Hybrid Peptide–Agarose Hydrogels for 3D Immunoassays

Recent advances in biosensing analytical platforms have brought relevant outcomes for novel diagnostic and therapy-oriented applications. In this context, 3D droplet microarrays, where hydrogels are used as matrices to stably entrap biomolecules onto analytical surfaces, potentially provide relevant advantages over conventional 2D assays, such as increased loading capacity, lower nonspecific binding, and enhanced signal-to-noise ratio. Here, we describe a hybrid hydrogel composed of a self-assembling peptide and commercial agarose (AG) as a suitable matrix for 3D microarray bioassays. The hybrid hydrogel is printable and self-adhesive and allows analyte diffusion. As a showcase example, we describe its application in a diagnostic immunoassay for the detection of SARS-CoV-2 infection. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Trace determination of the hydrogen sulfide biomarker thiosulfate in human urine by HPLC coupled with element selective ICPMS/MS detection

Hydrogen sulfide is a toxic gas but also established as a naturally occurring gaseous signaling molecule in humans, playing key physiological roles with particular involvement in lung disease including COVID-19. Thiosulfate is the conventional biomarker of hydrogen sulfide and is excreted in human urine at low micromolar levels. Thiosulfate is amenable to detection by the element-selective inductively coupled plasma tandem mass spectrometry (ICPMS/MS), but sulfur speciation in human samples at trace levels is challenging due to the high complexity of human sulfur metabolome and the utility of this detector under such settings has not been demonstrated. We report a method for thiosulfate determination in human urine at trace physiological levels by HPLC-ICPMS/MS. The method involved one-step derivatization to improve chromatographic behavior followed by direct injection. The instrumental limit of detection was 1.4 μg S L−1 (0.02 μM or 0.1 pmol). In a group of samples from volunteers (n = 24), measured thiosulfate concentrations in the diluted urine matrix were down to 8.0 μg S L−1 with a signal-to-noise ratio >10. The method was validated for recovery (80–110%), repeatability (RSD% <5%), and linearity (r2 = 0.9999, at a tested working concentration range of 0.01–1.0 mg S L−1), and the accuracy was assessed by comparing with HPLC-ESIMS/MS which showed agreement within ±20%. This work demonstrates the applicability of HPLC-ICPMS/MS for sulfur speciation at trace levels in a matrix with complex sulfur metabolome as human urine and provides a sensitive method for the determination of the hydrogen sulfide biomarker. © 2022 The Authors

Integration of a hybrid scan approach and in-house high-resolution MS2 spectral database for charactering the multicomponents of Xuebijing Injection

Xuebijing (XBJ) Injection is a reputable patent Chinese medicine widely used to cure sepsis, among the Chinese ″Three Medicines and Three Prescriptions″ solution to fight against COVID-19. We were aimed to achieve the comprehensive multicomponent characterization from the single drugs to traditional Chinese medicine (TCM) formula, by integrating powerful data acquisition and the in-house MS2 spectral database searching. By ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS), a hybrid scan approach (HDMSE-HDDDA) was developed, while the HDMSE data for five component drugs and 56 reference compounds were acquired and processed to establish an in-house MS2 spectral database of XBJ. Good resolution of the XBJ components was accomplished on a Zorbax Eclipse Plus C18 column within 24 min, while a fit-for-purpose HDMSE-HDDDA approach was elaborated in two ionization modes for enhanced MS2 data acquisition. XBJ MS2 spectral library was thus established on the UNIFITM platform involving rich structure-related information for the chemicals from five component drugs. We could identify or tentatively characterize 294 components from XBJ, involving 81 flavonoids, 51 terpenoids, 42 phthalides, 40 organic acids, 13 phenylpropanoids, seven phenanthrenequinones, six alkaloids, and 54 others. In contrast to the application of conventional MS1 library, this newly established strategy could demonstrate superiority in the accuracy of identification results and the characterization of isomers, due to the more restricted filtering/matching criteria. Conclusively, the integration of the HDMSE-HDDDA hybrid scan approach and the in-house MS2 spectral database can favor the efficient and more reliable multicomponent characterization from single drugs to the TCM formula. © 2022 The Author(s)

Identification and high-throughput quantification of baicalein and its metabolites in plasma and urine

Ethnopharmacological relevance: Scutellaria baicalensis Georgi. contains varieties of function compounds, and it has been used as traditional drug for centuries. Baicalein is the highest amount of flavonoid found in Scutellaria baicalensis Georgi., which exerts various pharmacological activities and might be a promising drug to treat COVID-19. Aim of the study: The present work aims to investigate the metabolism of baicalein in humans after oral administration, and study the pharmacokinetics of BA and its seven metabolites in plasma and urine. Materials and methods: The metabolism profiling and the identification of baicalein metabolites were performed on HPLC-Q-TOF. Then a column-switching method named MPX™-2 system was applied for the high-throughput quantificationof BA and seven metabolites. Results: Seven metabolites were identified using HPLC-Q-TOF, including sulfate, glucuronide, glucoside, and methyl-conjugated metabolites. Pharmacokinetic study found that BA was extensively metabolized in vivo, and only 5.65% of the drug remained intact in the circulatory system after single dosing. Baicalein-7-O-sulfate and baicalein-6-O-glucuronide-7-O-glucuronide were the most abundant metabolites. About 7.2% of the drug was excreted through urine and mostly was metabolites. Conclusion: Seven conjugated metabolites were identified in our assay. A high-throughput HPLC-MS/MS method using column switch was established for quantifying BA and its metabolites. The method has good sensitivity and reproducibility, and successfully applied for the clinical pharmacokinetic study of baicalein and identified metabolites. We expect that our results will provide a metabolic and pharmacokinetic foundation for the potential application of baicalein in medicine. © 2022

Extraction and characterization of metabolites from Olea europaea pulp and their molecular docking against SARS-CoV-2 main-protease (Mpro).

The present study is the first to extract the bioactive metabolites from Olea europaea fruit using the Soxhlet-maceration extraction method. The preliminary phytochemical; Fourier transform-infrared spectroscopy (FT-IR); gas chromatography-mass spectrometry (GC-MS) analyses, and their potential against SARS-CoV-2 Mpro through molecular docking were studied. The preliminary qualitative phytochemical analyses showed coumarin glycosides, tannins, terpenoids, cholesterol, carbohydrates, and proteins. FT-IR spectroscopy revealed C-H, C = O, O-H, C-N, C-O-C, C-O, CO-O-CO, C = C, and C-Br functional groups in the extract. GC-MS analysis was done and the compounds detected were docked against SARS-CoV-2 Mpro using AutoDock Vina.The squalene (ΔG = -6.2 kcal/mol) posed the best inhibition potential and was comparable with the control drug remdesivir. The compounds possessed excellent pharmacokinetic and toxicity properties and are safe and reliable. Thus, the present research unveiled the valuable metabolites from O. europaea and their antiviral potential against the SARS-CoV-2.

Alternate end-game strategies towards Nirmatrelvir synthesis: Defining a continuous flow process for the preparation of an anti-COVID drug

Scalable alternate end-game strategies for the synthesis of the anti-COVID drug molecule Nirmatrelvir (1, PF-07321332) have been described. The first involves a direct synthesis of 1 via amidation of the carboxylic acid 7 (suitably activated as a mixed anhydride with either pivaloyl chloride or T3P) with the amino-nitrile 10·HCl. T3P was found to be a more practical choice since the reagent promoted efficient and concomitant dehydration of the amide impurity 9 (derived from the amino-amide contaminant 8·HCl invariably present in 10·HCl) to 1. This observation allowed for the development of the second strategy, namely a continuous flow synthesis of 1 from 9 mediated by T3P. Under optimized conditions, this conversion could be achieved within 30 min in flow as opposed to 12–16 h in a traditional batch process. The final API had quality attributes comparable to those obtained in conventional flask processes.

Fabric phase sorptive extraction combined with high performance liquid chromatography for the determination of favipiravir in human plasma and breast milk

A fast procedure obtained by the combination of fabric phase extraction (FPSE) with high performance liquid chromatography (HPLC) has been developed and validated for the quantification of favipiravir (FVP) in human plasma and breast milk. A sol-gel polycaprolactone-block-polydimethylsiloxane-block-polycaprolactone (sol-gel PCAP-PDMS-PCAP) coated on 100% cellose cotton fabric was selected as the most efficient membrane for FPSE in human plasma and breast milk samples. HPLC-UV analysis were performed using a RP C18 column under isocratic conditions. Under these optimezed settings, the overall chromatographic analysis time was limited to only 5 min without encountering any observable matrix interferences. Following the method validation procedure, the herein assay shows a linear calibration curve over the range of 0.2–50 µg/mL and 0.5–25 µg/mL for plasma and breast milk, respectively. The method sensitivities in terms of limit of detection (LOD) and limit of quantification (LOQ), validated in both the matrices, have been found to be 0.06 and 0.2 µg/mL for plasma and 0.15 and 0.5 µg/mL for milk, respectively. Intraday and interday precision and trueness, accordingly to the International Guidelines, were validated and were below 3.61% for both the matrices. The herein method was further tested on real samples in order to highlight the applicability and the advantage for therapeutic drug monitoring (TDM) applications. To the best of our knowledge, this is the first validated FPSE-HPLC-UV method in human plasma and breast milk for TDM purposes applied on real samples. The validated method provides fast, simple, cost reduced, and sensitive assay for the direct quantification of favipiravir in real biological matrices, also appliyng a well-known rugged and cheap instrument configuration. © 2022 Elsevier B.V.

Presence and toxicity of drugs used to treat SARS-CoV-2 in Llobregat River, Catalonia, Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the on-going COVID-19 pandemic. In March 2020, it was declared global pandemic, causing millions of deaths. An evident tendency of global pharmaceutical consumption due to COVID-19 pandemic should be seen worldwide, and this increase might suppose an environmental threat. Pharmaceuticals administrated at home or in pharmacies are excreted by faeces and urine after consumption, and wastewater treatment plants (WWTPs) are not able to remove all pharmaceuticals residues that eventually will end up in the aquatic media (rivers and sea). For this reason, analytical techniques such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have become prominent to identify and quantify pharmaceuticals residues in aquatic matrices. In view of the scarce data on the occurrence of pharmaceuticals used as COVID-19 treatment, the aim of the present study was to evaluate the presence of these class of pharmaceuticals in river water which were dexamethasone, prednisone, ciprofloxacin, levofloxacin, remdesivir, ritonavir, lopinavir, acetaminophen, hydroxychloroquine, chloroquine and cloperastine, their toxicity in the aquatic environment using D. magna and to perform an exhaustive risk assessment in seven points of the Llobregat river basin. Dexamethasone, cloperastine and acetaminophen were the pharmaceuticals with higher concentrations, showing mean levels between 313 and 859 ng L-1.

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein.

Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein.

Impact of the loss of Laboratory Developed Mass Spectrometry testing at a major academic medical center.

Background: Our laboratory historically performed immunosuppressant and definitive opioid testing in-house as laboratory developed (LDT) mass spectrometry-based tests. However, staffing constraints and supply chain challenges associated with the COVID-19 pandemic forced us to refer this testing to a national reference laboratory. The VALID Act could impose onerous requirements for laboratories to develop LDTs. To explore the potential effect of these additional regulatory hurdles, we used the loss of our own LDT tests to assess the impact on patient care and hospital budgets. Methods: Laboratory information systems data and historical data associated with test costs were used to calculate turnaround times and financial impact. Results: Referral testing has extended the reporting of immunosuppressant results by an average of approximately one day and up to two days at the 95th percentile. We estimate that discontinuing in-house opioid testing has cost our health system over half a million dollars in the year since testing was discontinued. Conclusions: Barriers that discourage laboratories from developing in-house testing, particularly in the absence of FDA-cleared alternatives, can be expected to have a detrimental effect on patient care and hospital finances.

Increasing yield of in vitro transcription reaction with at-line HPLC monitoring.

The COVID-19 pandemic triggered an unprecedented rate of development of mRNA vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of IVT depends on understanding of the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line HPLC monitoring of consumption of NTPs with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/mL for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an ARCA-capped mRNA construct, which requires an approximate 4:1 ARCA:GTP ratio, the optimized fed-batch approach achieved productivity of 9 mg/mL with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thereby paving the way towards continuous manufacturing of mRNA. This article is protected by copyright. All rights reserved.

A Brief Review on Guduchi (Tinospora Cordifolia)

Guduchi (Tinospora cordifolia) is an essential drug of the Ayurvedic medicine system used in different Ayurvedic formulations to treat a variety of ailments. Guduchi is a member of the Menispermaceae family and is widely produced in tropical and sub-tropical countries such as India, Sri Lanka, China, Myanmar, Philippines, South Africa, Thailand, Bangladesh, and several south-east Asian continents such as Indonesia, Malaysia. All parts of Guduchi have nutritional value and medicinal importance, including the roots, stem, bark, and leaves. A different class of phytochemicals like alkaloids, glycosides, aliphatic compounds, diterpenoids, sesquiterpenoids, phenolic compounds, steroid and polysaccharides, etc., are found in Guduchi. Tinosporaside, tinosporine, magnosporine, berberine, choline, Jatrorrhizine, palmatine, beberine, giloin, giloinsterol, and other beneficial biomarkers are present in this herb. Guduchi is used to treat cold, fever, headache, jaundice, digestive disorder, among other things, and it shows several proven pharmacological activities such as anti-oxidant, anti-inflammatory, antidiabetic, immunomodulatory activity, anti-toxic, hepatoprotective, anticancer, cardioprotective activity, radioprotective, antimicrobial, anti-stress, anti-HIV and many more. This review article majorly highlights the phytochemical present in Guduchi, analytical works and pharmacological activities of Guduchi. Copyright © 2022 are reserved by International Journal of Pharmaceutical Sciences and Research.

Tsukamurella and mycobacterium tuberculosis pneumonia co-infection

Case Report: Tsukamurella species are aerobic, partially acid fast saprophytes commonly isolated from soil and water. They are opportunistic pathogens known to infect multiple organs and can contribute to significant pathologies such as bacteremia, peritonitis, and respiratory tract infections. Moreover, Tsukamurella shares certain characteristic properties to Mycobacterium tuberculosis and Actinomyces species, including the acid fast stain, which can contribute to misdiagnosis of patients. A 68 year old female patient presented to the ED for shortness of breath, fatigue, and weight loss for 6 months. The patient's past medical history includes pulmonary fibrosis, type 2 diabetes, coronary artery disease with stent, hyperlipidemia, hypertension, and M. tuberculosis infection when she was 3 years old in Finland. On admission, labs revealed thrombocytosis (reactive 555 000/microL), leukocytosis (14 450/microL), and microcytic anemia (9.4 microg/dl). Moreover, C reactive protein was elevated and procalcitonin was normal (0.06 microg/l);a COVID-19 PCR was negative. An X-ray revealed severe patchy and interstitial infiltrates throughout both lungs with parenchymal scarring and pleural thickening in the periphery of the left mid-lung zone with multifocal pneumonia. Blood and sputum cultures were performed under the impression of pneumonia, and treatment with azithromycin and ceftriaxone was started. A M. tuberculosis infection was suspected due to a positive AFS. Further chest CT suggested multifocal pneumonia within the left lung in addition to apparent cavitary lesions versus bulla, a chronic interstitial lung disease with traction bronchiectasis, calcified right lower lung nodule, and calcified hilar lymph nodes suggesting a history of granulomatosis diseases. A bronchoscopy with Bronchoalveolar lavage was performed. The initial sputum specimen direct smear showed acid-fast stain positive with Actinomyces growth, and Penicillin G was added to the treatment. Samples were sent to the state department lab, and biopsy revealed granulomatous inflammation negative for malignant cells. One month later, the patient's sputum culture showed Tsukamurella for High-performance liquid chromatography (HPLC). Moreover, a rifampicin sensible M. tuberculosis complex by NAA was also positive six weeks later. The patient was started on a complete TB regimen and continued in the outpatient pulmonology clinic with the addition of levofloxacin for three months and rifampicin substituted for rifabutin. As demonstrated in the case above, a Tsukamurella infection can present similarly to a Mycobacterium infection. Patients may be misdiagnosed or potentially be co-infected. Our patient was further tested and appropriately treated for Tsukamurella after further extensive diagnostic screenings. Due to a high rate of missed cases, it is important to keep Tsukamurella infection on the differential diagnosis as the patient presentation may initially appear to be a Mycobacterium or other pulmonary infection. Copyright © 2023 Southern Society for Clinical Investigation.

Shilajit : Extraction, Enrichment & Formulation

Incidences and mortality from cardiovascular illnesses have grown during the last few decades. In patients with pre-existing cardiac disease, the COVID-19 pandemic has exacerbated cardiovascular problems. Researchers observed that rates of many conditions, comprising coronary heart failure and stroke, had been drastically higher in those who had recovered from COVID-19 as compared to those who did not have the disease. Herbal medicine is becoming more and more popular due to its relatively high safety and low toxicity. Shilajit has several beneficial properties because it primarily comprises fulvic acid and more than 84 minerals. It demonstrates antioxidant activity, which is a key factor in cardioprotection. Shilajit is appropriately referred to as "Rasayana/ Rasayanam";it translates as "rejuvenator" in Ayurvedic and Siddha scriptures since it prevents illnesses and improves the quality of life. The current study involved the collection, authentication, purification, and use of several extractive solvents to achieve high yield, then the extract was enriched by using organic solvents. UV-Vis spectroscopy was performed on each of the enriched samples, and the results showed that the enriched samples had increased fulvic acid concentrations. Fulvic acid was used as a standard in thin-layer chromatography. The extract's in-vitro antioxidant activity was assessed using the DPPH assay, and the results indicated good antioxidant activity. phytosomes were formulated by rapid injection methods for extract and enriched extract, and they were characterized using several spectroscopic techniques. According to the study's findings, combining novel formulation with conventional drug may improve its therapeutic potential and bioavailability.

Optimization of ultrasonication-assisted extraction conditions using RSM-I-Optimal experimental design to recover vitamin D2 and K1 from selected green leafy vegetable samples

This study employed the response surface methodology to optimize the extraction conditions for recovering vitamins D2 and K1 from green leafy vegetables using ultrasonication-assisted extraction. The vitamin content was determined using an Accucore C18 column and a UPLC-Q-ToF/MS method. An RSM-I-Optimal design was used for designing the experiment to find the best combination of solvent level (mL), sonication time (min), sonication frequency (kHz), and temperature (°C). The experimental data from a 25-sample set were fitted to a second-order polynomial equation using multiple regression analysis. The extraction models had R2 values of 0.895 and 0.896, respectively, and the probability values (p < 0.0001) indicated that the regression model was highly significant. The optimal extraction conditions were: solvent level of 65 mL, sonication time of 45 min, sonication frequency of 70 kHz, and temperature of 45 °C. Under these conditions, the predicted recovery (%) values for vitamins D2 and K1 were 90.7% and 90.4%, respectively. This study has the potential to use the reported extraction method for routine quantification of vitamins D2 and K1 in the laboratory using UPLC-Q-ToF/MS.

Characteristics of elderly patients with rheumatoid arthritis who have not responded to methotrexate therapy within 24 weeks

Objective: To describe the clinical, laboratory and pharmacokinetic features of elderly patients with rheumatoid arthritis (RA), with insufficient response to methotrexate (MTX) therapy for 24 weeks compared with patients with a good response. Material(s) and Method(s): The study included 32 patients with RA, according to the older age category according to WHO criteria, 65 (82%) women and 14 (18%) men, BMI was 27 +/- 4 kg/m2, DAS28 was 5.9 +/- 1. In each case, MTX was administered parenterally, at the rate of 10-15 mg/m2 of body surface. therapeutic drug monitoring was carried out, it was the determination of the concentrations of MTX monoglutamate (initial form) and MTX compounds: polyglutamates and 7-hydroxymethotrexate (7-OH- MT) in erythrocytes (ER) and mononuclear cells (MO) after 4, 12 and 24 weeks. We used high performance liquid chromatography with mass spectrometric detection. The MTX metabolite index was calculated (the ratio of the metabolite concentration to the initial concentration of unchanged MT). Achievement of therapy targets (good response to therapy) was established in accordance with the EULAR criteria. The lack of achievement of therapy goals corresponded to an insufficient response to therapy. Result(s): By week 24, 12 patients (36%, group 1) achieved therapy targets, 17 patients (53%, group 2) did not reach treatment targets, and in 3 more, MTX was discontinued due to Adverse reactions (ARs) and/or the development of COVID-19. A comparison was made of clinical and laboratory parameters before the start of MTX treatment and during MTX therapy. At all stages of the study the groups did not differ in terms of: sex, age, BMI, disease duration, VAS (pain), DAS28 index, creatinine, taking glucocorticoids, statins, the presence and frequency of comorbid pathology (arterial hypertension, diabetes mellitus, chronic autoimmune thyroiditis). The 7-OH- MTX( ER) metabolic index after 12 weeks of treatment was higher in group 1 (1.35 [0.8;2.1] versus 0.35 [0.19;0.73] in group 2). Metabolic indices of other MTX metabolites did not differ. ARs were less common in group 1 (in 1 (18%) versus 6 (35%) in group 2), P = 0.09. Conclusion(s): Clinical and laboratory characteristics of patients of the older age group did not differ in groups with different responses to methotrexate therapy. The 7-OH- MT( ER) metabolism index after 12 weeks of treatment was higher in the group of patients with a good response to therapy, which most likely indicates a more rapid catabolism of MTX in this group of patients.

Analytical approaches for determination of COVID-19 candidate drugs in human biological matrices.

Since the outbreak of the COVID-19 pandemic, the use of antiviral and other available drugs has been considered to combat or reduce the clinical symptoms of patients. In this regard, it would be necessary to choose sensitive and selective analytical techniques for pharmacokinetic and pharmacodynamic studies, monitoring of drug concentration in biological fluids, and determination of the most appropriate dose to achieve the desired effect on the disease. In the present study, the analytical techniques based on spectroscopy and chromatography with different detectors for diagnosis and determination of candidate drugs in the treatment of COVID-19 in human biological fluids are reviewed during the period 2015-2022. Moreover, various sample preparation and extraction techniques, are being used for this purpose, such as protein precipitation (PP), solid-phase extraction (SPE), liquid-liquid extraction (LLE), and QuEChERS (quick, easy, cheap, effective, rugged, and safe) are investigated.

Red Blood Cell Substitutes: Liposome Encapsulated Hemoglobin and Magnetite Nanoparticle Conjugates as Oxygen Carriers.

The established blood donation and transfusion system has contributed a lot to human health and welfare, but for this system to function properly, it requires a sufficient number of healthy donors, which is not always possible. Pakistan was a country hit hardest by COVID-19 which additionally reduced the blood donation rates. In order to address such challenges, the present study focused on the development of RBC substitutes that can be transfused to all blood types. This paper reports the development and characterization of RBC substitutes by combining the strategies of conjugated and encapsulated hemoglobin where magnetite nanoparticles would act as the carrier of hemoglobin, and liposomes would separate internal and external environments. The interactions of hemoglobin variants with bare magnetite nanoparticles were studied through molecular docking studies. Moreover, nanoparticles were synthesized, and hemoglobin was purified from blood. These components were then used to make conjugates, and it was observed that only the hemoglobin HbA1 variant was making protein corona. These conjugates were then encapsulated in liposomes to make negatively charged RBC substitutes with a size range of 1-2 µm. Results suggest that these RBC substitutes work potentially in a similar way as natural RBCs work and can be used in the time of emergency.

Urinary Glycosaminoglycans: Characterization and Quantification.

The growing body of evidence supports the potential of using urinary glycosaminoglycans (uGAGs) levels as biomarkers to guide diagnosis and as predictive biomarkers of treatment efficacy. Recently, studies have shown that, in addition to MPS, the prognosis and treatment of cancers and viral infections, including COVID-19, are enabled by characterization and/or traits by GAGs. Reliable and accessible detection and assay protocols of urinary GAGs are therefore of great support for laboratory workers and clinicians. Here we describe a semiquantitative and quantitative urinary glycosaminoglycans determination using 1,9-dimethylmethylene blue (DMB) and the characterization of uGAGs using thin layer chromatography (TLC).

Systematic characterization of components of Makyo-kanseki-to granule and serum metabolomics for exploring its protective mechanism against acute lung injury in lipopolysaccharide-induced rats.

Makyo-kanseki-to has been used for the treatment of pneumonia, becoming a basic formula for coronavirus disease 2019. However, the chemical profile of Makyo-kanseki-to granule and its possible mechanism against acute lung injury from terminal metabolic regulation have been unclear. The aim of this study was to characterize the constituents in Makyo-kanseki-to granule and reveal the potential related mechanism of Makyo-kanseki-to granule treatment for acute lung injury using a rat model of lipopolysaccharide-induced acute lung injury. Totally, 78 constituents were characterized based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Makyo-kanseki-to granule could alleviate acute lung injury through modulating rectal temperature, pulmonary edema, histopathology, and processes of inflammatory and oxidative stress. Twenty-two potential biomarkers in acute lung injury rats were identified by metabolomics based on ultra-performance liquid chromatography coupled with quadrupole exactive high-field mass spectrometry. They were mainly involved in amino acids and glycerophospholipid metabolism, which were regulated by Makyo-kanseki-to granule. The present results not only increase the understanding of the chemical profile and molecular mechanism of Makyo-kanseki-to granule mediated protection against acute lung injury but also provide an experimental basis and new ideas for further development and clinical application of Makyo-kanseki-to granule.