Established Assay Research Articles

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

A-317 Development of a patient-focused volumetric absorptive microsampling strategy to assess urinary amino acid excretion

Abstract Background Biochemical determinations of urinary amino acid excretion patterns can diagnose specific inborn errors of metabolism (IEM). Clinical management of these genetic metabolic patients may require frequent urine amino acid tests. A simplified process for transporting home collected urine specimens would benefit the IEM community. Volumetric absorptive microsampling (VAMS) is an emergent alternative sampling technique for the collection of dried biological specimens. We describe the development and preliminary validation results of a VAMS strategy for the analysis of creatinine and amino acids (N=22) from microsampled dried urine. Methods Urine creatinine and amino acids were respectively measured in a clinical laboratory setting using established assays on a CobasPro (Roche) and a MassTrak ultra-performance liquid chromatography (UPLC) amino acid analysis (AAA) solution (Waters). Two 30 µL VAMS tips from a Mitra device (Neoteryx) were used to sample each random urine specimen. An ultrasonication-based solvent extraction procedure was optimized to extract the amino acids and creatinine from the VAMS tips. Solvent extracts were subsequently dried by centrifugal evaporation. Extract from one tip was re-dissolved in water and tested for creatinine using an enzymatic method. The other extract was re-dissolved in 0.02 N sodium hydroxide and tested according to the MassTrak AAA procedure, which deploys pre-column AccQTag derivatization with reversed-phase UPLC on a C18 column (2.1 × 150 mm; 1.7 µm) and UV detection at 260 nm. Accuracy, within-batch precision and short-term stability of the optimized VAMS protocol were determined. Results Extraction solvent with a 60:40 (v/v) water:methanol mixture provided the highest average amino acid recovery. This extraction solvent produced higher average amino acid recoveries at pH 11 (89±13%), than pH 3 (82±10%) or pH 7 (72±11%). Creatinine measured from the VAMS tips (N=44) had a consistent negative bias of 20% relative to neat urine (R=0.9905, slope=0.802, intercept=0.02). The creatinine corrected urine amino acid results from the Mitra device were corrected for this observed concentration bias. The accuracy of the amino acid results derived from the VAMS tip vs neat urine was 104 ± 14%. The average within-batch precision (CV%) of the amino acids was 9.8% (range=6.7% (cystine) to 17.6% (arginine)) and 6.2% for creatinine. The average 1 week storage stability at ambient temperature was 91% (range=73% (ornithine) to 107% (glycine)) for the amino acids and 107% for creatinine. The devised VAMS strategy was applied to the analysis of microsampled dried urine from a patient with cystinuria (OMIM no. 220100). Marked elevations in arginine, cystine, lysine and ornithine were respectively observed and are consistent with the biochemical diagnosis of the patient. Conclusions Creatinine corrected amino acid results from the devised VAMS strategy exhibit good precision and accuracy when compared to tradition analyses of neat urine. The potential use of microsampled dried urine for the diagnosis and monitoring of selected IEM has been demonstrated. The analytical and clinical performance of this VAMS protocol must be further validated, including the use of microsampled dried urine from home specimen collections.

Analyzing high-throughput assay data to advance the rapid screening of environmental chemicals for human reproductive toxicity.

While high-throughput (HTP) assays have been proposed as platforms to rapidly assess reproductive toxicity, there is currently a lack of established assays that specifically address germline development/function and fertility. We assessed the applicability domains of yeast (S. cerevisiae) and nematode (C. elegans) HTP assays in toxicity screening of 124 environmental chemicals, determining their agreement in identifying toxicants and their concordance with reproductive toxicity in vivo. We integrated data generated in the two models and compared results using a streamlined, semi-automated benchmark dose (BMD) modeling approach. We then extracted and modeled relevant mammalian in vivo data available for the matching chemicals included in the Toxicological Reference Database (ToxRefDB). We ranked potencies of common compounds using the BMD and evaluated correlation between the datasets using Pearson and Spearman correlation coefficients. We found moderate to good correlation across the three data sets, with r = 0.48 (95 % CI: 0.28-1.00, p<0.001) and rs = 0.40 (p=0.002) for the parametric and rank order correlations between the HTP BMDs; r = 0.95 (95 % CI: 0.76-1.00, p=0.0005) and rs = 0.89 (p=0.006) between the yeast assay and ToxRefDB BMDs; and r = 0.81 (95 % CI: 0.28-1.00, p=0.014) and rs = 0.75 (p=0.033) between the worm assay and ToxRefDB BMDs. Our findings underscore the potential of these HTP assays to identify environmental chemicals that exhibit reproductive toxicity. Integrating these HTP datasets into mammalian in vivo prediction models using machine learning methods could further enhance their predictive value in future rapid screening efforts.

Comparative evaluation of Point of Care assay with ELISA techniques for quantifying serum concentrations of ustekinumab in inflammatory bowel disease patients

Objectives: to evaluate the analytical performance and clinical utility of the POC-AFIAS assay in comparison with two ELISA established assays for quantifying serum concentrations of ustekinumab.Methods: A prospective study was conducted. Consecutive serum samples from adult patients undergoing treatment with ustekinumab were collected. Three analytical techniques were compared for the quantification of ustekinumab serum concentrations: the AFIAS-10® POC assay (POC-AFIAS), the Promonitor® ELISA assay (ELISA-PRO), and the ELISA Ridascreen® assay (ELISA-RDSC).Ustekinumab concentrations were evaluated within three therapeutic ranges: <1 µg/mL, 1-4.5 µg/mL, and >4.5 µg/mL. Statistical analysis included Pearson correlation, intra-class correlation coefficient, and Bland-Altman analysis.Results: A total of 104 patients were included in the study. The median ustekinumab concentrations measured were 5.22 µg/mL (POC-AFIAS), 3.99 µg/mL (ELISA-PRO), and 4.50 µg/mL (ELISA-RDSC). Strong correlations were observed between techniques (POC-AFIAS and ELISA-PRO: r=0.921, POC-AFIAS and ELISA-RDSC: r=0.940, ELISA-PRO and ELISA-RDSC: r=0.976). The Bland-Altman analysis revealed a bias difference of 1.81 µg/mL between POC-AFIAS and ELISA-PRO, and 1.27 µg/mL between POC-AFIAS and ELISA-RDSC. Agreement rates varied by therapeutic range, with the highest agreement observed within the therapeutic range (97.3%) and lower agreement for supra-therapeutic concentrations (74.6%).Conclusion: This study demonstrated that the POC-AFIAS assay provides comparable results to established ELISA techniques for quantifying serum concentrations of ustekinumab, particularly within the therapeutic range. The findings suggest that the POC-AFIAS assay offers a rapid and effective tool for managing ustekinumab therapy in clinical practice.

A CRISPR mediated point‐of‐care assay for the detection of mucosal calprotectin in an animal model of ulcerative colitis

AbstractInflammatory bowel disease (IBD) is a chronic disorder associated with inflammation in the gastrointestinal tract, leading to a range of debilitating symptoms. Fecal calprotectin is an established biomarker for ulcerative colitis (UC), one of the main IBD diseases, which provides indications of the presence and severity of inflammation in the digestive tract. Enzyme‐Linked Immunosorbent Assay (ELISA) as a gold standard approach for fecal calprotectin detection is time‐consuming and impractical in point‐of‐care settings. Moreover, obtaining fecal samples from patients is challenging and inhibits longitudinal monitoring. To address these specific problems, we have developed a novel approach for detecting calprotectin which leverages clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology. We successfully developed a portable tube‐based CRISPR/Cas assay for point‐of‐care testing of calprotectin. This assay showed a detection range from 1 to 10,000 ng/ml (over 4 log units), using both fluorescent and colorimetric analytical techniques. The established assay was further validated through measurements in mucosal samples obtained in an anesthetised preclinical rodent model of UC, with 2–3 times higher calprotectin concentration detected in UC rat samples compared to that of healthy control animals. This point‐of‐care test may provide a rapid, precise, and user‐friendly approach for the diagnosis and monitoring of IBD through mucosal sample testing.

Evaluation of the feasibility and efficacy of point-of-care antibody tests for biomarker guided management of COVID-19.

Biomarker guided therapy could improve management of COVID-19 inpatients. Although some results indicate that antibody tests are prognostic, little is known about patient management using point-of-care (POC) antibody tests. COVID-19 inpatients were recruited to evaluate 2 POC tests: LumiraDX and RightSign. Ease of use data was collected. Blood was also collected for centralized testing using established antibody assays (GenScript cPass). A nested case-control study assessed if POC tests conducted on stored specimens were predictive of time to sustained recovery, mortality, and a composite safety outcome. While both POC tests exhibited moderate agreement with the GenScript assay (both agreeing with 89% of antibody determinations), they were significantly different from the GenScript assay. Treating the GenScript assay as the gold standard, the LumiraDX assay had 99.5% sensitivity and 58.1% specificity while the RightSign assay had 89.5% sensitivity and 84.0% specificity. The LumiraDX assay frequently gave indeterminant results. Both tests were significantly associated with clinical outcomes. Although both POC tests deviated moderately from the GenScript assay, they predicted outcomes of interest. The RightSign test was easier to use and was more likely to detect those lacking antibody compared to the LumiraDX test treating GenScript as the gold standard.

Clinical performance of a rapid RT-PCR assay using STANDARD™ M10 SARS-CoV-2 between July 2022 and January 2023 in Korea

Rapid detection of SARS-CoV-2 is essential for clinical management in the emergency department during the COVID-19 pandemic. We evaluated the clinical performance of the recently developed cartridge-based rapid RT-PCR assay (STANDARD M10 SARS-CoV-2) in patients visiting the emergency department from July 2022 to January 2023, which was when the Omicron BA.5 sublineage was predominant in Korea. A total of 534 specimens were subjected to the STANDARD M10 and standard RT-PCR (Allplex SARS-CoV-2) assays. The overall, positive, and negative percent agreements between these two assays were 99.6%, 100%, and 99.6%, respectively. The results showed that compared with the established RT-PCR assay, the STANDARD M10 SARS-CoV-2 assay is a reliable and useful tool for SARS-CoV-2 detection during the study period. The new rapid RT-PCR will expand the diversity in rapid diagnostics and can help resolve the global imbalance associated with the supply of diagnostic resources.

Pharmacokinetic profiles of methylcobalamin in rats after multiple administration routes by a simple LC-MS/MS assay with a small volume of plasma

Methylcobalamin (MBL) is a vitamin B12 coenzyme and is effective for treating peripheral neuropathies. Little is known about pharmacokinetics (PK) of MBL in animals, we have developed a simple assay for MBL by using only 0.01 mL of plasma for PK of MBL in rats. Under minimal light exposure (<5 lx), MBL was extracted by a simple protein precipitation using methanol and detected by liquid chromatography with tandem mass spectrometry. MBL in rat plasma at 20–10,000 ng/mL was quantified using only 0.01 mL of plasma. Relative error and relative standard deviation met the acceptance criteria in reproducibility assessments, indicating the robustness of the assay. PK of MBL was evaluated after intravenous, intramuscular, and subcutaneous administration. PK of MBL was dose proportional at 5–20 mg/kg in both intramuscular and subcutaneous administrations. Bioavailability after the two dosing routes was complete (ca. 100 %). The incurred sample reanalysis also supported that the assay is robust. The established assay was successfully applied to PK studies in rats to find that MBL showed high bioavailability after intramuscular and subcutaneous administrations.

Multiplex qPCR development for the simultaneous and rapid detection of largemouth bass virus and infectious spleen and kidney necrosis virus in aquaculture

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R2) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3 %. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early.

Integration of highly sensitive large-area graphene-based biosensors in an automated sensing platform

Graphene-based biosensors, featuring exceptional electronic, mechanical, and surface properties, have emerged as frontrunners in advanced sensing technologies. However, to achieve widespread industrial adoption, advancements in the fabrication and integration of large-area graphene devices are essential. Critical parameters such as enhanced sensitivity, scalable production methods, economic viability, integration capabilities, and consistent uniformity must be meticulously addressed. In this work, we demonstrate that our ultra-clean, chemical wet transfer protocol of large-area graphene enables a scalable, smooth integration of graphene into an established assay platform for transporter protein drug discovery. Furthermore, we demonstrate sensitive detection of electrolytic buffers, varying pH, bovine serum albumin (BSA) and single-stranded DNA (ssDNA) adsorption, using our large-area graphene solution-gated field-effect transistor (SGFET) sensors, thereby proving their robust and reliable performance. The sensors’ biocompatibility and ion sensitivity, down to the picomolar range, substantiate their suitability for the investigation of electroactive transport in ion channels and membrane transporters.

Assessment of in vivo chemical mutagenesis by long-read sequencing.

Evaluating the mutagenic properties of chemicals is crucial for understanding their potential cancer risks. Recent Illumina-based error-corrected sequencing techniques have enabled the direct detection of mutations induced de novo by mutagens. However, as the Illumina platform lacks intrinsic error-correction capabilities, complex library preparations and bioinformatic processes are necessary to identify these rare mutations. In this study, we evaluated whether long-read PacBio-based HiFi sequencing (HiFi seq), which has integrated error-correction, can detect de novo mutations induced by mutagens in C57BL/6 mouse tissues. Using HiFi seq, dose-dependent increases in mutation frequencies were found in tissues from mice exposed to 7,12-dimethylbenz[a]anthracene, procarbazine, and N-propyl-N-nitrosourea. Furthermore, the mutational signatures derived from these exposures were consistent with those previously reported for these mutagens. This study demonstrates that HiFi seq can complement established mutation detection assays to facilitate the identification of hazardous compounds.

Small molecules targeting GRP78 mitigate anti-GRP78 autoantibody–mediated tissue factor procoagulant activity in cultured endothelial cells

BackgroundThe 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor (TF) procoagulant activity (PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. ObjectivesDetermine the effects of the engagement of the anti-GRP78 autoantibody to csGRP78 on ECs and the underlying mechanisms that impact TF PCA. MethodsImmunofluorescent staining was used to determine the presence of csGRP78 in tumor necrosis factor α–treated ECs. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay (Abcam) was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind GRP78 were identified using artificial intelligence. Enzyme-linked immunosorbent assays were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. ResultsIn tumor necrosis factor α–treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by endoplasmic reticulum stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin attenuated anti-GRP78 autoantibody–induced TF PCA. We identified B07∗, as a GRP78 binder that diminished anti-GRP78 autoantibody–induced TF PCA on ECs. ConclusionThese findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification.

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7μM; Ki = 37.1 ± 0.85μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

Analysis of tumor abnormal protein expression and epidermal growth factor receptor mutation status in non-small cell lung cancer

BackgroundThe level of tumor abnormal protein (TAP) level has a significant impact on tumor growth, recurrence, and metastasis. Previous studies have highlighted the influence of the mutations in exons 19 and 21 of the epidermal growth factor receptor (EGFR), particularly the sensitivity displayed by tumor cells to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. Our study is centered on exploring the clinical relevance of TAP and EGFR mutations in patients with non-small cell lung cancer (NSCLC).Material and methodsIn this study, tissue samples were collected from a total of 176 patients diagnosed with non-small cell lung cancer (NSCLC). Real-time PCR technology was utilized to detect mutations within exons 19 and 21 of the epidermal growth factor receptor (EGFR) gene in these samples. This approach enables precise identification of EGFR mutations associated with NSCLC. Furthermore, the study investigated the impact of various tumor markers, including tumor abnormal protein (TAP) and carcinoembryonic antigen (CEA), on EGFR mutation status. Established assays were employed to evaluate TAP and CEA levels, aiming to ascertain their potential correlation with EGFR mutation in NSCLC patients.ResultsEGFR exhibited mutation rates of 23.86% and 12.50% in exons 19 and 21, respectively. EGFR mutations were more prevalent in younger women (< 60 years old) and in cases with pleural invasion, vessel invasion, CEA > 6.5 ng/mL, and TAP > 228 µm2 for both genders. Increased TAP levels independently predicted EGFR mutations (P = 0.001 for males; P = 0.000 for females). An area under the curve (AUC) of 0.833 indecated EGFR mutation prediction with sensitivity and specificity of 79.7% and 87.0%, respectively. For females, the sensitivity increased to 89.7% and specificity increased to 93.8%.ConclusionsTAP effectively predicts EGFR mutations in NSCLC patients with moderate accuracy, particularly benefiting diagnosis in females with high sensitivity and specificity. Integrating TAP assessment into EGFR mutation testing can significantly enhance diagnostic precision, especially in female NSCLC cases.

Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Comparison of soil eDNA to camera traps for assessing mammal and bird community composition and site use.

Species detections often vary depending on the survey methods employed. Some species may go undetected when using only one approach in community-level inventory and monitoring programs, which has management and conservation implications. We conducted a comparative study of terrestrial mammal and bird detections in the spring and summer of 2021 by placing camera traps at 30 locations across a large military installation in northern Michigan, USA and testing replicate soil samples from these sites for environmental DNA (eDNA) using an established vertebrate metabarcoding assay. We detected a total of 48 taxa from both survey methods: 26 mammalian taxa (excluding humans, 24 to species and two to genus) and 22 avian taxa (21 to species and one to genus). We detected a relatively even distribution of mammalian taxa on cameras (17) and via eDNA analysis (15), with seven taxa detected from both methods. Most medium-to-large carnivores were detected only on cameras, whereas semi-fossorial small mammals were detected only via eDNA analysis. We detected higher bird diversity with camera traps (18 taxa) compared to eDNA analysis (eight taxa; four taxa were detected with both methods), but cameras alone were most effective at detecting smaller birds that frequently occupy arboreal environments. We also used Bayesian spatial occupancy models for two widely distributed game species (white-tailed deer, Odocoileus virginianus, and ruffed grouse, Bonasa umbellus) that were moderately detected with both survey methods and found species-specific site use (occupancy) estimates were similar between cameras and eDNA analysis. Concordant with similar studies, our findings suggest that a combination of camera trap and eDNA surveys could be most useful for assessing the composition of terrestrial mammal communities. Camera traps may be most efficient for assessing bird diversity but can be complemented with eDNA analysis, particularly for species that spend considerable time on the ground.

Quantitatively measuring the cytotoxicity of viscous hydrogels with direct cell sampling in a micro scale format “MicroDrop” and its comparison to CCK8

Tissue engineering holds promise for developing therapeutic applications using viscous materials e.g. hydrogels. However, assessing the cytotoxicity of such materials with conventional assays can be challenging due to non-specific interactions. To address this, we optimized a live/dead staining method for quantitative evaluation and compared it with the conventional CCK8 assay. Our MicroDrop method involved seeding droplets containing 5000 cells in 10 µl medium on 12-well plates. After allowing them to adhere for 4 h, various viscous samples were applied to the cells and measurements were conducted using a fluorescence microscope immediately and at daily intervals up to 72 h. A sodium dodecyl sulfate (SDS) dilution series compared the MicroDrop with the CCK8 assay. The findings revealed a cell-type specific pattern for 10 mg/ml hyaluronic acid (HA), wherein MC3T3-E1 cells maintained 95% viability until 72 h, while L929 cells experienced a gradual decline to 17%. 2 mg/ml HA exhibited consistent viability above 90% across all time points and cell lines. Similarly, fibrin demonstrated 90% viability across dilutions and time points, except for undiluted samples showing a decrease from 85% to 20%. Gelatin-methacrylol sustained viability above 70% across all time points at both 5% and 10% concentrations. The comparison of the SDS dilution series between viability (MicroDrop) and metabolic activity (CCK8) assay showed a correlation coefficient of 0.95. The study validates the feasibility of the established assay, providing researchers with an efficient tool for assessing cytotoxicity in viscous materials. Additionally, it holds the potential to yield more precise data on well-known hydrogels.Graphical

A method of identifying the high-risk mutations of sudden cardiac death at KCNQ1 and KCNH2 genes

Sudden Cardiac Death (SCD) often shows negative anatomy results after a systemic autopsy and the gene mutations of potassium channel play a key role in the etiology of SCD. We established a feasible system to detect SCD-related mutations and investigated the mutations at KCNQ1 and KCNH2 genes in the Chinese population. We established a mutation detection system combined with multiplex PCR, SNaPshot technique, and capillary electrophoresis. We genotyped 101 putative mutations at KCNQ1 and KCNH2 genes in 60 SCD of negative anatomy and 50 controls using the established assay and compared Odd Ratio (OR). Four coding variants were identified in the KCNQ1 gene: S546S, I145I, P448R, and G643S. The mutations of I145I and S546S did not differ significantly in the SCD compared with controls. 21 SCD individuals (35 %) and 1 control individual (2 %) showed a genotype of C/G at P448R (OR = 17.5, 95 % CI [2.40–127.82]). 24 SCD individuals (40 %) and 1 control individual (2 %) showed a genotype of C/G at G643S (OR = 20.0, 95 % CI [2.75–145.25]). We established a robust assay for rapid screening the putative SCD-related mutations in KCNQ1 and KCNH2 genes. The new assay in our study is easily amenable to the majority of laboratories without the need for new specialized equipment. Our method will meet the increasing requirement of mutation screening for SCD in regular DNA laboratories and will help screen mutations in those dead of SCD and their relatives.

A multi-assay liquid biopsy approach to improve detection of bone and soft tissue sarcomas.

10019 Background: Bone and soft tissue sarcomas are among the most common extra-cranial solid tumors of childhood, yet advances in liquid biopsies lag behind what has been achieved across adult cancers. Solid tumors that lack recurrent genetic mutations or translocations, such as bone and soft tissue sarcomas, are more difficult to detect in circulation using established assays, such as ddPCR. We have developed a pipeline to analyze cell-free DNA (cfDNA) using two molecular profiling techniques that we hypothesized would improve detection of circulating tumor DNA at diagnosis for patients with bone and soft tissue sarcomas. Methods: cfDNA was isolated from plasma of patients with osteosarcoma (OS) (n = 36) and fusion-negative rhabdomyosarcoma (FN-RMS) (n = 7) at multiple timepoints. Methods for detecting tumor DNA in cfDNA included ultra-low coverage whole genome sequencing (ULC-WGS, 0.5x) and ultra-deep targeted sequencing (UDP, ~1000x) using a panel of 11 and 22 tumor-associated genes for OS and FN-RMS, respectively. ULC-WGS data underwent ichorCNA analysis to define percent tumor content of each sample. UDP analysis identified somatic variants through variant callers, Mutect2, Lancet, strelka, and DELLY2, with ClinVar to annotate and define tumor-specific variants in each sample. Combining ULC-WGS and UDP data, an integrated tumor score was developed and assessed in diagnostic samples. Results: 136 OS and 30 FN-RMS cfDNA samples were collected during the patients’ disease courses. Circulating tumor material was detected in 18/23 (78%) OS and7/7 (100%) FN-RMS samples at diagnosis. The most frequent copy number aberrations were seen in the region of chromosome 8q containing the cMYC gene. cMYC amplification was found in 11/23 (48%) of diagnostic OS samples and 2/7 (29%) of diagnostic FN-RMS samples. UDP analysis identified variants in 17/23 (73%) and 7/7 (100%) of diagnostic OS and FN-RMS samples respectively. The most commonly detected variants included TP53, BRCA2, MET, and BRAF. Of the 23 diagnostic OS samples, four had TP53 translocations. The integrated tumor score detected the presence of tumor associated material within diagnostic samples with a sensitivity of 25/30 (83%), compared to the sensitivity of ULC-WGS or UDP alone, which was 15/30 (50%) and 24/30 (80%) respectively. Conclusions: A multi-assay liquid biopsy has promise to improve disease detection and monitoring for patients with solid tumors that lack recurrent driver mutations. We plan to integrate additional molecular profiling technologies to improve the sensitivity and specificity of our cfDNA assays and aim to adapt our pipeline across additional cancers.

Fully automated viability and toxicity screening—A reliable all‐in‐one attempt

AbstractBackgroundThe CRISPR/Cas9 technology is nowadays a common tool for genome editing to achieve new insights into, for example, diagnostics and therapeutics in cancer and genetic disorders. Cell proliferation and anticancer drug response studies are widely used to evaluate the impact of editing. However, these assays are often time‐consuming, expensive, and reproducibility is an issue. To overcome this, we developed a fast and cheap assay that combines a fully automated multispectral fluorescence microscopy platform with a nuclei staining and open‐source software analysis.MethodsHere, we generated different LEDGF/p75 model cell lines to validate the effect on proliferation and chemosensitivity. Therefore, a fast protocol for an optimized all‐in‐one attempt for cytotoxicity screenings and proliferation analysis of adherent cells in a 96‐well plate format was established using differential staining with two fluorescent dyes (Hoechst 33342 and propidium iodide) for live/dead cell discrimination. Subsequently, an automated cell nuclei count and analysis were performed using bioimage informatics.ResultsWith the new established assay technology, up to 50,000 cells/well can be detected and analyzed in a 96‐well plate, resulting in a fast and accurate verification of viability and proliferation with consistency of 98% compared to manual counting. Our screening revealed that LEDGF depletion using CRISPR/Cas9 showed a diminished proliferation and chemosensitivity independent of cell line origin. Moreover, LEDGF depletion caused a significant increase in 𝛾H2AX foci, indicating a substantial increase in DNA double strand breaks. LEDGF/p75 overexpression enhanced proliferation and chemoresistance underlining the role of LEDGF in DNA damage response.ConclusionIndependent of cancer cell type, LEDGF/p75 is a central player in DNA damage repair and is implicated in chemoresistance. Moreover, our automated fluorescence biosensor technology allowed fast and reliable data acquisition without any fixation or additional washing steps. Additionally, data analysis was implemented using the modular open‐source software that can be adapted as needed.