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1.
Sci Rep ; 12(1): 5968, 2022 Apr 08.
Article in English | MEDLINE | ID: covidwho-1784028

ABSTRACT

Skin antiseptics have important implications for public health and medicine. Although conventional antiseptics have considerable antimicrobial activity, skin toxicity and the development of resistance are common problems. Plasma-treated water has sterilization and tissue-regenerative effects. Therefore, the aim of this study was to identify whether plasma-activated water (PAW) manufactured by our microwave plasma system can be used as a novel antiseptic solution for skin protection. PAW was produced by dissolving reactive nitrogen oxide gas using microwave plasma in deionized water. The antibacterial effects of PAW against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Salmonella typhimurium and effective concentrations were investigated by a solid agar plate assay. The factors mediating the effects of PAW were evaluated by the addition of reactive species scavengers. Cytotoxicity and cell viability assays were performed to examine the protective effect of PAW on normal skin cells. PAW exhibited excellent sterilization and no toxicity in normal skin cells. Experiments also confirmed the potential of PAW as a sanitizer for SARS-CoV-2. Our findings support the use of PAW as an effective skin disinfectant with good safety in the current situation of a global pandemic.


Subject(s)
Anti-Infective Agents, Local , COVID-19 , Disinfectants , Anti-Infective Agents, Local/pharmacology , Disinfectants/pharmacology , Escherichia coli , Humans , Microwaves , Pandemics , SARS-CoV-2 , Water/pharmacology
2.
Int J Environ Res Public Health ; 19(7)2022 Apr 04.
Article in English | MEDLINE | ID: covidwho-1776226

ABSTRACT

In the COVID-19 pandemic context, numerous concerns have been raised regarding the hygienic status of certain objects we interact with on a daily basis, and especially cash money and their potential to harbor and transmit pathogenic bacteria. Therefore, in the present study, we analyzed different currency bills represented by British pounds (5 £, 10 £ and 20 £), Romanian lei (1 leu, 5 lei and 10 lei), U.S. dollars (1 $, 5 $ and 10 $) and Euros (5 €, 10 € and 20 €) in order to evaluate the bacterial survival rate and bacterial adherence. We used five reference microorganisms by American Type Culture Collection (ATCC, Manassas, VA, USA): Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Enterococcus sp. ATCC 19952, Salmonella enterica subsp. enterica serovar Typhi ATCC 6539, and Listeria monocytogenes ATCC 7644. Microorganisms were selected in accordance with the criteria of prevalence, pathogenicity, opportunism, and incidence. However, Maldi-TOF analysis from samples taken from the banknotes revealed only a few of the common pathogens that are traditionally thought to be found on banknotes. Some of the most important factors for the survival of pathogenic agents on surfaces are the presence of organic matter, temperature and humidity. Our data showed that Salmonella enterica survived 72 h on every banknote tested, while L. monocytogenes tended to improve persistence in humid conditions. Survival rate is also influenced by the substrate composition, being lower for polymer-based banknotes especially for Salmonella enterica, Listeria monocytogenes and Enterococcus sp. The adherence of bacterial strains was lower for polymer-based banknotes British pounds and Romanian Leu, in contrast to the cotton-based U.S dollars and Euro banknotes. The risk of bacterial contamination from the banknote bills is high as indicated by both a strong survival capacity and low adherence of tested bacteria with differences between the two types of materials used for the tested banknotes.


Subject(s)
COVID-19 , Listeria monocytogenes , Salmonella enterica , Escherichia coli , Humans , Pandemics , Polymers , Survival Rate
3.
Front Public Health ; 10: 794513, 2022.
Article in English | MEDLINE | ID: covidwho-1775996

ABSTRACT

Aquatic environments, under frequent anthropogenic pressure, could serve as reservoirs that provide an ideal condition for the acquisition and dissemination of antibiotic resistance genetic determinants. We investigated the prevalence and diversity of antibiotic-resistant Escherichia coli by focusing on their genetic diversity, virulence, and resistance genes in anthropogenic-impacted Larut River. The abundance of E. coli ranged from (estimated count) Est 1 to 4.7 × 105 (colony-forming units per 100 ml) CFU 100 ml-1 to Est 1 to 4.1 × 105 CFU 100 ml-1 with phylogenetic group B1 (46.72%), and A (34.39%) being the most predominant. The prevalence of multiple antibiotic resistance phenotypes of E. coli, with the presence of tet and sul resistance genes, was higher in wastewater effluents than in the river waters. These findings suggested that E. coli could be an important carrier of the resistance genes in freshwater river environments. The phylogenetic composition of E. coli and resistance genes was associated with physicochemical properties and antibiotic residues. These findings indicated that the anthropogenic inputs exerted an effect on the E. coli phylogroup composition, diversification of multiple antibiotic resistance phenotypes, and the distribution of resistance genes in the Larut River.


Subject(s)
Drug Resistance, Bacterial , Escherichia coli , Rivers , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Malaysia , Phylogeny , Prevalence , Rivers/microbiology
4.
Sci Rep ; 12(1): 5468, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1768860

ABSTRACT

This study investigated the performance of 24 commercial disinfectants present on the market during last year according to the manufacturer's instructions. Recently, national and international organizations of public health performed studies on disinfection products due to the increasing awareness of the potential and growing risks on human health, such as skin damage and reactions in the mucosal lining, especially for the healthcare workers in their frequent daily use. However, there are many limitations in the common cleaning/disinfection products on market as in the selection of effective disinfectants to decontaminate inanimate surfaces. We analyzed the disinfection power of hydrogen peroxide, quaternary ammonium compounds, alcohols, phenols and aldehydes used as active principles according to international guidelines. The antimicrobial properties were assessed by broth microdilution, and antibiofilm properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus); their virucidal efficacy was tested against Herpes simplex virus type 1 (HSV-1) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The quaternary ammonium compounds demonstrated better efficacy than others and in some cases ready to use products had also virucidal and antimicrobial activities after dilution at 0.125%. The scientific evidence indicates that many commercial products are used at high concentrations and high doses and this could have deleterious effects both on human health and the environment. A lower concentration of active ingredients would avoid the excessive release of chemicals into the environment and improve skin tolerance, ensuring the health and safety protection of workers, including the healthcare operators at their workplace.


Subject(s)
COVID-19 , Disinfectants , COVID-19/prevention & control , Disinfectants/pharmacology , Escherichia coli , Humans , Pandemics/prevention & control , SARS-CoV-2 , Staphylococcus aureus , Workplace
5.
Sensors (Basel) ; 22(6)2022 Mar 19.
Article in English | MEDLINE | ID: covidwho-1765836

ABSTRACT

Detection of bacterial pathogens is significant in the fields of food safety, medicine, and public health, just to name a few. If bacterial pathogens are not properly identified and treated promptly, they can lead to morbidity and mortality, also possibly contribute to antimicrobial resistance. Current bacterial detection methodologies rely solely on laboratory-based techniques, which are limited by long turnaround detection times, expensive costs, and risks of inadequate accuracy; also, the work requires trained specialists. Here, we describe a cost-effective and portable 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5α, BL21, TOP10, and JM109) within 15 min using 500 µL of sample, and costs only USD 2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 cfu, limit of quantification (LOQ) of 270 cfu, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a strong candidate for frequent testing at point of care; it also has application in various fields and industries where pathogen detection is of interest.


Subject(s)
Biosensing Techniques , Escherichia coli , Bacteria , Biosensing Techniques/methods , Limit of Detection , Printing, Three-Dimensional
6.
Int J Mol Sci ; 23(5)2022 Feb 23.
Article in English | MEDLINE | ID: covidwho-1736941

ABSTRACT

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.


Subject(s)
/chemistry , Escherichia coli/enzymology , Lipid Bilayers/metabolism , Ubiquinone/metabolism , Cardiolipins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Phosphatidylcholines/metabolism , Protein Conformation , Protein Domains
7.
Int J Environ Res Public Health ; 19(5)2022 02 24.
Article in English | MEDLINE | ID: covidwho-1736897

ABSTRACT

The impact of globalization on beekeeping brings new economic, scientific, ecological and social dimensions to this field The present study aimed to evaluate the chemical compositions of eight propolis extracts from Romania, and their antioxidant action and antimicrobial activity against seven species of bacteria, including pathogenic ones: Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. The phenolic compounds, flavonoids and antioxidant activity of propolis extracts were quantified; the presence of flavones and aromatic acids was determined. Quercetin and rutin were identified by HPLC analysis and characterized using molecular descriptors. All propolis samples exhibited antibacterial effects, especially against P. aeruginosa and L. monocytogenes. A two-way analysis of variance was used to evaluate correlations among the diameters of the inhibition zones, the bacteria used and propolis extracts used. Statistical analysis demonstrated that the diameter of the inhibition zone was influenced by the strain type, but no association between the propolis origin and the microbial activity was found.


Subject(s)
Propolis , Anti-Bacterial Agents/pharmacology , Antioxidants/chemistry , Antioxidants/pharmacology , Bacillus cereus , Escherichia coli , Microbial Sensitivity Tests , Plant Extracts/pharmacology , Propolis/pharmacology , Pseudomonas aeruginosa , Romania
8.
Sci Rep ; 12(1): 2803, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1735270

ABSTRACT

The COVID-19 pandemic has demonstrated the real need for mechanisms to control the spread of airborne respiratory pathogens. Thus, preventing the spread of disease from pathogens has come to the forefront of the public consciousness. This has brought an increasing demand for novel technologies to prioritise clean air. In this study we report on the efficacy of novel biocide treated filters and their antimicrobial activity against bacteria, fungi and viruses. The antimicrobial filters reported here are shown to kill pathogens, such as Candida albicans, Escherichia coli and MRSA in under 15 min and to destroy SARS-CoV-2 viral particles in under 30 s following contact with the filter. Through air flow rate testing, light microscopy and SEM, the filters are shown to maintain their structure and filtration function. Further to this, the filters are shown to be extremely durable and to maintain antimicrobial activity throughout the operational lifetime of the product. Lastly, the filters have been tested in field trials onboard the UK rail network, showing excellent efficacy in reducing the burden of microbial species colonising the air conditioning system.


Subject(s)
Air Filters/microbiology , Anti-Infective Agents/chemistry , Antiviral Agents/chemistry , Air Filters/virology , Anti-Infective Agents/pharmacology , Antiviral Agents/pharmacology , COVID-19/epidemiology , COVID-19/virology , Candida albicans/drug effects , Chlorhexidine/analogs & derivatives , Chlorhexidine/chemistry , Chlorhexidine/pharmacology , Escherichia coli/drug effects , Humans , Methicillin-Resistant Staphylococcus aureus/drug effects , SARS-CoV-2/drug effects , Time Factors
9.
Nat Commun ; 13(1): 988, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1713165

ABSTRACT

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.


Subject(s)
Frameshifting, Ribosomal , RNA, Messenger/chemistry , Bacterial Proteins/genetics , DNA Polymerase III/genetics , Escherichia coli/genetics , Fluorescence Resonance Energy Transfer , HIV/genetics , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Single Molecule Imaging , Thermodynamics
10.
J Am Chem Soc ; 144(9): 3761-3765, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1713117

ABSTRACT

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N-hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N-hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5'-acylation using Novozym 435.


Subject(s)
Antiviral Agents , COVID-19/drug therapy , Cytidine Deaminase/metabolism , Cytidine/analogs & derivatives , SARS-CoV-2 , Biocatalysis , Cytidine/biosynthesis , Cytidine/metabolism , Cytidine Deaminase/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Hydroxylamines , Metabolic Engineering , Protein Engineering , Uridine/metabolism
11.
STAR Protoc ; 3(2): 101238, 2022 06 17.
Article in English | MEDLINE | ID: covidwho-1704569

ABSTRACT

The immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome is largely unknown. Here we describe a protocol for analyzing sera samples with SARS-CoV-2 proteome microarray. The proteins were expressed by either E. coli expression system or eukaryotic cell expression systems and obtained by affinity purification. The protocol includes microarray fabricating and sera profiling, which will be used to build an antibody response landscape for IgG and IgM. The protocol may help to facilitate a deeper understanding of immunity related to SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Li et al. (2021c).


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Escherichia coli , Humans , Proteome
12.
Biophys J ; 121(7): 1276-1288, 2022 Apr 05.
Article in English | MEDLINE | ID: covidwho-1693832

ABSTRACT

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.


Subject(s)
Escherichia coli , Uracil-DNA Glycosidase , Escherichia coli/metabolism , Mutation , Uracil-DNA Glycosidase/chemistry , Uracil-DNA Glycosidase/genetics , Uracil-DNA Glycosidase/metabolism
13.
Signal Transduct Target Ther ; 7(1): 44, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1683982

ABSTRACT

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Immunoglobulin Fc Fragments/pharmacology , Recombinant Fusion Proteins/pharmacology , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/genetics , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , Antibody Affinity , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Models, Molecular , Neutralization Tests , Protein Binding/drug effects , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
14.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Article in English | MEDLINE | ID: covidwho-1671749

ABSTRACT

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.


Subject(s)
COVID-19/drug therapy , Interferon-alpha/pharmacology , SARS-CoV-2/drug effects , Transcriptome , Virus Replication/drug effects , Animals , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Disease Models, Animal , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Mice , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/pharmacology , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Signal Transduction , Vero Cells
15.
Int J Mol Sci ; 23(3)2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1667197

ABSTRACT

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.


Subject(s)
Antibodies, Viral/blood , Escherichia coli/growth & development , Peptides/administration & dosage , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Cloning, Molecular , Disulfides/metabolism , Escherichia coli/genetics , Female , Humans , Immune Sera/metabolism , Immunization , Mice , Mice, Inbred ICR , Peptides/genetics , Peptides/immunology , Protein Domains , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Th2 Cells/metabolism
16.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1666759

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
17.
Microb Cell Fact ; 21(1): 21, 2022 Feb 05.
Article in English | MEDLINE | ID: covidwho-1666655

ABSTRACT

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Viral/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mass Spectrometry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
18.
J Nanobiotechnology ; 19(1): 458, 2021 Dec 28.
Article in English | MEDLINE | ID: covidwho-1577211

ABSTRACT

Bio-inspired Topographically Mediated Surfaces (TMSs) based on high aspect ratio nanostructures have recently been attracting significant attention due to their pronounced antimicrobial properties by mechanically disrupting cellular processes. However, scalability of such surfaces is often greatly limited, as most of them rely on micro/nanoscale fabrication techniques. In this report, a cost-effective, scalable, and versatile approach of utilizing diamond nanotechnology for producing TMSs, and using them for limiting the spread of emerging infectious diseases, is introduced. Specifically, diamond-based nanostructured coatings are synthesized in a single-step fabrication process with a densely packed, needle- or spike-like morphology. The antimicrobial proprieties of the diamond nanospike surface are qualitatively and quantitatively analyzed and compared to other surfaces including copper, silicon, and even other diamond surfaces without the nanostructuring. This surface is found to have superior biocidal activity, which is confirmed via scanning electron microscopy images showing definite and widespread destruction of E. coli cells on the diamond nanospike surface. Consistent antimicrobial behavior is also observed on a sample prepared seven years prior to testing date.


Subject(s)
Anti-Bacterial Agents/chemistry , Coated Materials, Biocompatible/chemistry , Diamond/chemistry , Nanostructures/chemistry , Anti-Bacterial Agents/pharmacology , Coated Materials, Biocompatible/pharmacology , Copper/chemistry , Copper/pharmacology , Diamond/pharmacology , Escherichia coli/drug effects , Escherichia coli/growth & development , Nanostructures/ultrastructure , Nanotechnology , Surface Properties
19.
J Virol Methods ; 302: 114486, 2022 04.
Article in English | MEDLINE | ID: covidwho-1654882

ABSTRACT

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/genetics , Escherichia coli/genetics , Humans , Immunoassay/methods , Luminescence , Mice , Phosphoproteins/analysis , Phosphoproteins/genetics , Sensitivity and Specificity
20.
Int J Biol Macromol ; 200: 428-437, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1633983

ABSTRACT

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.


Subject(s)
COVID-19 Testing/methods , Coronavirus Nucleocapsid Proteins/chemistry , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Circular Dichroism , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Dimerization , Epitopes/chemistry , Escherichia coli/genetics , Humans , Immunoglobulin G/immunology , Models, Molecular , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity
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