Detection Of Low Levels Research Articles

Mutational and Copy Number Profiling of Circulating Tumor DNA in Acute Myeloid Leukemia Using Targeted Next Generation Sequencing

The assessment of circulating tumor DNA (ctDNA), released by tumor cells undergoing apoptosis or necrosis, has established utility in solid tumors due to the advantage of a non-invasive "liquid biopsy" replacing multiple site-specific biopsies. However, its role in acute myeloid leukemia (AML) is uncertain, where a significant proportion of variants detected in the bone marrow (BM) may not be detected in ctDNA (Short, Blood Adv 2020). We have previously demonstrated the possibility of comprehensive genomic characterization of lymphoid malignancy from ctDNA using a single targeted next generation sequencing (NGS) hybridization-based panel (Blombery, BJH 2017). We aimed to assess the performance of this same genomic approach in ctDNA and to compare it against BM in AML. In addition, we aimed to assess the integration of a sensitive variant caller (Mutect2; Benjamin, bioRxiv 2019) to the bioinformatics suite in an attempt to improve low-level variant detection. Nineteen patients were identified from sequential patients with AML treated at our institutions where paired ctDNA and BM aspirate DNA were available. ctDNA was analyzed using a hybridization-based NGS panel targeting genes recurrently mutated in hematological malignancy followed by a suite of bioinformatics tools including HaplotypeCaller (GATK)/Mutect2 (GATK) for variant calling, CNSpector/CNSpectorX (Markham, Sci Reports 2019) for copy number variation (CNV) assessment and GRIDSS (Cameron, Genome Res 2017) for structural variant detection. The cohort clinical details are summarized in Table 1; none had documented extramedullary disease at time of collection. A total of 66 unique variants in 27 genes were detected, summarized in Figure 1. Median number of variants detected was 3 per patient sample, including NPM1 (n=6), IDH1/2 (n=4) and FLT3 point mutation (n=2). Three patient samples had FLT3-ITD detected by fragment length analysis; none were detected by the NGS panel in either ctDNA or BM. Variant allele frequency (VAF) from both compartments were highly correlated (R2 0.87). Higher VAFs in ctDNA were more commonly observed for kinase activating mutations (12/17 variants) and TP53 (5/6). Using HaplotypeCaller alone, 58 and 61 variants were detected in the BM and ctDNA, respectively. Of the 2 variants "specific" to the BM, both (IDH1 and NRAS) were called by Mutect2 in ctDNA and confirmed by visual inspection of sequence read alignments. Of the 5 variants "specific" to the ctDNA, 4 were detected in the BM at low VAF: CBL (n=2), KRAS and TET2. One discrepant case was patient #15 with prior breast cancer: KRAS G13D 21% in ctDNA but absent in the BM. Analysis by Mutect2 additionally detected 3 variants not called by HaplotypeCaller: NRAS and KIT in both ctDNA and BM, and TP53 P278R (VAF 6%) specific to the ctDNA in patient #9 with normal karyotype AML without history of prior malignancy. Overall, 3/5 variants in ctDNA and 6/6 in BM with low VAF were kinase activating mutations. We then performed genome-wide alignment of off-target reads to generate a low-resolution digital karyotype and CNV from ctDNA (Figure 2) which was compared with CNV and conventional karyotyping from BM. CNVs were detected in ctDNA in 10/11 patients with abnormal karyotype (Figure 1). Of these, 6/7 with non-complex abnormal karyotypes had consistent ctDNA CNVs including (i) 3 patients (#1, #10 and #15) with either rearranged/amplified KMT2A by FISH were all found to have gains at 11q23.3-qter, and (ii) 1 patient (#4) had 2 marker chromosomes of unknown origin on karyotyping which were resolved as additional copies of 4p using ctDNA sequencing. Although no CNVs were detected in all 8 patients with normal karyotype, analysis of B-allele frequency from ctDNA revealed one patient (#5) with copy neutral loss of heterozygosity (CN-LOH) in 7q (with a concurrent EZH2 mutation >95% VAF). In summary, we have demonstrated the ability to detect sequence variants, perform low-resolution digital karyotyping, CNV detection and CN-LOH detection from ctDNA in AML using a single hybridization based NGS assay. When using this approach, we show a high degree of concordance for both sequence variant and CNV detection, supporting the use of ctDNA as an alternative to BM (e.g. in cases of dry tap, hypocellular AML or failed karyotyping). Finally, using a sensitive variant caller, additional mutations were able to be detected in the ctDNA, the significance of which require evaluation in future studies. Disclosures Tiong: Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Servier: Consultancy. Wilson:Illumina: Other: Illumina Diagnostic Genomics Scholarship. Bajel:Novartis: Honoraria; Astellas: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria; Amgen: Consultancy.

Highly sensitive room temperature liquefied petroleum gas sensor based on CoSnO2 nanoislands deposited graphene layers

In this present work, an effective Liquefied Petroleum Gas (LPG) detecting nanocomposite of r-GO-CoSnO2 operable at room temperature was exhibited. Nanoparticles of Cobalt (Co) doped Tin Oxide (SnO2) nanoparticles were decorated uniformly over the reduced graphene oxide (rGO) layers. Reduction of graphene oxide (GO) into rGO and combination of metal oxide decoration was synthesized by one step using cost effective hydrothermal method using reductant hydrazine hydrate. Nanoparticles of CoSnO2 were self assembled over rGO via high pressure built inside the Teflon autoclave chamber. Structural and surface morphological characterizations confirmed the structure, size and shape of the prepared materials with distinct properties which are essential for a reliable sensing device. Distribution of metal oxide throughout the graphene layers act as an active site for the LPG molecules adsorption and desorption processes. For selectivity various analyte gases such as Hydrogen, Oxygen and Ammonia were examined. The proposed unique nanocomposite has exhibits low level detection of 2 ppm with quick response time of 4 s and recovery time of 5 s at room temperature. From these results it suggests that the proposed material will be a promising potential candidate for the fabrication of LPG sensor devices.

A Highly Sensitive Square-wave Adsorptive Anodic Stripping Voltammetry for Trace Determination and Speciation of Palladium at Bismuth Film Modified Glassy Carbon Electrode using 1,2 Dihydroxy-cyclobut-1,2-ene 3,4 dione Schiff Base Reagent

The detection of precious metal ion moieties is of utmost significance for environmental control. Thus, the current study provides a low cost and highly-accurate square wave adsorptive anodic stripping voltammetric procedure (SW-AdASV) at a Bi film (BiF/GCE) for the analysis of the lower levels of palladium (II) ions. The proposed SW-AdASV methodology was based on the selective accumulation of the palladium (II) chelat of the newly Schiff base reagent of 1,2 dihydroxy-cyclobut-1,2-ene-3,4-dione (DCED) in a universal buffer of pH=2-3. Under the optimized analytical parameters, the plot of the palladium (II) concentration versus the corresponding anodic peak current was linear over a wide range of [5×10-9 – 2.5×10-6 molL-1 Pd] with a correlation coefficient of R2=0.9995 and excellent repeatability with RSDs of (3.3-3.8%) (n=5). In the SW-AdASV approach, the lower limits of detection (LOD) and quantitation (LOQ) as calculated were discovered to be 1.07×10-9 and 3.57×10-9 molL-1, respectively. The current sensor offered interference from several active metal ions, such as Hg2+ and Cd2+. Lastly, the developed SW-AdASV method with (BiF/GCE) was successfully applied for the detection of lower levels of Pd2+ in water. Furthermore, this procedure presents a sensitive, accurate process with short analytical time, ruggedness, robustness, and reproducibility.

Introducing Machine Learning to Environmental Health Research; Exploring Random Forest in Prediction Modeling

BackgroundAccurately assessing tobacco smoke exposure in early life is important to understanding and preventing childhood asthma. In populations with relatively low smoking exposure, it becomes challenging to characterize the main exposure sources of tobacco smoke exposure. When creating a prediction model of questionnaire items to explain variability in nicotine metabolite concentrations, traditional approaches include using directed acyclic graphs, change-in-estimate procedure, comparing AIC and/or R2 statistics, and stepwise selection. These approaches may be improved upon with machine learning. MethodsUsing the CHILD Cohort Study, we measured urinary concentrations of nicotine biomarkers (cotinine and trans-3’-hydroxycotinine (3HC)) at low detection levels (0.03ng/mL) and questionnaire responses related to smoking and other lifestyle factors. Urine samples were collected along with questionnaires at 3-4 months of age. The concentrations were corrected for specific gravity. Random forest regression was used to assign variable importance scores to questionnaire items based on how well they predicted the urinary cotinine and 3HC concentrations. Combining knowledge from this machine learning technique with traditional model selection strategies, a multivariable linear regression prediction model was selected to assess how well questionnaires explained urinary cotinine and 3HC concentrations. Results76% and 89% of the infants had detectable urinary cotinine and 3HC levels, with the geometric mean levels consistent with light or intermittent second-hand smoke, third-hand smoke or possibly from diet. Final models explained 32% and 41% of cotinine and 3HC concentration variation in our sample (n=2,017). Questions related to prenatal smoking exposure, second-hand smoke, housing, breastfeeding, and socioeconomic factors were most important in predicting urinary concentrations at 3 months. ConclusionsTo facilitate complex exposure assessment in interdisciplinary projects, researchers have more sensitive tools from outside their traditional disciplines to improve models and generate new hypotheses. Variable importance plots are a relatively easy and interpretable way to incorporate machine learning into environmental health research.

Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events

Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples.

Colorimetric Detection of Ammonia Using Synthesized Silver Nanoparticles from Durian Fruit Shell

There has been increased interest in the production of nanoparticles (NP) through green chemistry. This article used durian fruit shell aqueous solution that acts as a reductive preparation of silver NPs. The silver nanoparticles have a size of approximately 25 nm. The NP size uniformity was determined by the SEM and TEM analysis. X-ray diffraction technique was used to characterize crystalline silver nanoparticles face-centered cubic structure. XPS spectrum showed distinct silver peaks on the nanoparticles’ surface. An optical method that was based on surface plasmon resonance (SPR) was used to perform the green Ag NPs aqueous ammonia sensing study. Optical measurement facilitated the ammonia sensing study of Ag NPs that had been prepared. The study also investigated the performance of the optical sensor, thus adding validity to the study. Also, the research sought to determine how the concentration of ammonia in ammonia sensing affects the Ag NPs that had been obtained. The study observed a linear relationship with R2 as the correlation factor which was equal to 0.9831. This was observed from the ammonia concentration plot versus absorption ratio that suggested that there was a linear increase in absorption ratio with increase in ammonia concentration. The study significance is that the room temperature optical ammonia sensor can be used in future for medical diagnosis in the detection of low levels of ammonia in biological fluid like sweat, cerebrospinal fluid, saliva, plasma, or biological samples. This enhances the application of the technique in human biomedical applications.

Improved intact peptide and protein quantitation by LC-MS: Battling the deleterious effects of analyte adsorption.

Peptide and protein quantitation by liquid chromatography-mass spectrometry relies on the assumption of linear signal response with concentration. At low concentrations, analyte adsorption to pipette tips, sample vials and equipment can have significant deleterious effects on signal response. Meanwhile at high concentrations, linearity breaks down due to competitive ionization, signal suppression, and the formation of peptide or protein multimers. These effects result in calibration curves that are more sigmoidal than linear. Linearity at low protein levels for identification and quantitation is of paramount importance in the discovery and validation of biomarker molecules. Herein, we demonstrate the benefits of using commercial low-bind microcentrifuge tubes and LC vials on the response of a 27-mer peptide, Vn96, and the intact proteins apomyoglobin and carbonic anhydrase. Linear curves were acquired for Vn96 while apomyoglobin required the addition of intact carbonic anhydrase as an adsorption competitor to achieve linearity. A linear calibration curve for carbonic anhydrase was also acquired by using the polypeptide ubiquitin as an adsorption competitor and internal standard. Linear response was recorded for approximately two orders of magnitude for apomyoglobin and carbonic anhydrase and three orders of magnitude for Vn96 with detection limits ranging from 0.33 to 19 fmol/µL. Finally, we used low-bind vials for the online enzymatic digestion of apomyoglobin where a high concentration of apomyoglobin acted as an adsorption blocker for the low level trypsin enzyme. Fortunately, the liberated tryptic peptides showed no affinity for the walls of the low-bind vials. In this study, we take a comprehensive approach to combat analyte adsorption by showing the significance of utilizing low-bind vials and adsorption competitors to greatly improve upon signal sensitivity at low concentrations of target molecules. The use of these methodologies should improve the low-level detection of molecules by mass spectrometry.

K-carrageenan/PVA/nano-eggshell biocomposite-based non-enzymatic electrochemical biosensor for low-level urea detection

As a comprehensive survey on both health and biosensor technologies, in this study, a non-enzymatic electrochemical biosensor based on kappa-carrageenan/PVA/nano-eggshell (K-carrageenan/PVA/nano-eggshell) biocomposite was prepared to detect low-level urea in phosphate-buffered solution (PBS). Novel K-carrageenan/PVA/nano-eggshell biocomposite was prepared by ultrasonics sonochemistry. The electrochemical biosensor showed a sensitivity of 0.018 μA μM−1 cm−2 in a linear range of [250–1000] μM urea. Urea detection limit of the biosensor was 60 μM at room temperature.

Magnetic beads modified with Pt/Pd nanoparticle and aptamer as a catalytic nano-bioprobe in combination with loop mediated isothermal amplification for the on-site detection of Salmonella Typhimurium in food and fecal samples

Concentration of pathogens directly from food samples by using magnetic beads is a potential strategy in the on-site food sample analysis. In this study, magnetic beads, double modified with platinum/palladium nanoparticle (Pt/Pd NP) and DNA aptamer, is presented as catalytic nano-bioprobes for the on-site detection of Salmonella enterica serovar Typhimurium in food and fecal samples. Combination of the developed catalytic nano-bioprobes with loop mediated isothermal amplification (LAMP) enabled rapid detection of low levels of S. Typhimurium in food and chicken fecal samples without culture enrichment. S. Typhimurium-specific DNA aptamer immobilized on the magnetic bead could efficiently concentrate S. Typhimurium with a capturing efficiency higher than 76% in phosphate-buffered saline (PBS). Further, DNA-mediated inhibition of peroxidase-mimic activity of Pt/Pd NP in combination with LAMP was used as a unique approach to detect S. Typhimurium. With this unique approach, it was possible to capture and detect S. Typhimurium as low as 10–15 CFU/mL in chicken meat sample and 3–10 CFU/mL in both whole egg and chicken fecal samples within less than 3 h. Analysis of S. Typhimurium-spiked chicken meat, whole egg and chicken fecal materials have confirmed the precision of the method. A relative accuracy of 90% with an intra and inter assay precision of 8.36% and 9.92% respectively was achieved in the S. Typhimurium-spiked food samples. This unique approach has the potential for the integration in to Lab-on-a-chip based biosensors for on-site monitoring of foodborne pathogens in future.

Epigenetic research in lung cancer diagnostics:
 A literature review

Relevance: According to the International Agency for Research on Cancer (IARC), lung cancer (LC) currently ranks first in
 cancer incidence and mortality worldwide. The gold standard of LC diagnostics is the histological verification, the determination of the degree of invasion and tumor phenotype. At first glance, epigenetic methods seem to be secondary after determining the patient’s genetic profile. However, standard genetic analysis reveals only the DNA nucleotide sequence. Thus, epigenetic
 analysis is the only method that allows detecting potential abnormalities in cells. An important difference between genetic and
 epigenetic changes is that drugs are efficient against epigenetic changes but absolutely powerless against genetic mutations.
 The purpose of the study was to review and analyze the available molecular genetic methods for DNA methylation profiling in lung cancer.
 Results: All these observations support the hypothesis that methylation profiling in body fluids can help determine the people predisposed to or affected by LC. Circulating acellular DNA in the blood plasma contains tumor-specific mutations and disease-related DNA methylation patterns. Identifying new biomarkers-precursors of a potential cancer susceptibility or aggressiveness in such DNA would be a considerable advancement in prognostic medicine for patients at high risk of developing LC.
 Conclusion: A low level of LC detection might limit the number of DNA samples of patients with LC included in the studies.
 This is also the reason why specific methylation biomarkers have not yet been confirmed for clinical use. Future research on
 a larger number of blood samples, combined with the entire epigenome studies, may contribute to finding a group of LC biomarkers to improve LC detection.

Oxidative Stability at Different Storage Conditions and Adulteration Detection of Prickly Pear Seeds Oil

Lipid oxidation and adulteration have a negative impact on functionality and notoriety of foods especially vegetable oils and cause economic losses. The present study investigates the control of two commercial quality aspects of prickly pear seeds oil (PPSO): oxidative stability during storage and detection of adulteration. Peroxide index, specific extinction coefficients K232 and K270, free acidity, and fatty acids composition were evaluated during different periods of incubation (6, 12, and 18 months) at various temperatures (4°C, 25°C, 40°C, and uncontrolled room temperature ranging between 4°C and 40°C) with different packaging (protected and unprotected from sunlight, with and without nitrogen gas bubbling). Based on the physicochemical and biochemical parameters evolution, this study has shown that PPSO stored at 4°C for 18 months preserves the initial quality. However, at 40°C, an intense lipid oxidative process occurred after 6 months of storage. The changes have also affected fatty acids composition, especially rates of linoleic and oleic acids. The shelf-life of oils stored at 25°C and at uncontrolled room temperature can be limited to 6 months. Regarding the impact of light and nitrogen bubbling, sunlight has affected seriously the oxidative stability of oils after 12 months of storage and the bubbling with nitrogen has improved their stability when they have been stored in clear glass bottles. The levels of adulteration detection using fatty acids as markers are relatively high. The detection of oil adulteration can be depicted by fatty acids composition up to 15% of olive and almond oils and up to 20% of rapeseed oil. The iodine value could also be an indicator of the sunflower oil presence in PPSO. Therefore, other minor compounds including sterols and tocopherols should be investigated to depict PPSO adulteration with cheaper oils and to determine lower levels of detection in order to ensure the authenticity of PPSO.

Impedimetric, PEDOT:PSS-Based Organic Electrochemical Sensor for Detection of Histamine for Precision Animal Agriculture

High concentrations of histamine are associated with subacute ruminal acidosis, a common disease in high-producing lactating dairy cows. Therefore, the accurate detection of low histamine levels is a strategy to monitor and diagnose ruminal acidosis in early stages. In this letter, we have developed an impedimetric histamine biosensor based on an organic semiconductor: poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). It can be utilized to detect low concentrations of histamine in a phosphate-buffered saline (PBS) buffer and McDougall's buffer solution (MDBS) with an impedimetric readout technique. With PEDOT:PSS film as the sensing medium, the device displayed a limit of detection of 0.1 μM and an impedance of ∼82 Ω at low frequencies in MDBS. The impedance is correlated with the concentration of histamine: the R2 values in the PBS (pH = 5.5 and pH = 7.4) and the MDBS (pH = 8.4) are 0.95, 0.95, and 0.99, respectively. The sensor is fabricated using large-area solution processing technology, opening the door to low-cost, high-volume commercial sensor production.

Novel dual HIV maintenance therapy with nevirapine plus lamivudine retain viral suppression through 144 weeks-A proof-of-concept study.

The aim of this proof-of-concept study is to test feasibility and efficacy of NVP plus Lamivudine (3TC) as novel simplified HIV maintenance dual therapy (DT) strategy. Patients under combined antiretroviral treatment (cART) with fully suppressed HIV plasma viral load (pVL) >24 months-whereof >6 months on an NVP- containing regimen-were switched to oral NVP plus 3TC for 24 weeks. Patients could then decide whether to continue DT or return to the previous cART. HIV pVL was monitored monthly until week 144. The primary outcome was confirmed viral failure (RNA >100 copies/ml). Low-level detection of HIV-RNA in plasma was compared in each patient with pre-study viral load measurements. Twenty patients were included, switched to DT and all completed week 24. One patient decided thereafter to discontinue study participation for personal reasons. After a total of 144 observation weeks, none of the patients failed. The frequency of low- level HIV-RNA detection was not different from the period before randomization. Our findings are surprising but given the nature of a proof-of-concept study, the results do not support the use of this dual regimen. However, as this dual HIV maintenance strategy was feasible and effective, over a period of 144 weeks, we suggest NVP plus 3TC warrants further evaluation as potential maintenance option in patients tolerating nevirapine. A properly sized multicentre non-inferiority trial is ongoing to further evaluate the value of this DT maintenance strategy.

Highly sensitive, selective and naked-eye detection of bromide and bromate using distance–based paper analytical device

Bromine (Br) can usually be found as a bromide (Br‾) form contained in drinking water. Bromate (BrO3‾) formation often occurred during the ozonation process in the presence of Br‾. BrO3‾ is classified as a potential human carcinogen, so both the Br‾ and BrO3‾ concentrations must be strictly controlled before and after an ozone-based treatment procedure, respectively. This work reports on distance-based paper analytical devices (dPADs) that have been modified with silver hexagonal nanoprisms (AgNPrs) for highly sensitive and selective determination of both Br‾ and BrO3‾. The measurement of BrO3‾ is based upon its conversion to bromine vapor (Br2) when gauged with a paper-based headspace extractor (PAD-HS) that is coupled with dPADs (PAD-HS-dPADs). For Br‾ analysis, O2 plays an important role in the change from Br‾ to Br2 within an acid media. Br2 changes rapidly in water to give us HBrO which is a strong oxidizing agent of AgNPrs. Then, the oxidative reaction of the AgNPrs (pink color) within the presence of Br‾ and BrO3‾ establishes both silver bromide (AgBr) and the silver nanosphere (AgNPs, yellow color), which can then be easily observed as a change of a pink color band to a yellow color band by the naked eye. Quantification of Br‾ and BrO3‾ is then achieved by measuring the length of the yellow color band. Under the optimal conditions, the calibration curve will be linear in the range of 25 μg L−1 to 2 mg L−1, and from 0.5 to 50 μg L−1 for Br‾ and BrO3‾, respectively. The naked-eye detection limits were found to be 10 and 0.5 μg L−1 for Br‾ and BrO3‾, respectively. The proposed dPADs for the Br‾ and BrO3‾ detection exhibited an exceptional sensor performance combined with a low detection limit. They also have the benefits of ease of use, an instrument-free convenience, coupled with portability and a low-cost efficiency. Consequently, our sensing device should be applied to the low-level detection of Br‾ and BrO3‾ in real samples, including drinking water, rice, and flour.

Abstract 4028: Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia

Abstract Introduction: Anemia is a condition characterized by low Hemoglobin levels. Anemia is observed during tumor development through the release of inflammatory cytokines, which will affect iron availability, a key component of red blood cells. In recent years, consequences of anemia of cancer on patients is getting growing recognition from a quality of life perspective, as well as its impact on the treatment scheme and hospitalization duration. Cancer-induced anemia has been identified in 30% of treatment-naive cancers (ECAS study, 2011 - WHO methodology). The proportion of anaemic patients reaches 70% following treatments, such as chemotherapies. Current approaches consider erythropoietin derivatives, formulated iron or transfusions, which does not address underlying chronic inflammation. Moreover, their use is limited as a consequence of toxicities (EPO, formulated iron) or availability (transfusion). Increased expression of Hepcidin, a peptidic hormone regulating the storage of bioavailable iron, has been demonstrated as downstream effector of key inflammatory cytokines activation of ALK2/Smad axis. ALK2 (Activin-like receptor kinase 2) plays a critical role in the Smad signaling pathway and the production haemoglobin, through the regulation Hepcidin production in the liver. As a consequence, the selective inhibition of ALK2 as emerged as a valuable target for the treatment of Hepcidin-driven anemia of cancer to provide an alternative to current treatments. Results: OD52, a potent and selective macrocyclic ALK2 inhibitor identified from Nanocyclix® technology platform, was used as in vitro pharmacological tool to investigate the impact of ALK2 inhibition on Hepcidin expression level in HepG2 cells, as well as in mice hepatocytes. BMP6-induced expression of Hepcidin mRNA was completely inhibited by LDN-193189 to low detectable levels, whereas OD52 normalized its expression. Further validation was performed in mice hepatocytes, where OD52 decreased Hepcidin expression to a lower extend as compared with LDN-193189. These experiments confirmed the importance of selectivity within BMP-kinase receptor family, where ALK3 component represents an undesired off-target. In vivo evaluation of the compound in the acute model of turpentine-induced anemia demonstrated a normalization of haemoglobin level. In contrast, LDN-193189 increased haemoglobin above vehicle group to a level mimicking hemochromatosis, likely due to its ALK3 component. Through further medicinal chemistry optimization, OD66 was identified as suitable in vivo pharmacological tool and was evaluated in a cancer-induced anemia mouse model. The compound demonstrated a normalization of haemoglobin levels to comparable levels with the tumor-free group without displaying toxicity. Conclusion: The identification of potent and selective Nanocyclix® ALK2 inhibitors and their evaluation in relevant in vitro and in vivo models demonstrated a normalization of haemoglobin levels through the modulation of Hepcidin expression. This collaborative work warrants further optimization towards the identification of a preclinical candidate. Citation Format: Pascal Benderitter, Josselin Caradec, Edwige Nicodeme, Anne Bouillot, Sophie Vaulont, Benoit Viollet. Nanocyclix® ALK2 Inhibitors to overcome cancer-induced anemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4028.

Participation in the College of American Pathologists Laboratory Accreditation Program Decreases Variability in B-Lymphoblastic Leukemia and Plasma Cell Myeloma Flow Cytometric Minimal Residual Disease Testing: A Follow-up Survey.

Minimal residual disease (MRD) testing by flow cytometry is ubiquitous in hematolymphoid neoplasm monitoring, especially B-lymphoblastic leukemia (B-ALL), for which it provides predictive information and guides management. Major heterogeneity was identified in 2014. Subsequently, new Flow Cytometry Checklist items required documentation of the sensitivity determination method and required lower level of detection (LLOD) inclusion in final reports. This study assesses Laboratory Accreditation Program (LAP) participation and new checklist items' impact on flow cytometry MRD testing. To survey flow cytometry laboratories about MRD testing for B-ALL and plasma cell myeloma. In particular, enumerate the laboratories performing MRD testing, the proportion performing assays with very low LLODs, and implementation of new checklist items. Supplemental questions were distributed in the 2017-A mailing to 548 flow cytometry laboratories subscribed to the College of American Pathologists FL3 Proficiency Testing Survey (Flow Cytometry-Immunophenotypic Characterization of Leukemia/Lymphoma). The percentage of laboratories performing MRD studies has significantly decreased since 2014. Wide ranges of LLOD and collection event numbers were reported for B-ALL and plasma cell myeloma. Most laboratories determine LLOD by using dilutional studies and include it in final reports; a higher proportion of LAP participants used these practices than nonparticipants. Several MRD testing aspects vary among laboratories receiving FL3 Proficiency Testing materials. After the survey in 2014, new checklist items were implemented. As compared to 2014, fewer laboratories are performing MRD studies. While LLOD remains heterogeneous, a high proportion of LAP subscribers follow the new checklist requirements and, overall, target LLOD recommendations from disease-specific working groups are met.

Validation Using Diverse, Difficult-to-Detect Salmonella Strains and a Dark Chocolate Matrix Highlights the Critical Role of Strain Selection for Evaluation of Simplified, Rapid PCR-Based Methods Offering Next-Day Time to Results

Modifications to pathogen detection kits to accomplish simplified protocols with reduced time to results may impact method performance, particularly when combining shortened enrichment times and simplified enrichment procedures. We used Salmonella detection in dark chocolate as a model to test the impact of different enrichment times (minimum and maximum validated times) and procedures on detection of low levels of difficult-to-detect Salmonella strains, for three PCR kits that were AOAC International Performance Tested Method certified for detection of Salmonella spp. in dark chocolate. Initial inclusivity studies with pure cultures showed that all three kits detected 70 of 70 Salmonella spp. strains at 1 log above the theoretical limit of detection, with some strains yielding later cycle threshold values or having variable detection among technical replicates, indicating reduced assay performance for these strains. Based on these data, we selected a S. enterica subsp. enterica serovar Poona strain as well as three non-subsp. enterica strains to test the ability of the three kits to detect Salmonella in dark chocolate inoculated at low levels (0.06 to 1.18 most probable number per 25 g). With primary enrichment in skim milk at 35°C, detection frequency for all assays did not significantly differ from the reference method for both the minimum and maximum validated enrichment times. However, a pilot study that used primary enrichment in buffered peptone water at 42°C yielded significantly fewer positive samples (13 of 80) than were obtained with the U.S. Food and Drug Administration Bacteriological Analytical Manual method using enrichment in skim milk at 35°C (40 of 80 positive samples); strains representing subsp. houtenae and salamae were detected in significantly fewer chocolate samples than enrichment with skim milk. Our data indicate that continued efforts to simplify rapid pathogen detection kits may reduce kit performance in a way that can only be detected with stringent evaluation protocols that are designed to identify kit failure modes.

On-Line Gaseous Formaldehyde Detection Based on a Closed-Microfluidic-Circuit Analysis

This paper describes a compact microfluidic analytical device in a closed-circuit developed for the detection of low airborne formaldehyde levels. The detection is based on the passive trapping of gaseous formaldehyde through a microporous tube into the acetylacetone solution, the derivative reaction of formaldehyde with acetylacetone to form 3,5-Diacetyl-1,4-dihydrolutidine (DDL) and the detection of DDL by fluorescence. The recirculation mode of the analytical device means that the concentration measurement is carried out by quantification of the signal increase in the liquid mixture over time, the instantaneous signal increase rate being proportional to the surrounding gaseous formaldehyde concentration. The response of this novel microdevice is found to be linear in the range 0–278 µg m−3. The reagent volume needed is flexible and depends on the desired analytical resolution time and the concentration of gaseous formaldehyde in the environment. Indeed, if either the gaseous concentration of formaldehyde is high or the reagent volume is low, the fluorescence signal of this recirculating liquid solution will increase very rapidly. Consequently, the sensitivity simultaneously depends on both the reagent volume and the temporal resolution. Considering a reagent volume of 6 mL, the hourly and daily detection limits are 2 and 0.08 µg m−3, respectively, while the reagent autonomy is more than 4 days the airborne formaldehyde concentration does not exceed 50 µg m−3 as it is usually the case in domestic or public indoor environments.

Quantification of solid-state impurity with powder X-ray diffraction using laboratory source

The application of powder X-ray diffraction (PXRD) for the detection and quantification of low levels of a solid-state chemical impurity, BrettPhos oxide, in an active pharmaceutical ingredient is discussed. It is demonstrated that with appropriate methodology and experimentation, the impurity levels of as low as 0.07% w/w could be detected reliably and limit of quantification of 0.10% w/w could be achieved by PXRD, using a laboratory X-ray source. Method development, validation, and benchmarking using conventional high-performance liquid chromatography are presented in the manuscript highlighting the robustness and reproducibility of such measurements.

Novel 2‐hydroxynaphthalene‐based fluorescent turn‐on sensor for highly sensitive and selective detection of Al3+ and its application in imaging in vitro and in vivo

Excessive aluminum exposure in the human body has been held responsible for multiple adverse effects, and existing data underscore the significance of aluminum detection in environmental and biological systems. Developing high‐performance Al3+ fluorescent chemosensors can revolutionize our understanding of the physiological and pathological processes of Al3+ ions. Herein, we reported a highly sensitive and selective Schiff base fluorescence sensor, bis‐NAPPD (1,1'‐((1E,1'E)‐(pyridine‐2,3‐diylbis(azanylylidene))bis(methanylylidene))bis(naphthalen‐2‐ol)), which can recognize Al3+ ions and exhibits a remarkable turn‐on dual emission response (by ~23 fold) with a low nanomolar level detection limit (1.67 × 10−8 M) in methanol. Furthermore, the binding behavior and the turn‐on fluorescence probing mechanism of bis‐NAPPD were illustrated in detail by UV–vis titration, 1H NMR, and ESI‐MS spectroscopy as well as density functional theory calculations. Notably, bis‐NAPPD showed great potential for tracing Al3+ distribution in cells and living zebrafish larvae, and can also be applied in the fluorimetric detection of aluminum in sucralfate tablets with good precision and satisfactory accuracy, which may represent a promising Al3+ probe in bioimaging and biomedical applications.