ABSTRACT
Increasing production capacity may necessitate the facility to cater for higher hazardous area category (e.g., H-Occupancy) design features, such as specialized building construction and potential blast zones. [...]an assessment should cover: * Quantification of flammable material use for production steps, including buffer preparation and LNP storage * Equipment and facility cleaning strategies that contribute to the facility flammable materials inventory * Impact of HVAC design to avoid hazardous atmospheres (e.g., full fresh air), use of local exhaust ventilation (LEV) or fume hoods * Solvent distribution methods (e.g., closed solvent delivery and waste removal systems) * Location of solvent bulk storage outside of the processing area/ facility, and piping in what is necessary plus removing spent solvent in a timely manner (e.g., piped transfer to a waste tank for removal by a specialist contractor). At present, the process cannot be fully single-use, so thought needs to be put into the cleaning and sterilization processes, plus the analytical support infrastructure needed for reusable product-contact surfaces. [...]it is recommended that for each mRNA project, consideration is given to the following aspects to determine the link between the equipment available and the facility design: * Need for custom/proprietary equipment * Independent production rooms with "through-wall" buffer transfer through iris ports in from logistics corridor (Buffer Prep/Hold) * Room electrical classification needs versus process step. * Equipment selection versus electrical and fire code requirements * Benefits and limitations of implementing single-use technologies, given that the process will be hybrid (with stainless steel). [...]the limited capacity for outsourcing of supporting functions, such as facility environmental monitoring or product sterility testing, should be considered during concept design.
ABSTRACT
Aside from evaporation of water from buffer solutions, precipitation of buffer salts can also easily occur when a buffer solution comes into contact with a pure organic solvent. [...]one should avoid- at all costs-a situation where a LC system component containing an aqueous buffer is flushed immediately with a pure organic solvent. Solvents and Buffers Figure 1 shows a picture of a buffer bottle I observed in an LC laboratory about a year ago. Many of the aqueous buffer solutions used in LC (and even high-performance liquid chromatography [HPLC] grade water, if given enough time and exposure to aboratory dust) are environments quite favorable to microbes, particularly those in the middle of the pH range.
ABSTRACT
Among Armstrong's many accomplishments is the development of ionic liquid stationary phases for capillary GC. [...]the American Chemical Society Division of Analytical Chemistry Satinder Ahuja New Investigator Award in Separation Science was presented to. Table III provides a listing of accessories and consumables launched over the past year. Besides several short courses associated with Pittcon, additional training and educational resources are available for 2020.
ABSTRACT
Recent advancements in nanotechnology have resulted in improved medicine delivery to the target site. Nanosponges are three-dimensional drug delivery systems that are nanoscale in size and created by cross-linking polymers. The introduction of Nanosponges has been a significant step toward overcoming issues such as drug toxicity, low bioavailability, and predictable medication release. Using a new way of nanotechnology, nanosponges, which are porous with small sponges (below one microm) flowing throughout the body, have demonstrated excellent results in delivering drugs. As a result, they reach the target place, attach to the skin's surface, and slowly release the medicine. Nanosponges can be used to encapsulate a wide range of medicines, including both hydrophilic and lipophilic pharmaceuticals. The medication delivery method using nanosponges is one of the most promising fields in pharmacy. It can be used as a biocatalyst carrier for vaccines, antibodies, enzymes, and proteins to be released. The existing study enlightens on the preparation method, evaluation, and prospective application in a medication delivery system and also focuses on patents filed in the field of nanosponges.Copyright © 2023 The Authors.
ABSTRACT
There are three main components manufactured from whole blood: red blood cells (RBCs), plasma, and platelets. Plasma contains a multitude of different proteins, peptides, and biologic substances. Approximately 53 million liters of plasma was collected in the United States in 2019. Following collection, plasma is frozen and manufactured into plasma-derived medicinal products (PDMPs). During the manufacture process, several thousand plasma units are pooled for Cohn fractionation, which is based upon cold ethanol precipitation of proteins. The PDMPs are further prepared using ion exchange or affinity chromatography and additional steps to inactivate and remove infectious diseases such as viruses. Almost 20 different therapeutic plasma proteins are purified from plasma via these multi-step manufacturing processes. Interestingly, the demand for pharmaceutical plasma products, particularly intravenous immunoglobulin (IVIG) products, has been increasing. The manufacture and therapeutic role of blood derivatives particularly immunoglobulin therapy, Rh immunoglobulin (RhIG), COVID-19 convalescent plasma (CCP) and hyperimmune globulins, albumin, clotting factors, fibrin sealants, and platelet rich plasma will be described.Copyright © 2022 AME Publishing Company. All Rights Reserved.
ABSTRACT
According to the World Health Organization, Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, we seek to develop a genetic prophylaxis for TB. We have determined D-cycloserine to be the optimal target for this approach due to its relatively short six-enzyme biosynthetic pathway. D-CS is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB in human cells, we endeavored to express DcsE in human cells and test its functionality. We overexpressed DcsE tagged with FLAG and GFP in A549 lung cancer cells as determined using fluorescence microscopy. We observed that purified DcsE catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, DcsE synthesized in human cells is a functional enzyme capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards DCS production in human cells.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Spike protein is a receptor protein that has e role in the entry step of SARS-CoV2. This protein will bind to the ACE2 receptor in the human body and activate TMPRSS2. Inhibition of this protein will prevent the binding of the virus to host cells to spread the infection. This study aims to identify the activity of bioactive compounds of Merremia mammosa (Lour) tuber obtained from LC-MS/MS QTOF analysis of a previous study against the Spike protein of SARS-CoV2 using molecular docking and ADMET analysis. Molecular docking was conducted using SARS-CoV2 spike protein (PDB id. 6M0J) using Maestro Schrodinger software. Results showed that from 206 compounds there are 8 compounds of Merremia mammosa (Lour) that have lower predictive binding energies than standard drugs arbidol, hydroxychloroquine, and chloroquine. Result(s): 206 compounds of Merremia mammosa (Lour) tuber were successfully docked, there were 8 compounds that have docking scores more negative than standard drugs. It indicates that 8 compounds are more active than the positive controls. ADMET study revealed all of those potential ligands had the possibility to be developed as drugs. Conclusion(s): Molecular docking simulations were successfully utilized to identify the potential compounds from Merremia mammosa (Lour) tuber with the activity as an inhibitor for spike protein of SARS-CoV2. Further in vitro assay and purification are needed for future research.Copyright © 2021 Muslim OT et al.
ABSTRACT
HbA1c measurement is widely used for diagnosis/ management/remission of diabetes with international schemes certifying comparability. A) 75 year-old Chinese female with type 2 diabetes was admitted in April 2020 with Covid-19 and diabetic ketoacidosis. Glucose was 35mmol/l and HbA1c 150mmol/mol with previous HbA1c of 45mmol/mol on metformin and alogliptin. She was treated for ketoacidosis and once-daily Lantus introduced along with supportive management of viral illness. B) 68 year-old Afro-Caribbean with type 2 diabetes on metformin before admission, presented with new onset, jerky ballistic movements of high amplitude in right arm, 10-15 movements every 5 min. Admission glucose was >33mmol/l, ketones 1.8mmol/l and HbA1c >217mmol/ mol. Hemichorea-hemiballism, a hyperglycaemia related movement was diagnosed and insulin commenced. Glucose decreased to 8-20mmol/ l, reaching 5-15mmol/ l by time of discharge. Ballistic movements resolved when glycaemic control improved with HbA1c 169mmol/mol, 25 days after discharge. C) Several days before admission, a female with diabetes over 20 years required attention from paramedics on four occasions for hypoglycaemia. Months beforehand metformin was replaced by gliclazide due to chronic kidney disease with HbA1c 50mmol/mol, and she was transfused six weeks before admission for microcytic anaemia. Gliclazide was discontinued and her diet modified which prevented further hypoglycaemic episodes. Variant haemoglobin, beta-thalassaemia which can overestimate glycaemia;undetected by HbA1c HPLC method, invalidated HbA1c as did the blood transfusion. These cases highlight that inadequate understanding of HbA1c can lead to acute presentations of dysglycaemia. As HbA1c accuracy can be affected by multiple factors, clinical assessment and triangulation are key to the management of such patients.
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The 6XS6 is the structure of the SARS-CoV-2 spike protein. The physiological role of the spike protein is relative to the respiratory syndrome coronavirus and has a stronger infect on the human body than the ancestor virus. The purification of the 6XS6 is in the homo sapiens cell by the affinity chromatography, PBS supplemented and Size Exclusion chromatography. At last, using the Cryo-Electron Microscopy to see the structure. This paper is using the D614G mutation to illustrate the structure of the 6XS6. The N-terminal domain and C-terminal domain of the 6XS6 protein are ALA27 and VAL1137. Furthermore, the mutation doesn't have the hydrogen bond because the Asp614 is substituted by the Gly614, and the molecule that interacts with the Ala 647 may occur. While the 6XS6 structure has lots of non-covalent and disulfide bonds. Comparing the structure of the 6XS6 and 6VXX, both are glycoproteins, have three monomers, have two subunits, and have the same category of expression and classification. The different conformations of the two structures can affect the binding ability with the ACE2. This paper can help the researchers to further understand the structure and function of the 6XS6 which can be used in future experiments. © 2023 SPIE.
ABSTRACT
The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Reducing the molecule complexity is achieved by reducing the molecule size after enzymatic digestion to produce smaller fragments more amenable to LC separation and tandem mass spectrometry (MS/MS) sequencing. Non-denaturing CEX chromatography, size-exclusion chromatogra- phy (SEC), hydrophobic interaction chromatography (HIC), and protein A modes can be easily coupled to reversed-phase LC (RPLC) because of the high aqueous conditions, enabling the versatile 4D-LC-MS systems with the use of alternative modes to 1D CEX, such as SEC or Protein A (6,7). [...]the nanopar-ticle size and free drug concentration are determined at the particle Level, whereas the encapsulated drug and lipids forming the layer are commonly characterized at the molecuar level after denaturing the lipid nanoparticle (LNP) via a surfactant. [...]MDLC-MS setups present a formidable opportunity to unify the characterization of drug delivery systems at the molecular and particle evels, which would enable their high throughput analysis.
ABSTRACT
The COVID19 pandemic accelerated opportunities for innovation within the decentralization process of clinical trials with opportunities for implementation of patient-centric workflows for efficiency and cost-reduction. Decentralized sample collection, particularly whole blood using dried blood spots (DBS) provides the ideal mechanism for patient driven sample collection with ease of access to sample generation, drug level assessments and metabolomic prMegofiling, providing longitudinal real-time measure of drug specific pharmacodynamic readout for safety and efficacy. In this study, we report the development of a protocol for the capture and comprehensive profiling of metabolomics using dried blood spots from a cohort of 49 healthy volunteer donors. Using liquid chromatography combined with mass spectrometric (UPLC-MS/MS) methods an untargeted metabolomic approach resulted in the identification of >800 biochemicals of which a significant subset was found to be presented in corresponding matched plasma (from whole blood) samples. The biochemicals identified from the DBS samples included metabolites that were part of the lipid, amino acid, nucleotide, peptide, cofactors, carbohydrate and energy super pathways. A significant number of metabolites identified in the DBS samples were xenobiotics including those representing the biotransformation products of drugs. The overall metabolite profiles were analyzed for precision and accuracy of measure, variability in performance and dynamic range to establish benchmarks for evaluation. An additional cohort with a longitudinal sampling as part of the protocol provided the reproducibility of the analytic method for inter-day variability of metabolite performance over time. Although metabolomic profiles varied between individuals from a population perspective, there was minimal variation observed within individuals when samples were profiled longitudinally over several weeks. Thus, the protocols for DBS collection and the corresponding capture of a large set of metabolites with reproducible performance provides an opportunity for its implementation in oncological clinical trials as part of a de-centralized clinical trial solution.
ABSTRACT
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.
ABSTRACT
The people who work at companies that manufacture chromatography instruments and consumables are often well positioned to be aware of developments, needs, and trends that not everyone else sees, because they serve customers in a range of areas of focus and with diverse demands-such as academic researchers investigating fundamental aspects of separation science techniques, industrial analysts solving problems that they may not be allowed to talk about at conferences, or chemists working in government laboratories in areas like environmental research. Inaccurate data can be generated from variable, glass vial surface chemistries, which can lead to investigations or flawed decisions can be made from these results. The Reduced Surface Activity (RSA(tm)) Glass Technology was developed to address these issues, and to provide chemists with sample containers for LC-MS, MS, HPLC, GC, and CE that deliver reliable, consistent results by not adsorbing basic analytes, or adding metals to, or changing the pH of the diluent. The stringent RSA manufacturing processes are continued through to final contaminate-free packaging and quality control, where they are tested for adsorption, metal content, and residual materials.
ABSTRACT
Plain language summaryMERS-CoV is a virus that causes a severe illness in the nose, mouth and throat of humans. It is a zoonotic virus, which means that it can spread from animals to humans. MERS-CoV was first found in Saudi Arabia in 2012 and continues to pose a threat to public health. Interactions between the virus and human cells and proteins are important to establishing infection. Understanding these interactions is important for the development of drugs to treat viral infections. Here, we have identified some proteins that interact with MERS-CoV. Tweetable A proteomic approach for the identification of cellular proteins that interact with the 5 '-terminal region of MERS-CoV RNA genome. #MERS-CoV #RNA_viruses. Aim: The aim of this study was to identify host factors that interact with the 5 ' end of the MERS-CoV RNA genome. Materials & methods: RNA affinity chromatography followed by mass spectrometry analysis was used to identify the binding of host factors in Vero E6 cells. Results: A total of 59 host factors that bound the MERS-CoV RNA genome in non-infected Vero E6 cells were identified. Most of the identified cellular proteins were previously reported to interact with the genome of other RNA viruses. We validated our mass spectrometry results using western blotting. Conclusion: These data enhance our knowledge about the RNA-host interactions of coronaviruses, which could serve as targets for developing antiviral therapeutics against MERS-CoV.
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Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and biomedical research. Here we propose combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition, and data analysis with the DIA-NN software suite, to achieve high-quality proteomic experiments from limited sample amounts, at a throughput of up to 400 samples per day. For instance, when benchmarking our workflow using a 500-µL/min flow rate and 3-min chromatographic gradients, we report the quantification of 5211 proteins from 2 µg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used this platform to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-min chromatographic gradient and alternating column regeneration on a dual pump system. The method delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates.
ABSTRACT
Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 µm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode.
ABSTRACT
The COVID-19 pandemic caused several educational challenges. Conducting laboratory experiments was an uphill task during the pandemic. Here, we developed a low-cost and reliable home-based experimental setup to teach column and thin layer chromatography (TLC) using silica gel granules available at home. Powdered silica gel, prepared by grinding silica gel granules, was used as the stationary phase. Iso-propyl alcohol, purchased from a pharmacy, was diluted with water and used as the mobile phase. A food coloring was chromatographically separated using the designed column. Moreover, TLC plates were prepared using powdered silica gel and a drop of food coloring was separated on TLC plates using the same mobile phase. In the article, we show our experiences by providing methods used to implement this experimental setup. We assume that this experimental setup will be helpful for other universities, research institutes and schools to develop online laboratory curricula to demonstrate basic chromatography techniques required for subjects such as chemistry, biochemistry and biology.
ABSTRACT
Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.