97,454 results on '"Indicators and Reagents"'
Search Results
2. Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format
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Chen, Yan, Gin, Jennifer W, Wang, Ying, de Raad, Markus, Tan, Stephen, Hillson, Nathan J, Northen, Trent R, Adams, Paul D, and Petzold, Christopher J
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Biochemistry and Cell Biology ,Biological Sciences ,Industrial Biotechnology ,Vaccine Related ,Biotechnology ,Acetone ,Proteomics ,Proteins ,Indicators and Reagents ,General Science & Technology - Abstract
Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5-2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.
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- 2023
3. Ferrobotic swarms enable accessible and adaptable automated viral testing
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Lin, Haisong, Yu, Wenzhuo, A. Sabet, Kiarash, Bogumil, Michael, Zhao, Yichao, Hambalek, Jacob, Lin, Shuyu, Chandrasekaran, Sukantha, Garner, Omai, Di Carlo, Dino, and Emaminejad, Sam
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Prevention ,Biotechnology ,Infectious Diseases ,Biodefense ,Bioengineering ,Emerging Infectious Diseases ,Vaccine Related ,Infection ,Humans ,COVID-19 ,COVID-19 Testing ,Molecular Diagnostic Techniques ,Nucleic Acid Amplification Techniques ,Pandemics ,RNA ,Viral ,SARS-CoV-2 ,Sensitivity and Specificity ,Algorithms ,Automation ,Magnets ,Robotics ,Indicators and Reagents ,General Science & Technology - Abstract
Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.
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- 2022
4. Improved ELISA for linoleate-derived diols in human plasma utilizing a polyHRP-based secondary tracer
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Singh, Nalin, Li, Dongyang, McReynolds, Cindy B, Morisseau, Christophe, and Hammock, Bruce D
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Analytical Chemistry ,Chemical Sciences ,Good Health and Well Being ,Chromatography ,Liquid ,Enzyme-Linked Immunosorbent Assay ,Humans ,Indicators and Reagents ,Linoleic Acid ,Tandem Mass Spectrometry ,Other Chemical Sciences ,Analytical chemistry ,Chemical engineering - Abstract
Dihydroxyoctadecenoic acids (DiHOMEs) are cytochrome P450 pathway-derived metabolites of linoleic acid, a highly abundant dietary fatty acid. They serve thermogenic functions at low concentrations but, at high concentrations, are involved in proinflammatory and deleterious outcomes in a wide range of pathologies. Hence, the development of a reliable analytical method is critical to elucidate their potential as biomarkers of health, and enzyme-linked immunoassay (ELISA)-based approaches offer unique benefits as alternatives to traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Accordingly, an earlier ELISA for DiHOMEs was dramatically improved employing new secondary tracers and geared towards use in human plasma, a universal matrix in biomedical applications, as well as urine. Three ELISA formats, two utilizing polyHRP-based secondary labels for signal amplification, were compared. The best format involved a biotinylated detection antibody and a polyHRP-conjugated streptavidin tracer. Assay detectability was enhanced 20-fold, relative to the original immunoassay, and performance assessments validated precision, selectivity, and robustness. Fast and easy extraction-clean up steps yielded high analytical recovery and permitted the assay to operate in moderate concentrations (up to 20%) of plasma, expanding its practical relevance. Finally, the ELISA was applied towards detection of DiHOMEs in clinical samples and authenticated with complementary LC-MS/MS analysis. Hence, the method provides a valuable analytical tool to investigate the diverse and extensive roles of DiHOMEs in regulatory biology.
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- 2022
5. Clot Waveform Analysis for Hemostatic Abnormalities.
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Hideo Wada, Katsuya Shiraki, Takeshi Matsumoto, Hideto Shimpo, and Motomu Shimaoka
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WAVE analysis ,PARTIAL thromboplastin time ,BLOOD coagulation factor IX ,THROMBOPOIETIN receptors ,THROMBIN time ,PROTHROMBIN time - Abstract
Clot waveform analysis (CWA) observes changes in transparency in a plasma sample based on clotting tests such as activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Evidence indicates that not only an abnormal waveform but also peak times and heights in derivative curves of CWA are useful for the evaluation of hemostatic abnormalities. Modified CWA, including the PT with APTT reagent, dilute PT (small amount of tissue factor [TF]-induced clotting factor IX [FIX] activation; sTF/FIXa), and dilute TT, has been proposed to evaluate physiological or pathological hemostasis. We review routine and modified CWA and their clinical applications. In CWA-sTF/FIXa, elevated peak heights indicate hypercoagulability in patients with cancer or thrombosis, whereas prolonged peak times indicate hypocoagulability in several conditions, including clotting factor deficiency and thrombocytopenia. CWA-dilute TT reflects the thrombin burst, whereas clot-fibrinolysis waveform analysis reflects both hemostasis and fibrinolysis. The relevance and usefulness of CWA-APTT and modified CWA should be further investigated in various diseases. [ABSTRACT FROM AUTHOR]
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- 2023
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6. The design of a quality improvement dashboard for monitoring spinal cord and column injuries
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Zahra Azadmanjir, Mohsen Sadeghi-Naini, Mohammad Dashtkoohi, Maziar Moradi-Lakeh, Jalil Arabkheradmand, James S. Harrop, and Vafa Rahimi-Movaghar
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Indicators and reagents ,Quality of health care ,Spinal cord injuries ,Disease registries ,Dashboard ,Computer applications to medicine. Medical informatics ,R858-859.7 - Abstract
Background: Interactive dashboards are a powerful tool for dynamic visualization and monitoring of patient performance and serve as a useful to for optimal decision-making. The National Spinal Column and Cord Injury Registry of Iran (NSCIR-IR) was designed to efficiently display and broadcast important patient care data. This has been achieved through an electronic dashboard display (graph and visual displays), rather than traditional static paper reports (text). Objectives: The objective of this study was to design and develop an electronic visual dashboard as a display system to monitor the quality of care in the NSCIR-IR collaborating centers. Methods: The indicators chosen were 20 pre-hospital and in-hospital quality of care (QoC) assessment tool indicators. A structured query was created from the NSCIR-IR system database to create the dashboard database. The Microsoft Power BI software was used. After data cleaning, filtering of erroneous records, and modeling, visual displays were designed and evaluated. Results: The dashboard reported on quality of care (QoC) for 2,745 patients registered in NSCIR-IR. 17% of registered cases had at least one data error in the quality of care indicators. These errors were automatically filtered by the system. The two most prominent weaknesses in (QoC indicators were delay in patient transfer by EMS (Mean and SD were 9.54 ± 13.8 h) and timing of surgical spinal cord decompression (114.5 ± 45.3 h). Conclusions: Electronic dashboards provide efficient and concise data summaries “at a glance”. However, their value and accuracy are dependent on the entered data quality. Identifying data source errors and correcting them continuously led to improved quality of data.
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- 2024
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7. Advanced Strategies for Proton-Transfer Reactions Coupled with Parallel Ion Parking on a 21 T FT-ICR MS for Intact Protein Analysis
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Weisbrod, Chad R, Anderson, Lissa C, Hendrickson, Christopher L, Schaffer, Leah V, Shortreed, Michael R, Smith, Lloyd M, Shabanowitz, Jeffrey, and Hunt, Donald F
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Analytical Chemistry ,Chemical Sciences ,Physical Chemistry ,Indicators and Reagents ,Ions ,Proteins ,Protons ,Tandem Mass Spectrometry ,Other Chemical Sciences ,Medical biochemistry and metabolomics ,Analytical chemistry ,Chemical engineering - Abstract
Proton-transfer reactions (PTRs) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to "concentrate", in the gas phase, nearly all the signals from an intact protein charge state envelope into a single charge state, improving the signal-to-noise ratio (S/N) by 10× or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides a tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call "PTR-MS1-PIP", generates a pseudo-MS1 spectrum derived from ions that are exposed to the PTR reagent and PIP waveforms but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in the spectral S/N of intact proteins. This permits the observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the PTR rate without compromise to the parking efficiency. We demonstrate that this process, called "rapid park", can improve reaction rates by 5-10× and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS1 and rapid park provides a 10-fold reduction in ion injection time, improving the rate of tandem MS sequencing.
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- 2021
8. Evaluation of multi-color genetically encoded Ca2+ indicators in filamentous fungi
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Kim, Hye-Seon, Kim, Jung-Eun, Hwangbo, Aram, Akerboom, Jasper, Looger, Loren L, Duncan, Randall, Son, Hokyoung, Czymmek, Kirk J, and Kang, Seogchan
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Microbiology ,Plant Biology ,Biological Sciences ,Genetics ,Ascomycota ,Calcium ,Calcium Signaling ,Fungi ,Fusarium ,Indicators and Reagents ,Luminescent Proteins ,Neurospora crassa ,Calcium signature ,Genetically encoded calcium indicators ,Signaling ,Time-lapse imaging ,Plant biology - Abstract
Genetically encoded Ca2+ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca2+ signaling in fungi, ranging from documenting Ca2+ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca2+ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca2+ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca2+ indicator. Wild-type and three Ca2+ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.
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- 2021
9. Inhibition of hyaluronan synthesis by 4-methylumbelliferone ameliorates non-alcoholic steatohepatitis in choline-deficient l-amino acid-defined diet-induced murine model
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Yang, Yoon Mee, Wang, Zhijun, Matsuda, Michitaka, and Seki, Ekihiro
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Pharmacology and Pharmaceutical Sciences ,Biomedical and Clinical Sciences ,Nutrition ,Hepatitis ,Chronic Liver Disease and Cirrhosis ,Digestive Diseases ,Liver Disease ,2.1 Biological and endogenous factors ,Aetiology ,Inflammatory and immune system ,Oral and gastrointestinal ,Amino Acids ,Animals ,Choline ,Choline Deficiency ,Hyaluronic Acid ,Hymecromone ,Indicators and Reagents ,Male ,Mice ,Mice ,Inbred C57BL ,Non-alcoholic Fatty Liver Disease ,CXCL1 ,Hyaluronic acid ,NASH ,TLR4 ,Pharmacology & Pharmacy ,Pharmacology and pharmaceutical sciences - Abstract
Hyaluronan (HA) as a glycosaminoglycan can bind to cell-surface receptors, such as TLR4, to regulate inflammation, tissue injury, repair, and fibrosis. 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, is a drug used for the treatment of biliary spasms. Currently, therapeutic interventions are not available for non-alcoholic steatohepatitis (NASH). In this study, we investigated the effects of 4-MU on NASH using a choline-deficient amino acid (CDAA) diet model. CDAA diet-fed mice showed NASH characteristics, including hepatocyte injury, hepatic steatosis, inflammation, and fibrogenesis. 4-MU treatment significantly reduced hepatic lipid contents in CDAA diet-fed mice. 4-MU reversed CDAA diet-mediated inhibition of Ppara and induction of Srebf1 and Slc27a2. Analysis of serum ALT and AST levels revealed that 4-MU treatment protected against hepatocellular damage induced by CDAA diet feeding. TLR4 regulates low molecular weight-HA-induced chemokine expression in hepatocytes. In CDAA diet-fed, 4-MU-treated mice, the upregulated chemokine/cytokine expression, such as Cxcl1, Cxcl2, and Tnf was attenuated with the decrease of macrophage infiltration into the liver. Moreover, HA inhibition repressed CDAA diet-induced mRNA expression of fibrogenic genes, Notch1, and Hes1 in the liver. In conclusion, 4-MU treatment inhibited liver steatosis and steatohepatitis in a mouse model of NASH, implicating that 4-MU may have therapeutic potential for NASH.
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- 2021
10. Optimization of Radiochemical Reactions using Droplet Arrays.
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Rios, Alejandra, Holloway, Travis S, Wang, Jia, and van Dam, R Michael
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Biochemistry and Cell Biology ,Biological Sciences ,Indicators and Reagents ,Radiochemistry ,Radiopharmaceuticals ,Silicon ,Solvents ,Temperature ,Psychology ,Cognitive Sciences ,Biochemistry and cell biology - Abstract
Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction optimization or novel radiopharmaceutical development since each data point involves significant reagent consumption, and contamination of the apparatus requires time for radioactive decay before the next use. To address these limitations, a platform for performing arrays of miniature droplet-based reactions in parallel, each confined within a surface-tension trap on a patterned polytetrafluoroethylene-coated silicon "chip", was developed. These chips enable rapid and convenient studies of reaction parameters including reagent concentrations, reaction solvent, reaction temperature and time. This platform permits the completion of hundreds of reactions in a few days with minimal reagent consumption, instead of taking months using a conventional radiosynthesizer.
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- 2021
11. Catalytic asymmetric addition of an amine N–H bond across internal alkenes
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Xi, Yumeng, Ma, Senjie, and Hartwig, John F
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Inorganic Chemistry ,Organic Chemistry ,Chemical Sciences ,Alkenes ,Amination ,Amines ,Aminopyridines ,Ammonia ,Catalysis ,Chemistry Techniques ,Synthetic ,Hydrogen ,Indicators and Reagents ,Iridium ,Ligands ,Nitrogen ,Phosphines ,General Science & Technology - Abstract
Hydroamination of alkenes, the addition of the N-H bond of an amine across an alkene, is a fundamental, yet challenging, organic transformation that creates an alkylamine from two abundant chemical feedstocks, alkenes and amines, with full atom economy1-3. The reaction is particularly important because amines, especially chiral amines, are prevalent substructures in a wide range of natural products and drugs. Although extensive efforts have been dedicated to developing catalysts for hydroamination, the vast majority of alkenes that undergo intermolecular hydroamination have been limited to conjugated, strained, or terminal alkenes2-4; only a few examples occur by the direct addition of the N-H bond of amines across unactivated internal alkenes5-7, including photocatalytic hydroamination8,9, and no asymmetric intermolecular additions to such alkenes are known. In fact, current examples of direct, enantioselective intermolecular hydroamination of any type of unactivated alkene lacking a directing group occur with only moderate enantioselectivity10-13. Here we report a cationic iridium system that catalyses intermolecular hydroamination of a range of unactivated, internal alkenes, including those in both acyclic and cyclic alkenes, to afford chiral amines with high enantioselectivity. The catalyst contains a phosphine ligand bearing trimethylsilyl-substituted aryl groups and a triflimide counteranion, and the reaction design includes 2-amino-6-methylpyridine as the amine to enhance the rates of multiple steps within the catalytic cycle while serving as an ammonia surrogate. These design principles point the way to the addition of N-H bonds of other reagents, as well as O-H and C-H bonds, across unactivated internal alkenes to streamline the synthesis of functional molecules from basic feedstocks.
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- 2020
12. Comparative Assessment of Detecting Bacterial Populations on the Surface of Medical Equipment in ICU by Standard Microbial Culture and Nanosensor
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Ali Ekrami, Mohammad Ali Hosseini, and Hasan Ekrami
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Cross Infection ,Nanotechnology ,Infection Control ,Indicators and Reagents ,Healthcare-Associated Infections ,Economic biology ,QH705-705.5 - Abstract
Abstract: A healthy, clean, and secure environment is necessary for the hospital, one of the fundamental foundations of the nation's healthcare system, to function well and sustain the general well-being of society. Timely detection of contaminated surfaces and efficient and timely disinfection will be helpful in hospital infection control. For this purpose, the level of surface contamination of medical equipment in intensive care units was to be determined and compared as part of this research. Method: The present study was carried out descriptively, with a sample size of 400 cases on ten types of medical equipment, for one month, with two method Nano sensors color indicator and standard microbial Culture. Results: According to the results obtained, we saw 182 cases of contamination (45.5%) in the samples obtained with the Nanosensor and 176 points of contamination (44%) in the samples obtained with the microbial culture medium, in both bedside instruments and ventilators as The most contaminated surfaces were identified. Also, E. coli, at 55.68%, Staphylococcus aureus, at 28.9%, and Salmonella 23.86%, were recognized as the most common microorganisms. Conclusion: Both methods have the necessary precision to identify contamination reservoirs, And the contamination reported in both methods is similar to what was expected. So in cases where the goal is not to distinguish the type of microorganisms and only to identify the general contamination, Nanosensors can be used as a fast, accurate, and low-cost method.
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- 2023
- Full Text
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13. A Physical Organic Approach to Tuning Reagents for Selective and Stable Methionine Bioconjugation
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Christian, Alec H, Jia, Shang, Cao, Wendy, Zhang, Patricia, Meza, Arismel Tena, Sigman, Matthew S, Chang, Christopher J, and Toste, F Dean
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Organic Chemistry ,Chemical Sciences ,Aziridines ,HEK293 Cells ,Humans ,Indicators and Reagents ,Methionine ,Molecular Probes ,Peptides ,Cyclic ,General Chemistry ,Chemical sciences ,Engineering - Abstract
We report a data-driven, physical organic approach to the development of new methionine-selective bioconjugation reagents with tunable adduct stabilities. Statistical modeling of structural features described by intrinsic physical organic parameters was applied to the development of a predictive model and to gain insight into features driving the stability of adducts formed from the chemoselective coupling of oxaziridine and methionine thioether partners through Redox Activated Chemical Tagging (ReACT). From these analyses, a correlation between sulfimide stabilities and sulfimide ν (C═O) stretching frequencies was revealed. We exploited the rational gains in adduct stability exposed by this analysis to achieve the design and synthesis of a bis-oxaziridine reagent for peptide stapling. Indeed, we observed that a macrocyclic peptide formed by ReACT stapling at methionine exhibited improved uptake into live cells compared to an unstapled congener, highlighting the potential utility of this unique chemical tool for thioether modification. This work provides a template for the broader use of data-driven approaches to bioconjugation chemistry and other chemical biology applications.
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- 2019
14. Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)
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Mishra, Manish, Tiwari, Shuchita, Gunaseelan, Anita, Li, Dongyang, Hammock, Bruce D, and Gomes, Aldrin V
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Analytical Chemistry ,Chemical Sciences ,Biotechnology ,Animals ,Blotting ,Western ,Electrophoresis ,Polyacrylamide Gel ,Horseradish Peroxidase ,Humans ,Indicators and Reagents ,Limit of Detection ,Linear Models ,Luminescent Measurements ,Myocardium ,Proteins ,Rats ,Reproducibility of Results ,Sensitivity and Specificity ,Streptavidin ,Chemiluminescence ,Horseradish peroxidase ,PolyHRP ,SDS PAGE ,Streptavidin-Biotin ,Western blotting ,Biochemistry and Cell Biology ,Chemical Engineering ,Biochemistry and cell biology ,Analytical chemistry - Abstract
Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.
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- 2019
15. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation
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Jia, Shang, He, Dan, and Chang, Christopher J
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Chemical Sciences ,Chlorides ,Histidine ,Indicators and Reagents ,Molecular Structure ,Phosphorus Compounds ,Phosphorylation ,General Chemistry ,Chemical sciences ,Engineering - Abstract
Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. We now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddition (CuAAC) chemistry. We establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purification, as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chemistry.
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- 2019
16. The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.
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Akter, Rehana, Zhyvoloup, Alexander, Zheng, Bingqian, Bhatia, Surita R, and Raleigh, Daniel P
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Humans ,Anilino Naphthalenesulfonates ,Rosaniline Dyes ,Trityl Compounds ,Amyloid ,Fluorescent Dyes ,Indicators and Reagents ,Biological Assay ,Drug Design ,Benzothiazoles ,Islet Amyloid Polypeptide ,Amylin Receptor Agonists ,General Science & Technology - Abstract
The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aβ peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aβ also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aβ amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aβ amyloid formation and inhibit Aβ induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured β-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aβ and h-amylin amyloid formation, illustrates the limitation of using Aβ inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.
- Published
- 2019
17. Readily Accessible Ambiphilic Cyclopentadienes for Bioorthogonal Labeling
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Levandowski, Brian J, Gamache, Raymond F, Murphy, Jennifer M, and Houk, KN
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Azides ,Chemistry Techniques ,Synthetic ,Cyclooctanes ,Cyclopentanes ,Halogenation ,Indicators and Reagents ,Staining and Labeling ,Chemical Sciences ,General Chemistry - Abstract
A new class of bioorthogonal reagents based on the cyclopentadiene scaffold is described. The diene 6,7,8,9-tetrachloro-1,4-dioxospiro[4,4]nona-6,8-diene (a tetrachlorocyclopentadiene ketal, TCK) is ambiphilic and self-orthogonal with remarkable stability. The diene reacts rapidly with a trans-cyclooctene and an endo-bicyclononyne, but slowly with dibenzoazacyclooctyne (DIBAC), allowing for tandem labeling studies with mutually orthogonal azides that react rapidly with DIBAC. TCK analogues are synthesized in three steps from inexpensive, commercially available starting materials.
- Published
- 2018
18. Constructing New Bioorthogonal Reagents and Reactions
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Row, R David and Prescher, Jennifer A
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Bioengineering ,Generic health relevance ,Chemistry Techniques ,Synthetic ,Cycloaddition Reaction ,Cyclopropanes ,Indicators and Reagents ,Triazines ,Chemical Sciences ,General Chemistry - Abstract
Chemical tools are transforming our understanding of biomolecules and living systems. Included in this group are bioorthogonal reagents-functional groups that are inert to most biological species, but can be selectively ligated with complementary probes, even in live cells and whole organisms. Applications of these tools have revealed fundamental new insights into biomolecule structure and function-information often beyond the reach of genetic approaches. In many cases, the knowledge gained from bioorthogonal probes has enabled new questions to be asked and innovative research to be pursued. Thus, the continued development and application of these tools promises to both refine our view of biological systems and facilitate new discoveries. Despite decades of achievements in bioorthogonal chemistry, limitations remain. Several reagents are too large or insufficiently stable for use in cellular environments. Many bioorthogonal groups also cross-react with one another, restricting them to singular tasks. In this Account, we describe our work to address some of the voids in the bioorthogonal toolbox. Our efforts to date have focused on small reagents with a high degree of tunability: cyclopropenes, triazines, and cyclopropenones. These motifs react selectively with complementary reagents, and their unique features are enabling new pursuits in biology. The Account is organized by common themes that emerged in our development of novel bioorthogonal reagents and reactions. First, natural product structures can serve as valuable starting points for probe design. Cyclopropene, triazine, and cyclopropenone motifs are all found in natural products, suggesting that they would be metabolically stable and compatible with a variety of living systems. Second, fine-tuning bioorthogonal reagents is essential for their successful translation to biological systems. Different applications demand different types of probes; thus, generating a collection of tools that span a continuum of reactivities and stabilities remains an important goal. We have used both computational analyses and mechanistic studies to guide the optimization of various cyclopropene and triazine probes. Along the way, we identified reagents that are chemoselective but best suited for in vitro work. Others are selective and robust enough for use in living organisms. The last section of this Account highlights the need for the continued pursuit of new reagents and reactions. Challenges exist when bioorthogonal chemistries must be used in concert, given that many exploit similar mechanisms and cannot be used simultaneously. Such limitations have precluded certain multicomponent labeling studies and other biological applications. We have relied on mechanistic and computational insights to identify mutually orthogonal sets of reactions, in addition to exploring unique genres of reactivity. The continued development of mechanistically distinct, biocompatible reactions will further diversify the bioorthogonal reaction portfolio for examining biomolecules.
- Published
- 2018
19. Mitochondria-targeted Probes for Imaging Protein Sulfenylation.
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Holmila, Reetta J, Vance, Stephen A, Chen, Xiaofei, Wu, Hanzhi, Shukla, Kirtikar, Bharadwaj, Manish S, Mims, Jade, Wary, Zack, Marrs, Glen, Singh, Ravi, Molina, Anthony J, Poole, Leslie B, King, S Bruce, and Furdui, Cristina M
- Subjects
Mitochondria ,Humans ,Sulfenic Acids ,Mitochondrial Proteins ,Indicators and Reagents ,Molecular Probes ,Cytological Techniques ,Staining and Labeling ,Protein Processing ,Post-Translational ,Oxidation-Reduction ,A549 Cells ,1.1 Normal biological development and functioning ,Cancer - Abstract
Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.
- Published
- 2018
20. Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein–Nanoparticle Interaction
- Author
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Duan, Yaokai, Liu, Yang, Shen, Wen, and Zhong, Wenwan
- Subjects
Nanotechnology ,Bioengineering ,Generic health relevance ,Binding Sites ,Fluorescamine ,Indicators and Reagents ,Molecular Structure ,Nanoparticles ,Protein Conformation ,Proteins ,Surface Properties ,Analytical Chemistry ,Other Chemical Sciences - Abstract
Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.
- Published
- 2017
21. Regioselective Termination Reagents for Ring-Opening Alkyne Metathesis Polymerization
- Author
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Jeong, Hyangsoo, von Kugelgen, Stephen, Bellone, Donatela, and Fischer, Felix R
- Subjects
Inorganic Chemistry ,Organic Chemistry ,Chemical Sciences ,Alkynes ,Caproates ,Cyclobutanes ,Indicators and Reagents ,Lactones ,Molybdenum ,Polymerization ,Polymers ,General Chemistry ,Chemical sciences ,Engineering - Abstract
Alkyne cross-metathesis of molybdenum carbyne complex [TolC≡Mo(OCCH3(CF3)2)3]·DME with 2 equiv of functional ynamines or ynamides yields the primary cross-metathesis product with high regioselectivity (>98%) along with a molybdenum metallacyclobutadiene complex. NMR and X-ray crystal structure analysis reveals that ynamides derived from 1-(phenylethynyl)pyrrolidin-2-one selectively cleave the propagating molybdenum species in the ring-opening alkyne metathesis polymerization (ROAMP) of ring-strained 3,8-dihexyloxy-5,6-dihydro-11,12-didehydrodibenzo[a,e][8]annulene and irreversibly deactivate the diamagnetic molybdenum metallacyclobutadiene complex through a multidentate chelate binding mode. The chain termination of living ROAMP with substituted ethynylpyrrolidin-2-ones selectively transfers a functional end-group to the polymer chain, giving access to telechelic polymers. This regioselective carbyne transfer strategy gives access to amphiphilic block copolymers through synthetic cascades of ROAMP followed by ring-opening polymerization of strained ε-caprolactone.
- Published
- 2017
22. New Architecture for Reagentless, Protein-Based Electrochemical Biosensors
- Author
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Kang, Di, Sun, Sheng, Kurnik, Martin, Morales, Demosthenes, Dahlquist, Frederick W, and Plaxco, Kevin W
- Subjects
Analytical Chemistry ,Chemical Sciences ,Bioengineering ,Biosensing Techniques ,Electrochemical Techniques ,Escherichia coli Proteins ,Indicators and Reagents ,Methyl-Accepting Chemotaxis Proteins ,General Chemistry ,Chemical sciences ,Engineering - Abstract
Here we demonstrate a new class of reagentless, single-step sensors for the detection of proteins and peptides that is the electrochemical analog of fluorescence polarization (fluorescence anisotropy), a versatile optical approach widely employed to this same end. Our electrochemical sensors consist of a redox-reporter-modified protein (the "receptor") site-specifically anchored to an electrode via a short, flexible polypeptide linker. Interaction of the receptor with its binding partner alters the efficiency with which the reporter approaches the electrode surface, thus causing a change in redox current upon voltammetric interrogation. As our first proof-of-principle we employed the bacterial chemotaxis protein CheY as our receptor. Interaction with either of CheY's two binding partners, the P2 domain of the chemotaxis kinase, CheA, or the 16-residue "target region" of the flagellar switch protein, FliM, leads to easily measurable changes in output current that trace Langmuir isotherms within error of those seen in solution. Phosphorylation of the electrode-bound CheY decreases its affinity for CheA-P2 and enhances its affinity for FliM in a manner likewise consistent with its behavior in solution. As expected given the proposed sensor signaling mechanism, the magnitude of the binding-induced signal change depends on the placement of the redox reporter on the receptor. Following these preliminary studies with CheY, we also developed and characterized additional sensors aimed at the detection of specific antibodies using the relevant protein antigens as the receptor. These exhibit excellent detection limits for their targets without the use of reagents or wash steps. This novel, protein-based electrochemical sensing architecture provides a new and potentially promising approach to sensors for the single-step measurement of specific proteins and peptides.
- Published
- 2017
23. Impact of M-protein detection on the response evaluations of patients undergoing treatment with the IgG-κ monoclonal antibodies daratumumab or isatuximab, and discrepancies between immunofixation electrophoresis (IFE) systems and reagents.
- Author
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Shirouchi Y, Kaihara K, Sekita T, Amano N, Nakayama K, Miyake K, Abe H, Oinuma H, and Maruyama D
- Subjects
- Humans, Female, Aged, Male, Middle Aged, Immunoelectrophoresis methods, Treatment Outcome, Immunoglobulin G blood, Immunoglobulin kappa-Chains blood, Indicators and Reagents, Antibodies, Monoclonal therapeutic use, Multiple Myeloma drug therapy, Multiple Myeloma blood, Antibodies, Monoclonal, Humanized therapeutic use, Myeloma Proteins analysis
- Abstract
Background: Immunofixation electrophoresis (IFE) is the standard method for confirming the presence of a monoclonal protein (M-protein) at multiple myeloma (MM) diagnosis. IFE is also essential at assessment of complete response (CR) and stringent CR during treatment. As the CR assessment is influenced by daratumumab and isatuximab, HYDRASHIFT assays were developed., Methods: Samples from patients under treatment that included daratumumab or isatuximab were tested and monitored by IFE on the HYDRASYS system using HYDRASHIFT assays (HYDRASYS/HYDRASHIFT) and by IFE on the Epalyzer2 system (Epalyzer)., Results: The IFE using HYDRASYS/HYDRASHIFT avoided a false positive caused by drug-related IgG-κ and contributed to accurate assessment of CR. Furthermore, HYDRASYS/HYDRASHIFT detected small M-proteins at early relapse and detected free light chains (FLCs) in patients with renal impairment exhibiting high serum FLCs despite being often missed on Epalyzer., Conclusion: Sensitivity and specificity of M-protein detection vary greatly depending on the IFE system and reagents used., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)
- Published
- 2024
- Full Text
- View/download PDF
24. APTT critical value establishment in four different reagent/instrument systems based on single factor deficiencies.
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Huang H, Qu T, Hu M, Chen Z, Fan K, and Mo X
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- Humans, Partial Thromboplastin Time, Retrospective Studies, Female, Male, Coagulation Protein Disorders blood, Coagulation Protein Disorders diagnosis, ROC Curve, Blood Coagulation Tests methods, Blood Coagulation Tests standards, Indicators and Reagents, Blood Coagulation Factors analysis
- Abstract
Introduction: There are significant differences in the activated partial thromboplastin time (APTT) critical values reported in different studies, most of which does not make recommendations for any specific clear detection systems. The International Council for Standardization in Hematology (ICSH) recommends that APTT critical values be established based on the reagent type, coagulation factor sensitivity and heparin response. The objective of this study was to establish APTT critical values by using different reagents and based on single coagulation factor deficiencies., Methods: The APTT values were determined in commercial endogenous coagulation factor-deficient plasma at concentrations of 1 IU/dL, 2 IU/dL, 5 IU/dL, 10 IU/dL, 20 IU/dL, and 30 IU/dL by using four assay systems. The retrospective collection of data from patients who lacked factor VIII (FVIII), FIX, or FXI alone was performed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of APTT for identifying patients with an endogenous coagulation factor activity < 5 IU/dL., Results: The APTT values in the plasma samples with the same concentrations of endogenous coagulation factors were significantly different among the four assay systems (P < 0.001). The suggested critical values of APTT were 40.0 s for Sysmex CS5100 (Actin FSL), 58.0 s for Sysmex CS5100 (Actin), 51.8 s for STA-R Evolution (STA-PTTA), and 64.8 s for ACL TOP 700 (HemosIL SynthasIL). On the basis of the ROC curve, the optimal threshold values for APTT (STA-PTTA) were 55.8 s in patients with a simple deficiency of FVIII (sensitivity = 100%, specificity = 85.7%, area under the ROC curve (AUC) = 0.982), 54.3 s in patients with a simple deficiency of FIX (sensitivity = 100%, specificity = 92.9%, AUC = 0.986), and 71.7 s in patients with a simple deficiency of FXI (sensitivity = 100%, specificity = 94.1%, AUC = 0.992), which were closer (difference of 0.6-2.5 s) to the cutoff points for commercial plasma at equal factor levels., Conclusions: APTT critical values need to be established for different reagents based on the presence of a single coagulation factor deficiency., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
- Published
- 2024
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25. Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.
- Author
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Lin Q, Takebayashi K, Torigoe N, Liu B, Namula Z, Hirata M, Nagahara M, Tanihara F, and Otoi T
- Subjects
- Animals, Swine, RNA, Guide, CRISPR-Cas Systems genetics, Embryo Culture Techniques methods, Indicators and Reagents, Mutation, Lipids, CRISPR-Cas Systems genetics, Gene Editing methods, Zygote metabolism, Transfection methods
- Abstract
The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency., (© 2024. The Society for In Vitro Biology.)
- Published
- 2024
- Full Text
- View/download PDF
26. Variability in combinations of APTT reagent and substrate plasma for a one-stage clotting assay to measure factor VIII products.
- Author
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Suzuki A, Suzuki N, Kanematsu T, Okamoto S, Suzuki N, Tamura S, Kikuchi R, Katsumi A, Kojima T, and Matsushita T
- Subjects
- Humans, Partial Thromboplastin Time methods, Partial Thromboplastin Time standards, Blood Coagulation Tests methods, Blood Coagulation Tests standards, Blood Coagulation, Indicators and Reagents, Reproducibility of Results, Factor VIII analysis
- Abstract
Introduction: An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma., Aim: A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP., Methods: FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP., Results: F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3., Conclusion: In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur., (© 2024 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)
- Published
- 2024
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- View/download PDF
27. Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection.
- Author
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Lopez MLD, Crichton EM, Allison MJ, Dema AH, Bonderud MT, and Helbing CC
- Subjects
- Indicators and Reagents, Freezing, DNA Primers genetics, Specimen Handling methods, Real-Time Polymerase Chain Reaction methods, Real-Time Polymerase Chain Reaction standards, DNA, Environmental analysis, DNA, Environmental genetics
- Abstract
Objective: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays., Results: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
28. Study on the methods of visualizing bloodstains after thermal exposure.
- Author
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Di J, Yan X, Wang P, Zang Z, Jin J, and Zhang J
- Subjects
- Humans, Infrared Rays, Luminol, Fluorescein, Indicators and Reagents, Calorimetry, Fluorescent Dyes, Forensic Medicine methods, Luminescent Agents, Blood Stains, Photography, Hot Temperature
- Abstract
To establish the correlation between thermal conditions imposed on bloodstains and visualizing effect of enhancement techniques, infrared photography and four chemical enhancement reagents were used to visualize bloodstains following thermal exposure. A black tile was selected as the substrate to intensify the visualization challenge, with a Cone Calorimeter serving as the standardized heating source to control thermal conditions. Compared with standard photography, infrared photography is proven to be a valuable complement to chemical reagents, showing significant advantages in visualizing bloodstains after thermal exposure. However, it is worth noting that infrared image fell short of standard image when bloodstains displayed raised, embossed morphology or when bloodstains almost disappeared under specific conditions. The enhancement effectiveness was found to be strongly correlated with thermal conditions imposed on bloodstains, and the morphology evolution of bloodstains during heating affected the chemical enhancement effect additionally, especially when the bulge morphology was formed, and it was observed that reagents were more effective after removing the dense shell of the bulge. Among the four selected chemical enhancement reagents, fluorescein performed exceptionally well, maintaining its effectiveness even for bloodstains heated at 641°C for 10 min. TMB demonstrated its visualizing ability for bloodstains heated at 396°C for 5 min and heated at 310°C for 20 min. BLUESTAR® followed afterwards, while luminol performed worst. The correlation between thermal conditions imposed on bloodstains and the corresponding visualizing effectiveness of enhancement techniques provides important references for detecting bloodstains at fire scenes., (© 2024 American Academy of Forensic Sciences.)
- Published
- 2024
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- View/download PDF
29. Enhanced cellular internalization of near-infrared fluorescent single-walled carbon nanotubes facilitated by a transfection reagent.
- Author
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Levin N, Hendler-Neumark A, Kamber D, and Bisker G
- Subjects
- Humans, Indicators and Reagents, Microscopy, Fluorescence, Polyethylene Glycols, Nanotubes, Carbon, Adenocarcinoma
- Abstract
Functionalized single-walled carbon nanotubes (SWCNTs) hold immense potential for diverse biomedical applications due to their biocompatibility and optical properties, including near-infrared fluorescence. Specifically, SWCNTs have been utilized to target cells as a vehicle for drug delivery and gene therapy, and as sensors for various intracellular biomarkers. While the main internalization route of SWCNTs into cells is endocytosis, methods for enhancing the cellular uptake of SWCNTs are of great importance. In this research, we demonstrate the use of a transfecting reagent for promoting cell internalization of functionalized SWCNTs. We explore different types of SWCNT functionalization, namely single-stranded DNA (ssDNA) or polyethylene glycol (PEG)-lipids, and two different cell types, embryonic kidney cells and adenocarcinoma cells. We show that internalizing PEGylated functionalized SWCNTs is enhanced in the presence of the transfecting reagent, where the effect is more pronounced for negatively charged PEG-lipid. However, ssDNA-SWCNTs tend to form aggregates in the presence of the transfecting reagent, rendering it unsuitable for promoting internalization. For all cases, cellular uptake is visualized by near-infrared fluorescence microscopy, showing that the SWCNTs are typically localized within the lysosome. Generally, cellular internalization was higher in the adenocarcinoma cells, thereby paving new avenues for drug delivery and sensing in malignant cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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- View/download PDF
30. Fast FRAP-SIA method to determine antioxidant capacity.
- Author
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Jiménez-Morales WA and Cañizares-Macias MDP
- Subjects
- Oxidation-Reduction, Indicators and Reagents, Iron, Antioxidants analysis, Food
- Abstract
One of the most used methods to measure antioxidant capacity in food is the ferric reducing antioxidant power (FRAP) test, which is simple, sensitive, and economical, nevertheless has long analysis times, causing measurement errors due to the instability of the FRAP reagent due to its precipitation sequential injection analysis (SIA) is a flow technique that can correct these disadvantages because it is more quickly. So, a novel FRAP-SIA method was developed to evaluate the antioxidant capacity. The system was optimized using a central composite design for hydrodynamic and chemical factors, resulting in a flow rate of 35 μL s
-1 , and aspirate volumes of 33 μL-38 μL-33 μL for the sequence (FRAP-Antioxidant-FRAP). FRAP reagent was prepared with an HCl solution at 0.005 mol L-1 , improving its stability 24 times, concerning when it is in acetate buffer at pH 3.6. The method showed excellent accuracy (RSD <3%) with a LOD of 1.0 μmol L-1 of Trolox for a linear range of 5-120 μmol L-1 . The reaction time was diminished by 96% concerning the FRAP-microplate assay (from 30 min to 1.2 min). The method was applied in beverages and extracts, obtaining recovery values ranging from 91.24 to 114.22%., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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- View/download PDF
31. An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.
- Author
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Sacco RE, Jensen ED, Sullivan YB, LaBresh J, and Davis WC
- Subjects
- Animals, Bottle-Nosed Dolphin immunology, Indicators and Reagents, Immunologic Techniques veterinary
- Abstract
There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination., Competing Interests: Declaration of Competing Interest The authors declare the following which may be considered as potential competing interests: Joanna LaBresh is the President and Founder of Kingfisher Biotech, Inc. Yvonne B. Sullivan is currently employed at Kingfisher Biotech, Inc as Director of Development. The mAbs listed in Table 1 from Washington State University Monoclonal Antibody Center are commercially available. Royalties are paid to Washington State University., (Copyright © 2024. Published by Elsevier B.V.)
- Published
- 2024
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32. Photo-sensitive peptide inducing targeted cross-linking in a one-step and reagent-, enzyme- and antibody-free detection of SARS-Cov-2 marker protein.
- Author
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Hou M, Hou W, Qin M, Wang Q, and Zhou L
- Subjects
- Humans, Indicators and Reagents, Antibodies, Viral, Proteins, Peptides, Peptide Hydrolases, SARS-CoV-2, COVID-19 diagnosis
- Abstract
Modern biosensing technology plays a crucial role in combating the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). However, the associated assays remain costly, considering their extensive daily use. In response, we developed a simplified one-step SARS-CoV-2 protease assay that reduces both time and financial expenses. This approach eliminates the need for extra reagents, enzymes, or antibodies. The simplification involves a photo-sensitive Bengal red-tagged substrate peptide, allowing specific cross-linking upon protease-substrate recognition. This process forms a di-tyrosine product with a distinctive fluorescence signal readout, enabling the detection of SARS-CoV-2 in patient serum samples. This method anticipates a major reduction in assay costs in the near future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)
- Published
- 2024
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33. DNA Reference Reagents for Genotyping RH Variants.
- Author
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Sippert E, Volkova E, Rippee-Brooks M, Denomme GA, Flegel WA, Lee C, Araojo R, Illoh O, Liu Z, and Rios M
- Subjects
- Humans, Genotype, Alleles, DNA genetics, Reference Standards, High-Throughput Nucleotide Sequencing methods, Polymorphism, Genetic, Indicators and Reagents, Rh-Hr Blood-Group System genetics, Genotyping Techniques methods, Genotyping Techniques standards
- Abstract
Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)
- Published
- 2024
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34. Back to Old Books Toward Affordable Research: Homemade Phenol-Based Reagent for Triphasic RNA Purification.
- Author
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Abdelfattah MM, Hosny Y, Elkady NA, Abbas EM, Farahat IG, Hassan NHA, and Helwa R
- Subjects
- Humans, Guanidines chemistry, COVID-19, SARS-CoV-2, RNA, Viral genetics, RNA, Viral isolation & purification, Thiocyanates, RNA isolation & purification, Animals, Indicators and Reagents, Phenol chemistry
- Abstract
Covid-19 crisis did hit many socio-economic aspects in the whole world. In the scientific research, the problem is getting even worse, since most of materials and consumable are allocated to the health sector. Many research laboratories around the world have big delay in receiving their purchases to accomplish their research projects. In the developing countries, the situation is much more difficult, since most of the funding resources are directed to the Covid-19 crisis and there is a notable increase in reagents' prices. Therefore, the aim of the present study is to make a homemade reagents for RNA purification from eukaryotic cells/tissues. The homemade phenol-based RNA extraction reagents were prepared using saturated phenol pH 4.3 (adjusted by 0.5 M citrate buffer) and guanidine thiocyanate. To validate the phenol-based reagent, RNA was purified from different biological samples (cell line, tissues, and fungi) using homemade phenol-based versus a commercial one. Concentration of RNA samples extracted from the same number of cells were compared to assess the homemade preparation of phenol-based reagent. In conclusion, homemade phenol-based reagent is cost effective and comparable to the commercial one. Using homemade phenol-based, RNA extraction was successfully purified from different biological sources., (© 2023. The Author(s).)
- Published
- 2024
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35. Patent Issued for Water absorbent agent having water-absorbent resin as main component and comprising a sulfur-containing reducing agent (USPTO 12053756).
- Published
- 2024
36. New Findings in Cancer Described from Michigan State University (EGFR- and Integrin a [ [V] ] b [ [3] ] -Targeting Peptides as Potential Radiometal-Labeled Radiopharmaceuticals for Cancer Theranostics).
- Published
- 2024
37. Researchers Submit Patent Application, "Sotorasib Dosing Regimen", for Approval (USPTO 20240261289).
- Published
- 2024
38. New Medical Radiology Findings from Chinese Academy of Sciences Described (The development of 177Lu-DOTA-CC-PSMA following a unified "Click Chemistry" protocol of synthesizing metal nuclide-conjugated radiopharmaceuticals).
- Subjects
CLICK chemistry ,RADIOLOGY ,RADIOPHARMACEUTICALS ,PROSTATE-specific membrane antigen ,METALS - Abstract
The article focuses on the development of a new radiopharmaceutical, 177Lu-DOTA-CC-PSMA, using a unified click chemistry approach. Topics include the challenges of traditional metal nuclide labeling methods, the advantages of click chemistry for synthesizing radiopharmaceuticals, and the efficacy of 177Lu-DOTA-CC-PSMA in targeting prostate-specific membrane antigens with high precision and stability.
- Published
- 2024
39. Study Data from Faculty of Mathematics Provide New Insights into Bacillus subtilis (A Comparative Analysis of Capping and Reducing Agents of Microbial Cell aspergillus Niger and bacillus Subtilis for Biosynthesis of Ag Doped Zno...).
- Abstract
A study conducted by researchers at the Faculty of Mathematics in Padang, Indonesia, focused on the green synthesis of Ag doped ZnO (AZNPR) using microbial cells as capping and reducing agents. The study found that the microbial cells, specifically Aspergillus niger and Bacillus subtilis, contained extracellular enzymes responsible for controlling the growth of AZNPR. The formation of AZNPR was characterized using various analyses, and the nanorods showed the ability to inhibit Candida albicans, a fungus causing infectious disease. The research was supported by the Basic Research Featured Professor Publication Research Cluster at Andalas University. [Extracted from the article]
- Published
- 2024
40. New Research on Health and Medicine from University of Pennsylvania Perelman School of Medicine Summarized (Optimizing Regulatory Reviews for Clinical Protocols that Use Radiopharmaceuticals: Findings of the University of Pennsylvania Radiation...).
- Abstract
A recent study conducted by the University of Pennsylvania Perelman School of Medicine examined the process of reviewing clinical protocols that involve the use of radiopharmaceuticals. The study found that institutional radiation safety committees often require revisions to protocols, leading to delays in protocol activations. The researchers analyzed 110 protocols submitted to the University of Pennsylvania's Radiation Research Safety Committee and identified factors associated with approval time. They concluded that changes to the review process, such as publishing standardized radiation safety language and encouraging timely responses to stipulations, could help expedite reviews without compromising quality. [Extracted from the article]
- Published
- 2024
41. Localized in vivo prodrug activation using radionuclides.
- Abstract
A preprint abstract from biorxiv.org discusses a strategy called "RAdionuclide induced Drug Engagement for Release" (RAiDER) that aims to deliver combined radiation and chemotherapy to tumor sites while minimizing exposure to off-target sites. The study tested the effectiveness of different radionuclides in chemically activating a model radiation-activated prodrug. Results showed that RAiDER with certain radionuclides increased the cytotoxicity of the prodrug and delayed tumor growth in mouse models of cancer. The study suggests that RAiDER has the potential to improve the efficacy of radiopharmaceutical therapies for cancer treatment. However, it is important to note that this preprint has not been peer-reviewed. [Extracted from the article]
- Published
- 2024
42. Findings on Breast Cancer Reported by Investigators at Stanford University Medical Center (Frequent Amplification and Overexpression of Psma In Basallike Breast Cancer From Analysis of the Cancer Genome Atlas).
- Abstract
A recent study conducted at Stanford University Medical Center analyzed the molecular basis of applying prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals in breast cancer subtypes. The study found that PSMA was frequently overexpressed in basallike breast cancer (BLBC) compared to other subtypes. Additionally, PSMA expression was associated with relapse-free and distant metastasis-free survival in BLBC patients. These findings suggest that PSMA-targeting radiopharmaceuticals may be effective in treating this aggressive subtype of breast cancer. The research has been peer-reviewed and published in the Journal of Nuclear Medicine. [Extracted from the article]
- Published
- 2024
43. Patent Application Titled "Systems For Radioisotope Generation And Methods Of Preparation And Administration" Published Online (USPTO 20240249856).
- Abstract
A patent application titled "Systems For Radioisotope Generation And Methods Of Preparation And Administration" has been published online by the US Patent and Trademark Office. The patent application, filed by inventors from Jubilant Draximage Inc., describes a radioisotope generator system that aims to produce customized, predictable, and reproducible supplies of radioisotopes for use in nuclear medicine. The system includes a shielded reactor housing with three ports, a radioisotope generator column, and an adapter disk. The invention addresses safety concerns and contamination issues associated with conventional generator systems. The patent application provides detailed claims and technical specifications for the system. [Extracted from the article]
- Published
- 2024
44. Researchers from University of California San Francisco (UCSF) Discuss Findings in Chemicals and Chemistry (Assessing Response To Psma Radiopharmaceutical Therapies With Single Spect Imaging At 24 Hours After Injection).
- Subjects
PROSTATE-specific membrane antigen ,PROSTATE-specific antigen ,NUCLEAR medicine ,COENZYMES ,REPORTERS & reporting ,PROSTATE cancer - Abstract
A study conducted by researchers from the University of California San Francisco (UCSF) explored the relationship between lesion-absorbed dose and tumor response in Lu-177-PSMA-617 radiopharmaceutical therapies (RPTs). The study evaluated 50 patients with 335 index lesions undergoing RPT and found that quantitative lesion-based response correlated with patient-level prostate-specific antigen (PSA) response. However, there was a limited relationship between baseline absorbed dose and lesion-based responses. The researchers concluded that establishing a dose-response relationship with a single time point at 24 hours presented some limitations. [Extracted from the article]
- Published
- 2024
45. Blue Earth Diagnostics, a Bracco Company, Announces Results Evaluating Diagnostic Performance of POSLUMA(R) Flotufolastat F 18 and Clinical Trial Enrollment in African American Men with Prostate Cancer.
- Subjects
MEDICAL personnel ,PROSTATE-specific membrane antigen ,AFRICAN American men ,TUMOR antigens ,ANDROGEN receptors ,PROSTATE cancer patients - Abstract
Blue Earth Diagnostics, a Bracco company, has announced the results of a study evaluating the diagnostic performance of POSLUMA (flotufolastat F 18) in patients with recurrent prostate cancer. The study included a sub-analysis of African American men, who have a higher mortality and prevalence of prostate cancer. The results showed a high detection rate among African American patients, with 93% having a positive POSLUMA scan. The study also found that the participation of African American men in the trial was twice the enrollment typically reported in other oncology clinical trials. These findings are important for clinical decision-making and addressing racial disparities in cancer outcomes. [Extracted from the article]
- Published
- 2024
46. Patent Application Titled "Method Of Generating Multifunctional Mycelium Products" Published Online (USPTO 20240247410).
- Subjects
CHROMOGENIC compounds ,TEXTILE product manufacturing ,BACTERIAL enzymes ,METHICILLIN-resistant staphylococcus aureus ,PATENT applications - Abstract
A patent application titled "Method Of Generating Multifunctional Mycelium Products" has been published online by the US Patent and Trademark Office. The patent application, filed by inventors Kimberly Zawi Carlson, Hyder Ali Khoja, Donald Steinberg, and Maqsad Suriev, and assigned to Mushrooms Inc., describes a method of producing mycelium-based products such as thread, foam, films, gels, and liquids. These products have the potential to inhibit bacterial growth, including antibiotic-resistant strains, and can be used in various fields such as healthcare, home comforts, and environmental filters. The method emphasizes eco-friendliness and sustainability. [Extracted from the article]
- Published
- 2024
47. Research from Candiolo Cancer Institute Provides New Study Findings on Diagnostics (The Potential Role of PSMA-Targeted PET in Salivary Gland Malignancies: An Updated Systematic Review).
- Subjects
PROSTATE-specific membrane antigen ,POSITRON emission tomography ,ADENOID cystic carcinoma ,MEDICAL screening ,SALIVARY glands - Abstract
A recent study conducted by researchers at the Candiolo Cancer Institute in Turin, Italy, explored the potential use of positron emission tomography/computed tomography (PET/CT) with prostate-specific membrane antigen (PSMA)-targeting radiopharmaceuticals for the detection of salivary gland malignancies (SGM), specifically adenoid-cystic carcinoma (ACC). The study reviewed six articles and found that PSMA-targeted PET/CT showed a high detection rate for ACC lesions, suggesting its potential as a diagnostic tool. The researchers also noted that PSMA could be a suitable target for radioligand therapy. However, further multicentric studies are needed to confirm these findings and establish the role of PSMA-targeting radiopharmaceuticals in ACC. [Extracted from the article]
- Published
- 2024
48. Patent Issued for Capsule array devices and methods of use (USPTO 12037634).
- Published
- 2024
49. Lower dose prostate cancer treatments retain efficacy while improving tolerability.
- Subjects
PROSTATE-specific antigen ,TECHNOLOGICAL innovations ,NUCLEAR medicine ,PATIENT experience ,MEDICAL technology ,PROSTATE cancer - Abstract
New research published in The Journal of Nuclear Medicine has found that lower doses of radiopharmaceutical therapy for advanced stage metastatic castrate-resistant prostate cancer are just as effective as the standard dose. Two reduced dose approaches, deescalated 225Ac-PSMA-617 and a cocktail therapy of 177Lu/225Ac-PSMA-617, showed similar median overall survival and prostate specific antigen (PSA) response rates compared to the standard dose. These lower dose treatments were also better-tolerated by patients, reducing uncomfortable side effects such as dry mouth. The study suggests that patients with late-stage prostate cancer can benefit from targeted radiopharmaceutical alpha-therapy. [Extracted from the article]
- Published
- 2024
50. Research on Biomedical Physics and Engineering Discussed by Researchers at Shiraz University of Medical Sciences (Evaluating the Radioactive Waste Produced per Patient by Radiopharmaceutical Sources and Measuring the Radioactive Contamination...).
- Abstract
Researchers at Shiraz University of Medical Sciences in Iran conducted a study to monitor radiation contamination in the nuclear medicine department of Bushehr. They measured the activity of radioisotopes, radiopharmaceuticals, and radioactive waste, as well as the absorbed doses of irradiators. Contamination was determined using wipe tests on various surfaces, and personnel were monitored for contamination. The study found that daily assessment of radiopharmaceutical administration and surface contamination monitoring are important for waste management and estimating radiation pollution levels. The research provides valuable insights into the field of nuclear medicine and its impact on occupational and environmental safety. [Extracted from the article]
- Published
- 2024
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