A1 activity, as well as a non-reactive matching control reagent (Ctrl-AlDeSense) to account for off-target staining. This isoform-selective tool has already been demonstrated to be a versatile chemical tool through the detection of ALDH1A1 activity in K562 myelogenous leukemia cells, mammospheres, and melanoma-derived CSC xenografts. In this article, the utility of the probe was showcased through additional fluorimetry, confocal microscopy, and flow cytometry experiments where the relative ALDH1A1 activity was determined in a panel of five ovarian cancer cell lines.

A Stable Dried Tube Specimen for Quality Assurance and Training Programs for HIV Rapid Test for Recent Infection.

The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.

Facile preparation of α-MnO2 nanowires for assembling free-standing membrane with efficient Fenton-like catalytic activity

Assembling MnO2 nanowires into macroscopic membrane is a promising engineered technology for catalyst separation and enhancement of Fenton-like reaction activity, yet its development is limited by the deficiencies in preparation and property modulation of the MnO2 nanowires. In this work, we developed a facile method using C2H5OH and CH3COOK as reductive and vital control reagents to react with KMnO4 by hydrothermal reaction at 140 °C for 12 h, to prepare the ultralong α-MnO2 nanowires up to tens of micrometers with high purity and aspect ratio. Such strategy not only had the advantages of being mild, easily controlled and environmental pollution-free, but also endowed α-MnO2 nanowires with excellent ability as a Fenton catalyst when assembled into free-standing membrane for degrading phenolic compounds (kobs = 0.0738 ∼ 0.1695 min−1) in a continuous flow reaction. The reactive oxygen species (i.e., •OH) from Fenton-like reaction were enriched within this α-MnO2 nanowire membrane via nanoconfinement effect, which further enhanced the mass transportation of •OH available for phenolic contaminants. MnO2 nanowire membrane using our method possessed the high practical potential for water purify due to its easy-preparation and enhanced catalytic performances.

Molecular dynamics simulation for quantitative characterization of wettability transition on silica surface

Surfactant controlling the wettability of reservoir rocks is one of the important ways to enhance oil recovery in low-permeability tight reservoirs. A large number of scholars have developed a series of excellent wettability control reagents through macro physical simulation experiments, but macro experiments are difficult to analyze the micro mechanism of liquid/liquid and liquid/solid interface action of reagents controlling rock wettability. In this paper, the wetting behavior of water molecules on silica surfaces modified with different hydroxylation, methylation and hydrophobic chain length was studied by molecular dynamics simulation, and the quantitative relationship between different modification degrees and wetting angle was established. The results show that the original wetting angle of silica is 58.5°, and the surface wettability can be changed from hydrophilic to hydrophobic through surface modification, with a wetting angle of 27.25°∼115.78°. With the increase of the coverage of hydrophobic methyl or hydrophilic hydroxyl modified groups, the wetting angle increases or decreases respectively. The quantitative relationship between modification degree and wetting angle was established by simulated data. With the increasing of the chain length of methyl group, the distance between water molecule and silica surface increases and the interaction decreases, which can improve the regulation effect of methyl group hydrophobicity. This paper provides a reference basis for the synthesis of rock wettability control reagent and the quantitative characterization of modification degree.

Quorum Quenching Bacteria Bacillus velezensis DH82 on Biological Control of Vibrio parahaemolyticus for Sustainable Aquaculture of Litopenaeus vannamei

Aquatic pathogens such as Vibrio parahaemolyticus cause a bacterial infection that reduces the economic benefits of aquaculture and affects the food quality and safety of human beings. Quorum quenching (QQ) is considered a novel strategy of microbial antagonism that inhibits pathogens and reduces the abuse of antibiotics. This study investigates a QQ bacterial strain, Bacillus velezensis DH82 from the deep sea Yap trench, in vitro to examine the effects of DH82 and its functional products against V. parahaemolyticus, focusing on the Quorum sensing (QS) regulation and the inhibition of pathogenicity and bacterial growth. The study also conducted in vivo investigation in the aquaculture of Litopenaeus vannamei challenged with V. parahaemolyticus by immersion and injection challenge. The results of the QS regulator transcription level demonstrated the multiple QQ enzymes in DH82 regulated the pathogenicity but could not fully control the biofilm formation; the effective antibacterial activity of extracellular peptides on microbial antagonism verified the inhibition on bacterial growth of V. parahaemolyticus. The in vivo experiment in aquaria demonstrated the effective enrichment of DH82 and inhibition of Vibrio in both the aquatic system and the shrimp intestine. The dietary DH82 relieved the negative effect of Vibrio on the activity of enzyme acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) under challenge of Vibrio pathogens, and was not harmful to host according to lysozyme (LZM) activity. DH82 also ameliorated the damage to the intestine and muscles induced by V. parahaemolyticus infection according to tissue imaging. Though DH82 did present some dose-dependent adverse effects to the host, the findings revealed the effective QQ and antibacterial activity of DH82 on emerging biocontrol against V. parahaemolyticus, therefore indicating the potential application of DH82 as a biological control reagent in the sustainable and green production of aquaculture.