Comparative Assessment of APTT Reagents for Evaluating Anticoagulant Sensitivity of Fucosylated Glycosaminoglycans (FGs) Derived from Sea Cucumbers.

Fucosylated glycosaminoglycans (FGs) derived from sea cucumbers exhibit potent intrinsic Xase (iXase) inhibition, anticoagulation, and antithrombosis. Plasma activated partial thromboplastin time (APTT), a widely used screening test worldwide, is crucial for evaluating anticoagulant efficacy. However, the applicability of these commercially available APTT reagents for assessing anticoagulation of FGs remains unreported. In this study, we investigated the disparity between ellagic acid and colloidal silica APTT reagents in evaluating anticoagulation of dHG-5 and dHLFG-4, two depolymerized FGs, and elucidated the underlying rationale. The results demonstrated that dHG-5 and dHLFG-4 exhibited heightened sensitivity to the ellagic acid APTT reagent both in vitro and in vivo, and did not significantly affect the activation of APTT reagents for plasma. In addition, both ellagic acid and colloidal silica APTT reagents inhibited the anti-iXase of dHG-5 and dHLFG-4, and the inhibition of the ellagic acid APTT reagent was less pronounced compared to the colloidal silica APTT reagent. These findings suggest that the reduced impact of the ellagic acid APTT reagent on the anti-iXase activity of dHG-5 and dHLFG-4 is responsible for the increased sensitivity in plasma APTT analysis. This study offers valuable insights into the characteristics of two APTT reagents applied for assessing the anticoagulant activity of FG-related compounds.

A low-cost efficient online derivatization system for the determination of saccharides by high-performance liquid chromatograph-ultraviolet detector.

In this study, a low-cost efficient online derivatization system was developed which allows for the detection of various types of mono- and oligo-saccharides only utilizing high-performance liquid chromatography (HPLC)-ultraviolet detector (UV) system. In the proposed method, phenylhydrazine was used as the derivatization reagent and directly spiked in the mobile phase, allowing for the separation and detection of mono- and oligosaccharides in an accessible instrument system (HPLC-UV). And the online derivatization design of the proposed method has significantly reduced the potential harm of derivatization reagents to the analysts. Furthermore, critical chromatographic parameters were optimized via the Box-Behnken design strategy, culminating in the ideal response for saccharides. Finally, the methodology validation of the proposed method was conducted. The proposed method showed satisfactory linear ranges with acceptable correlation coefficients (R2  > 0.99), outstanding accuracy (Recovery: 95.3%-105.6%), high intra-day precision (relative standard deviation [RSD]: 1.4%-7.1%) and inter-day precision (RSD: 2.0%-7.4%). The robustness and ruggedness of the proposed method were proved as the recovery values in the range of 95.0%-104.6% and 95.1%-104.8% for robustness and ruggedness, respectively. These satisfactory validation results confirm the applicability and reliability of the proposed method for the analysis of saccharides in various complex real-world samples.

Effective assessment of lanthanide ion delivery into live cells by paramagnetic NMR spectroscopy.

We report an effective assessment of lanthanide ion (Ln3+) delivery into live cells by paramagnetic NMR spectroscopy. Free Ln3+ ions are toxic to live cells resulting in a gradual leakage of target proteins to the extracellular media. The citrate-Ln3+ complex is an efficient and mild reagent over the free Ln3+ form for live cell delivery.

A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen-Based Diagnostic Tests for COVID-19.

Rapid diagnostic tests (RDTs) that detect antigen indicative of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative reverse transcription (RT)-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor Standard Q COVID-19 antigen (Ag) test, Abbott Panbio COVID-19 Ag rapid test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparisons between diagnostic tests. IMPORTANCE This study reports the results for severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) nucleocapsid (N) and spike (S) antigen quantification assays and their performance against clinical reverse transcription (RT)-PCR results, thus describing an open-access quantification method for two important SARS-CoV-2 protein analytes. Characterized N antigen panels were used to evaluate the limits of detection of four different rapid tests for SARS-CoV-2 against multiple sources of nucleocapsid antigen, demonstrating proof-of-concept materials and methodology to evaluate SARS-CoV-2 rapid antigen detection tests. Quantification of N antigen was used to characterize the relationship between viral count and antigen concentration among clinical samples and panel members of both clinical sample and viral culture origin. This contributes to a deeper understanding of protein antigen and molecular analytes and presents analytical methods complementary to clinical evaluation for characterizing the performance of both laboratory-based and point-of-care rapid diagnostics for SARS-CoV-2.

Platform headspace gas chromatography method for high-throughput determination of residual solvents in pharmaceutical materials.

Static headspace capillary gas chromatography (HSGC) has been employed to monitor the level of residual solvents in the pharmaceutical materials. Most of the HSGC methods, however, consume significant amounts of diluents and require considerable amount of sample preparation time. Accordingly, a HSGC method featured with fast turnaround time, and minimal amount of solvent use has been developed for the quantitative analysis of 27 residual solvents frequently used in the development and manufacturing processes of pharmaceutical industry. This HSGC-FID method employs a commercially available fused silica capillary column, a split injection (40:1), and a programmed temperature ramp. It was qualified for specificity, accuracy, repeatability/precision, linearity, LOQ, solution stability, and robustness using two representative sample matrices. The standards, samples and spiked samples were demonstrated to be stable for at least 10 days at room temperature in sealed headspace vials with a recovery of ≥ 93%. The method was also shown to be robust, and its performance was not affected by small changes of carrier gas flow rate, initial oven temperature or the headspace oven temperature. In this new approach, the analytical sample was prepared by dissolving the sample into 1 mL of the diluent and the standard solution was prepared by diluting 1 mL of the custom-made stock into 9 mL of the diluent whereas the traditional approach requires liters of the diluent, making the new approach environmentally friendly, sustainable, economical, agile, error-proofing and thus appropriate for a variety of pharmaceutical applications.

Comparative assessment of lyophilized and wet reagents for the molecular detection of H5N1 high pathogenic avian influenza virus and H9N2 low pathogenic avian influenza virus.

Global surveillance for Avian Influenza Virus (AIV) in birds is essential for assessing public and animal health risks and real-time polymerase chain reaction (RT-qPCR) is among the official methods recommended by the World Organisation for Animal Health (WOAH) to confirm the presence of the virus in laboratory specimens. Yet, in low-resource setting laboratories, the detection of AIV can be hampered by the need to maintain a cold chain for wet reagents. In such cases, alternatives should be ready to maximize surveillance capacities and mining of AIV. Therefore, we compared two lyophilized RT-qPCR reagents (1st - 5 × CAPITAL™ 1-Step qRT-PCR Probe Reagent, lyophilized kit, and 2nd - Qscript lyo 1-step-kit) to the WOAH recommended protocol by Nagy et al., 2020 using QuantiTect Probe RT-PCR-kit as wet reagent. The comparative study panel comprised 102 RNA samples from two AIV subtypes, i.e. H5 and H9 subtypes. Despite that the wet reagent exhibited the lowest limit of detection (LOD) compared to the two lyophilized reagents, the inter-assay agreement was substantial between the 1st lyophilized reagent and the comparator with 95.1% of shared positive results. Cohen's-kappa was fair between the 2nd lyophilized reagent and the comparator with 75.5% of shared positive results. Agreement using the statistical test Bland-Altman was good for samples with Cq-values < 25 for all reagents, revealing discrepancies when the viral load is low. This trend was especially evident while using the 2nd lyophilized reagent. Similar trends were obtained using the same lyophilized reagents but following the protocol by Heine et al., 2015 with AgPath-ID™ One-Step RT-PCR as a comparator, showing that Cq-values increase using lyophilized reagents but correlate strongly with the wet reagent. Further, inter-assay agreement between reagents improved when the protocol from Heine et al., 2015 was applied, suggesting a higher resilience to chemistry changes allowing easier reagents interchangeability.

Analytical optimization of liquid-liquid extractive spectrophotometric assessment protocol for tetravalent platinum: Analysis of environmental samples and cisplatin.

An easy and reliable method is optimized for extractive spectrophotometric assessment of platinum(IV) with 4-(4'-nitrobenzylideneimino)-3-methyl-5-mercapto-1,2,4-triazole as an extractant. The basis of this method is the formation of red platinum(IV) complex with the above reagent in acetate buffer medium (pH 5.0) and extraction in chloroform. Good linearity with regression equation as y = 1.011 × 104 ×  + 0.002 having correlation coefficient (R2) of 0.998 over concentration up to 17.5 µg mL-1 of platinum(IV) was achieved with apparent molar absorptivity of 1.011 × 104 L mol-1 cm-1. The limit of detection (0.22 µg mL-1), limit of quantification (0.73 µg mL-1) and Sandell's sensitivity (0.0193 µg cm-2) were also estimated. The interference of various cations was removed by using proper masking agents and consequently by using EDTA and citrate to mask certain transition metals, the method becomes highly specific for platinum(IV), including the effects of platinum group metals. The method is effectively used for determination of platinum(IV) from environmental and real samples such as alloys, catalysts, thermocouple wire and pharmaceutical sample.

An origami microfluidic paper device for on-site assessment of urine tampering. First use of Nessler's reagent for the colorimetric determination of creatinine.

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler's reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler's reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

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.

Preliminary forensic assessment of the visualised fingerprints on nonporous substrates immersed in water using the green and optimised novel nanobio-based reagent.

The discovery of forensic evidence (e.g. weapons) during forensic underwater investigations has seen an increasing trend. To date, small particle reagent (SPR) has been one of the routinely used methods for visualising fingerprints on wet, non-porous substrates. However, the long term use of SPR is detrimental to humans and environment due to the use of toxic chemicals. Although previously we have successfully developed and optimised a greener nanobio-based reagent (NBR), its suitable practical use in a more realistic scene (e.g. outdoor pond) was not evaluated. Therefore, this present research is aimed at (1) investigating the performance of NBR against the benchmark SPR in visualising fingerprints immersed in a natural outdoor pond and (2) evaluating the greenness of NBR against the analytical Eco-Scale. Results showed that the performance of the optimised NBR was mostly comparable (University of Canberra (UC) comparative scale: 0) with SPR at visualising fingerprints on three different non-porous substrates immersed in a natural outdoor pond. Observably, the NBR had higher preference towards aged fingerprints (up to 4 weeks of immersion). In addition, its greenness assessment revealed 76 points, indicating 'excellent green analysis'. The findings gathered here further supported the practical use of the NBR in forensic investigations.

Two Instrument Comparison of Reagents From a US FDA-Approved Assay for the Assessment of Ki-67 in High-Risk Early Breast Cancer.

The objective of this study was to measure concordance of results obtained from the US Food and Drug Administration-approved Ki-67 immunohistochemistry MIB-1 pharmDx assay performed on the Dako Omnis automated staining instrument (Omnis) versus results produced from the assay reagents applied using an optimized protocol on the more widely available Autostainer Link 48 (ASL48) platform. Tissue sections obtained from 40 formalin-fixed paraffin-embedded breast carcinoma samples, with available Oncotype DX Breast Recurrence Score (RS) results, were stained. Three certified pathologists scored slides at 3 timepoints, totaling 360 observations for each instrument (N=720 total) using the approved scoring approach. Using the ≥20% cutoff, agreement was calculated with corresponding 2-sided 95% percentile bootstrap confidence intervals (CIs). Pairwise comparisons (N=360) from the interinstrument evaluation, performed with all observers, resulted in 325 (90.3%) concordant outcomes (244 negative and 81 positive) and 35 (9.7%) discordant outcomes. The overall agreement was 90.3% (95% confidence interval, 85.6% to 94.4%). No significant systematic differences were observed between instruments. Specimens scored from the Omnis were on average <1% higher than ASL48, with high correlation and little bias between the continuous Ki-67 scores (concordance correlation coefficient=0.916). Most specimens with a Ki-67 score ≥20% had a RS >25. This study demonstrated that good concordance can be achieved with the reagents run on the ASL48 instrument when using an optimized protocol and standardized scoring.

As diferentes fases, os seus impactos e os desafios da pandemia de covid-19 no Brasil / The different stages, its impacts, and the challenges of the covid-19 pandemic in Brazil / Las diferentes fases, sus impactos y los desafíos de la pandemia de covid-19 e Brasil

A dinâmica da pandemia de covid-19 vem evoluindo segundo fases marcadas por uma maior ou menor taxa de infecção, e diferentes impactos sobre o sistema de saúde e a mortalidade. Essas fases foram estudadas segundo quatro indicadores disponibilizados no sistema MonitoraCovid-19: positividade de testes de diagnóstico, número de casos, número de óbitos e o número de pessoas com ao menos duas doses de vacinas ­ padronizados numa série histórica para permitir sua comparação. São distinguidas cinco fases, marcadas pela circulação de diferentes variantes do vírus, estratégias de implementação ­ ou mesmo boicote ­ de medidas de controle e a vacinação gradativa da população. Esses indicadores podem ser usados no futuro para monitorar tendências e apontar políticas públicas adequadas.

The dynamics of the covid-19 pandemic has evolved according to phases marked by a higher or lower rate of infection, the different impacts on the health system and mortality. These phases were studied according to four indicators made available by the MonitoraCovid-19 system: positivity rates of diagnostic tests, number of cases, number of deaths and number of people who received at least two vaccine doses, standardized in a historical series to allow their comparison. Five phases are discerned, evidenced by the circulation of different virus variants, strategies employed to implement -or even undermine ­ disease control measures and the gradual vaccination of the population. These indicators can be used in the future to monitor trends and point out appropriate public policies.

La dinámica de la pandemia de covid-19 ha evolucionado según fases marcadas por una mayor o menor tasa de contagios, sus diferentes impactos en el sistema de salud y mortalidad. Estas fases se estudiaron según cuatro indicadores disponibles en el sistema MonitoraCovid-19: positividad de las pruebas diagnósticas, número de casos, número de defunciones y el número de personas vacunadas con al menos dos dosis, estandarizados en una serie histórica para permitir su comparación. Se distinguen cuatro fases, marcadas por la circulación de diferentes variantes del virus, las estrategias de implementación - o mismo el boicoteode medidas de control y la paulatina vacunación de la población. Estos indicadores pueden utilizarse en el futuro para monitorear tendencias y señalar políticas públicas apropiadas.

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR.

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

An Atom-Economic Inverse Solid-Phase Peptide Synthesis Using Bn or BcM Esters of Amino Acids.

An atom-economic N-to-C-directed solid-phase peptide synthesis is reported that uses benzyl (Bn) or (benzhydryl-carbamoyl)-methyl (BcM) esters of amino acids as the building blocks, which facilitate efficient hydrazinolysis, convenient conversion to acyl azide, and robust amidation with the next amino acid ester. This method is free of coupling reagents and free of protection on the side-chain OH, CO2H, CONH2, etc., therefore exhibiting a significantly improved atom economy compared to those of BOC- or Fmoc-based C-to-N-directed approaches.