Reverse transcriptases (RTs) are enzymes synthesizing DNA using RNA as the template and serving as the standard tools in modern biotechnology and molecular diagnostics. To date, the most commonly used reverse transcriptase is the enzyme from Moloney murine leukemia virus, M-MuLV RT. Since its discovery, M-MuLV RT has become indispensable for modern RNA studies; the range of M-MuLV RT applications is vast, from scientific tasks to clinical testing of human pathogens. This review will give a brief description of the structure, thermal stability, processivity, and fidelity, focusing on improving M-MuLV RT for practical usage. Since the discovery in 1970, RTs have taken place in a common laboratory practice, which value hardly could be overestimated. RTs are applied to solve numerous tasks when RNA is a matter of concern, from pathogen detection or cloning to single-cell transcriptome analysis, allowing the possibility of working freely with fragile RNA molecules.Among all known RTs, the reverse transcriptase from a Moloney Murine Leukemia Virus (M-MuLV RT) is one of the most scrupulously studied and well-known. M-MuLV RT was one of the first discovered RTs and served as a model for researching RTs properties and catalysis. Structure, catalysis, temperature optimum, thermostability, fidelity of DNA synthesis, processivity, optimal buffer composition, bypass of DNA damage, RNase H activity, strand transfer, and strand displacement activities – all biochemical properties of M-MuLV RT has been extensively investigated and been described in an abundance of papers.Being an indispensable tool for RNA studies, M-MuLV RT underwent multiple attempts to improve different characteristics, primarily temperature optimum and thermostability, as they define for a great degree M-MuLV RT performance in practical applications. Higher reaction temperature disrupts RNA secondary structures allowing for continuous synthesis of cDNA. Other features of M-MuLV RT have also been targets for alterations, while less frequently. Thus, high processivity represents the ability to produce long cDNA molecules; fidelity – the number of errors  in the synthesized cDNA.In the present review, we tried to glimpse a structure, selected biochemical properties, and attempts to improve M-MuLV RT for practical usage. By that, we hope to provide information for future endeavors in the design of reverse transcriptases superior to currently used ones.2Murine oncoviruses were discovered in 1951 Moloney MLV and other murine leukemia viruses serve as a model to study virion structure, life cycle, and specific features of retroviral infections, providing a lot of information about how retroviruses operate both outside and inside the cell. It also should be noted that M-MuLV is a source of numerous retroviral vectors used in animal transgenesis, siRNA delivery, gene therapy Rauscher murine leukemia virus, together with Rous sarcoma virus, was an object of research when Howard Temin and David Baltimore discovered an RNA-dependent DNA synthesis or reverse transcription Dozens of reverse transcriptases have been discovered for the 50 years since the discovery of Temin and Baltimore, including telomerase, a crucial component of maintaining linear DNA molecules in cells The next chapter will provide a review of the structure, thermal stability, processivity, and fidelity of M-MuLV RT.33.1The reverse transcriptase of Moloney MuLV is a typical RT, being a single subunit enzyme with a molecular mass of 71 kDa, length 671 a.a., possessing 3 catalytic activities: RNA- and DNA-dependent DNA polymerase , and RNAse H 2+ cations than Mg2+ (3.5-fold more polymerase activity) and inhibited by 1.0 mM NaPPi2, and pH 8.4 in Tris–HCl buffer in the presence of 1 mM dNTPs. Substituting Na+ or NH4+ for K+ ions or replacing Cl– with CH3COO– salts did not alter the polymerase efficacy M-MuLV RT is more active in the presence of MnM-MuLV RT can switch templates during synthesis, making it possible to shift from one strand to another 3.2All known reverse transcriptases are members of a specific enzymatic family included in the DNA polymerase superfamily. Enzymes of this superfamily, despite a relatively low amino acid sequence homology and distinct origin, share a similar 3D structure, where DNA polymerase consists of 3 separate domains: palm, thumb, and fingers. In addition, reverse transcriptases with RNAse H activity reveal the unique RNAse H domain with a respective active site and connection domain. Thus, the spatial architecture of M-MuLV RT consists of 5 domains: palm, fingers, and thumb, forming the polymerase, connection domain, and RNAse H domain. The structure of partial N-terminus fragment of M-MuLV RT was resolved by M. Georgians et al. in 1995 First 23 a.a. residues of M-MuLV RT are dispensable for the enzyme function Fingers , palm , and thumb (276–361) domains comprise N-terminal part of M-MuLV RT, while connection domain (362–496 a.a.) and RNAse H (497–671 a.a.) are located at C-terminal part Fingers domain has been proposed to provide an intermediate binding site for template-primer in between phosphonucleotidyl transfer reactions. The surface which interacts with the template comprises S60-Q84, N95-D124, F156-C157, and Q190-N194 Conserved Y64, D114, R116, N119, and G191 residues participate in DNA binding; P65, Q113, K120 interact with blunt-end DNA. Residues R116, L115, and G191 form 4 hydrogen bonds with blunt DNA Residues K103 The thumb domain plays a crucial role in substrate binding and processivity and contains consecutive surface hydrophobic residues, F303-L304 and L432-V433-I434 2+ or Mn2+ cations for optimal catalysis. Distance between polymerase and RNAse H active sites on a substrate is around 17–20b. Separated fingers, palm, and thumb domains retain polymerase activity.M-MuLV RT possesses two active sites needed for the reverse transcription of viral RNA. RNAse H activity can be dependent or independent from the polymerase activity. Both active sites require MgPolymerase active site resides on a structurally conserved surface in the palm domain m·dNTP, KD·DNA, sensitivity to dideoxynucleotides, and processivity remained stable m·dNTP increased only 3–10-fold. Residue Q190 is believed to be involved in the catalysis at the conformational change beyond the chemical step and resistance of M-MuLV RT to ddNTP, participating in recognition of 3′-OH of incoming dNTP In the palm domain, regions I125-F155, L220-E233, and K257-E275 are located on the surface, interacting with a template m of binding dTTP and rUTP, while Vmax remained 100-fold higher for dTTP. As a result, F155V mutant can utilize rNTPs for both incorporation and elongation on either RNA or DNA template, while slowly than using dNTPs max for both activities increases 4-fold, allowing to synthesize longer RNA fragments. In addition, Q84A alone increases affinity to template 2-folds Residue F155 is crucial to prevent the incorporation of rNTPs in a nascent DNA strand; its substitution to F155V leads to an equivalent KThe connection domain comprises P360-K373, Y394-A436, and S453-A462 on the surface, which interacts with a template RNAse H domain can change its conformation and is believed to participate in the processive DNA synthesis. RNAse H domain also interacts with peptidyl release factor 1 (eRF1). It prevents the binding of peptidyl release factor 3 (eRF3) to eRF1, promoting stop-codon read-through and impeding mRNA degradation by a nonsense-mediated mRNA decay mechanismRNAse H active site comprises a conserved Asp-Glu-Asp motif, with catalytically active residues D524, E562, D583, D653. RNAse H primer grip includes V402-G403-W404, S557-A558-Q559-R560, Y586, T590; their substitutions are deleterious for polymerase activity Pro primer and stability of polypurine tract) is compromised E. coli, Bacillus halodurans, and H. sapiens2+ cations but are inactive in with Mg2+m and Vmax of polymerization are similar to the wild-type enzyme, which hints at the involvement of ΔC in the interaction of M-MuLV RT with a template The separate RNAse H domain retains catalytic activity; however, its specific activity is lower than that of the whole enzyme ribo-A and ribo-C of the RNA. This site of initial reaction is identical to that recognized by HIV-1 RT. Substitutions of D114 and R116 residues in the fingers domain involved in the biding of the template do not impair RNAse H activity of M-MuLV RT RNAse H of M-MuLV RT avoids cleavage of the polypurine tract, and the catalysis is performed in a 3′-OH end-dependent manner with the hydrolysis site at +18 nt of RNA from 3′-end of the primer 3.3The optimal working temperature for M-MuLV RT is 37–42 °C as it was measured on the homopolymer polynucleotide templates A:oligo(d)T). In practice, RNA templates have a complex secondary structure that can pause the reverse transcription process, resulting in undesirable truncated reaction products – M-MuLV RT also demonstrates the 4-fold increase of half-life at 50 °C in the presence of a primed template RNAse H deficient M-MuLV RT is more thermostable than wild-type enzyme − M-MuLV RT correlates with its tighter binding to the template than the wild-type enzyme. More thermostable than M-MuLV RT, AVL RT also demonstrates a higher affinity to a template. H− M-MuLV RT synthesizes full-length cDNA up to reaction temperature 50 °C, while H+ M-MuLV RT loses this ability when the temperature is higher than 45 °C. It was speculated that RNAse H cleaves RNA in primed template duplex, resulting in depletion of free 3′-OH ends available for RT. Thus, RNAse H could diminish the amount of substrate protecting RT from thermal inactivation. These findings became a ground for improving M-MuLV RT thermal stability, which will be described below.The increased thermal stability of H3.4Reverse transcriptases are meant to replicate viral genomes spanning several dozens of thousands of nucleotides. For this task, RTs should possess the ability to synthesize long stretches of DNA; in other words, RTs should be processive enzymes. In terms of DNA polymerases, processivity is defined as a mean number of nucleotides incorporated during synthesis into the nascent DNA strand per a single binding of an enzyme with a template; or as a probability of not terminating at a given position. Processivity is closely related to a high catalytic rate and fidelity of the synthesis. Thus, a decrease of polymerase activity automatically means a loss of processivity as the enzyme slows down its movement on the template. The processivity of RTs is inferior to replicative DNA polymerases, as the latter is supported by specific processivity factors .From the practical point of view, high processivity facilitates further cDNA analysis due to a decrease in the error number at sequencing. In addition, high-processive enzymes are less prone to template switching, thus reducing the amount of artificial chimeric transcripts and produce longer cDNA fragments, simplifying bioinformatical examination of sequencing results.− M-MuLV RT is speculated to result from loss of the DNA-binding sites of the RNAse H domain. H− M-MuLV RT is more prone to pause at G and G nucleotides of RNA template or dA nucleotides of DNA template than wild-type enzyme − M-MuLV RT is deficient in forming these active RT complexes.Overall processivity of MMTV RT is substantially higher than that with HIV-1 RT + and H− M-MuLV RT is 20–40 nt under single-hit conditions in the presence of a heparin trap. However, long cDNA products could be synthesized during several hours of incubation with the excessive amount of the enzyme The processivity of HAs expected, mutations of residues involving the interaction with template often lead to a decrease of M-MuLV RT processivity. Several mutations detrimental to processivity have been described in the fingers domain of M-MuLV RT. Thus, substitutions of residues D114, R116, and N119 result in a loss of ability for processive DNA synthesis, as the respective mutant enzymes do not produce long cDNA fragments 3.5Fidelity is a characteristic defining the enzyme’s ability to copy a DNA or RNA template without introducing any errors, e.g., mismatches, deletion, insertions, and undesirable template switching. Fidelity could be described as the ability of RT to incorporate an incompatible nucleotide into the nascent DNA strand and to extend the already existing mismatch. Practical applications, such as a single cell transcriptome analysis or search for somatic mutations, require high fidelity of reverse transcriptases, which will render the possibility of technical errors.In general, reverse transcriptases have been reported to have lower intrinsic fidelity comparing to replicative DNA polymerases. The reason is that RTs do not possess proof-reading exonuclease activity reducing error numbers. Notably, many factors should be considered in infidelity assessment, from the type (RNA or DNA) and nucleotide context (including modified bases) of the template to the concentration of dNTP; the influence of reaction temperature on the RTs fidelity remains unknown. The difference in all mentioned parameters leads to conflicting results in the studies of M-MuLV RT fidelity.M-MuLV RT is reported to be 15-fold more faithful than HIV RT −4, 1.0 × 10−4 and 8.5 × 10−4, 2.6 × 10−4, respectively, on different RNA templates). Deletions and insertions are 10 times less frequent than substitutions; among all substitutions the most frequent are C:A (32.2%), T:C (16.3%), A:G (14.3%), and T:G (10.4%) K. Yasukawa et al., using the bar-coded NGS technique (Ion Torrent), demonstrated that M-MuLV RT mutant (E286R/E302K/L435R/D524A) has a lower mutation frequency than HIV-1 RT  for the RDDP, and 8.4 ± 1.9 × 10−5 total error rate  for the DDDP. The most frequent errors in a first-strand synthesis are substitutions of dT, in a second strand – dA:dG, dT:dC, dC:dT. Half of the deletions arising during RDDP are single nucleotide (49%), 2 nucleotides – 38%, the most frequent insertions are 3-nucleotide (50%), and 1-nucleotide (42%). For DDDP, 90% of deletions are 1-nucleotide, 6% − 2-nucleotide, 85% of insertions are 1-nucleotide, 9% − 2-nucleotide. The presence of N6-methyladenosine (m6A), pseudouridine (Ψ), and 5-hydroxymethylcytidine (hm5C) in RNA template significantly increase the number of errors V. Potapov et al., using Pacific Biosciences Single Molecule Real-Time sequencing, demonstrated a 6.3 ± 1.2 × 10Numerous studies demonstrated the importance of different functional sites – polymerase active site, template-binding site, dNTP-binding pocket, primer grip of RNAse domain – on the overall fidelity of DNA synthesis by vMuLV RT. Thus, the integrity of the polymerase active site and its YVDD moiety significantly affect the fidelity. Mutations of V223 lead to a 2-fold decrease of the enzyme’s fidelity on an RNA template, while DNA template results contradict the underlying importance of template context for fidelity studies Mutations in the DNA-binding site in the fingers domain also affect the fidelity of M-MuLV RT. Thus, mutants Y64A, D114A, R116A show decreased fidelity with an increased rate of misincorporation (7.4–27.6-fold) and elongation of mismatches (18.6–70.8-fold) In the RNAse H domain, mutations in primer grip  decrease M-MuLV RT fidelity, ranging from 2.1- to 3.8-fold 3.6As it was mentioned earlier, M-MuLV RT is one of the most commonly used enzymes in modern molecular biology and related fields, including medical diagnostics. However, biochemical characteristics of native M-MuLV RT are insufficient in many cases, which leaves a place for the improvement of the enzyme. For instance, the thermostability of M-MuLV is a crucial parameter limiting the enzyme’s application, as reverse transcription could be stalled by a complex secondary structure of an RNA template. Therefore, many researchers have focused on improving M-MuLV RT characteristics. Several different approaches have been applied to change the properties of a specific protein. Among them, site-directed mutagenesis, random mutagenesis, directed evolution, and protein chimerization methods; a detailed description of the results is given in the respective chapters.Site-directed mutagenesis is based on using knowledge about a protein structure and functions to alter its properties. The selected single mutations could be combined to design more suitable complex variants. Regarding M-MuLV RT, most studies focus on substituting residues in catalytically important sites or regions, interacting with a template. Directed evolution is several techniques for protein alterations intended to mimic the process of natural selection under the pressure of a specific important factor important for practical applications. The process could be repeated to enrich the screened library with desirable variants. For M-MuLV RT, the variables were optimal reaction temperature and reaction buffer composition. Random mutagenesis is based on a selection of variants with desirable properties among libraries of randomly created mutants. As in the case of directed evolution, several rounds of screening could be applied to obtain the variants with better performance.Protein chimerization assumes the fusion of protein’s genes to get a hybrid polypeptide. Thus, several DNA polymerases have been improved by fusing with DNA-binding proteins. In the case of M-MuLV RT, several similar attempts have been reported.In this chapter, we provide a short review of the achievements made in the field. The reviewed results are grouped based on the altered characteristic solubility, RNAse H activity, thermal stability, affinity to a template, and processivity; the brief summary is given in 3.6.1Protein solubility is an important factor defining the convenience of purification and the yield of the active enzyme, as insoluble proteins tend to lose their intrinsic enzymatic activities. However, despite numerous studies, increasing the solubility level remains a challenging task, and reverse transcriptases are not the exception, prone to form inclusion bodies or aggregate during purification procedures. Studies describing successful attempts to improve M-MuLV RT solubility are listed below.J. Gu et al. demonstrated that truncation of the 24 N-terminal residues leaves intact M-MuLV RT’s enzymatic properties and improves its solubility 3.6.2As was mentioned above, the RNAse H domain is involved in the processive synthesis of DNA, and its truncation leads to decreased processivity of M-MuLV RT. Thus, the RNAse H domain became a target for several studies intended to improve the processivity and the synthesis yield of M-MuLV RT.− M-MuLV RT at 48–56 °C. D524A mutant shows increased thermal stability either in the presence or absence of a template, while T50 values for both wild-type and H- enzymes increase by the same margin with a template (2.6 and 2.4 °C). Km*RNA values for both WT and H− M-MuLV RTs are similar . It was suggested that D524A alters the structure of RNAse H and its interaction with polymerase domains M. Mizuno et al. rendered RNAse H activity of M-MuLV RT by mutation D524A and demonstrated increased relative polymerase activity of HK. Yasukawa et al. constructed and characterized four chimeric RTs , which comprise one of the fingers, palm, thumb, and RNase H domains originated from AMV RT, respectively. The other three and the connection domains originated from M-MuLV RT. Chimeric RTs almost lose polymerase activity while retaining RNAse H activity, though significantly decreased. The enzyme’s relative RNAse H activity is higher at 52–60 °C than the parental enzymes, with the optimal temperature also 2–4 °C higher. Therefore, a decrease of RNAse activity in the chimeric M-MuLV RT with the RNAse H domain from AMV RT implies the interaction between this domain and polymerase domains m to dNTP. The concentration of dNTP in reaction buffer lower than Km of wild-type M-MuLV RT leads to a selection of mutants with lower Km. It should be noted that methionine is the most frequent residue in the YXDD motif among lentiviral RTs, operating in a nondividing cell with a relatively low concentration of dNTP.S. Paliksa et al. used the compartmentalized ribosome display to obtain M-MuLV RT mutant working at conditions optimal for Taq-polymerase. Among 29 randomly picked genes, 2/3 had mutations in the active site motif YVDD and substitutions of Q221. A substantial number of mutations were also found in the RNAse H domain; several of them rendered RNAse H activity . Mutants Q221R, V223A/M show processivity 2-fold lower or 2-fold higher than the wild-type enzyme, respectively, while possessing lower K2+ ions. Surprisingly, the lower fidelity of Q221R substitution is amended by a second mutation.V223A/M has fidelity similar to the wild-type enzyme, while Q221 has 5-fold lower, double mutants – 2-fold lower. The apparent difference with previously observed lower fidelity of V223 mutants and the present study could be explained by a different reaction buffer with a 2–3-fold lower concentration of MgResidues C635 and G637 are located in the His-loop of the RNase H domain, encompassing the sequence CPGHQK; residues P636-K640 of this functionally important loop are conserved in all RNases H. Residues C635 and G637 are close to the putative position of the highly conserved active site residue H638. Mutations in these positions affect the interaction of the RNase H domain and a substrate. Residue I597 is located near a positively charged C-helix in the RNAse H domain. However, substitution I597A impairs viral replication and is prone to mutate spontaneously to I597V, and the latter does not affect RNAse H activity 3.6.3Thermostability affects the overall performance of RTs, defining the ability of RT to synthesize cDNA at elevated temperatures for bypassing complex RNA templates structures. For that reason, most studies dedicated to RTs improvement are touching the subject of thermal stability.K. Yasukawa et al. mutated 12 residues in M-MuLV RT that have been shown previously as interacting with a template or located in surfaces that interact with a template in the fingers, thumb, and connection domains. Substitutions of residues D108, D114, and Q84, near important residues K103, R110, and R116, lead to a significant decrease or a complete loss of polymerase activity. The reason behind the inactivation after substitutions of residue W313 remains unknown. Triple (E286R/E302K/L435R) and quadruple (E286R/E302K/L435R/D524A) mutants demonstrate 70% of wild-type enzyme polymerase activity. Ehe stabilization effect of mutations is additive; combined mutants are more stable at 50 °C in both presence or absence of template and synthesize cDNA at 6 °C higher temperature than the wild-type enzyme A. Konishi et al. mutated 5 residues in hydrophobic clusters on the M-MuLV RT surface, replacing them with either lysine or arginine. Polymerase activity of L304R and L304K is close to zero; L432R and I434R retain 40–60% of polymerase activity. Mutants V433K and V433R are 3–5-fold more stable at 50 °C than the wild-type enzyme. Combinations of V433R with D108R/E286R or D108R/E286R/D524 further improve the thermal stability of M-MuLV RT. V433K and V433R are thought to stabilize enzymes via disrupting the interaction between hydrophobic surfaces M. Baba et al. performed scrupulous mutagenesis of 29 residues: 10 hydrophobic residues to increase a surface charge, 8 hydrophobic or polar residues inside the molecule to strengthen its hydrophobic core, 8 hydrophobic or polar residues near charged residues to create an additional salt bridge, 3 cysteine residues to abolish a disulfide bond. All mutations in the thumb decrease polymerase activity, while mutations in the finger affect the activity less crucially. Most mutations advantageous for thermostability are located in the palm than in other domains. Among all possible combinations of beneficial mutations, A32V , L72R (fingers), W388R (connection) are compatible with E286R, E302K, and L435R providing a sextuple mutant, able to produce cDNA at 55–65 °C Y. Narukawa et al. introduced in M-MuLV RT the additional disulfide bonds. As a result, among five designed variants, A551C/T662C is the most thermostable A. Baranauskas et al. used the compartmentalized ribosome display and selected the most frequent 28 mutations. Among the most thermostable clones, 3 are in the palm, connection, and RNAse H domains, 1 – in the fingers and thumb, and only 4 clones can synthesize full-length cDNA. In most cases, replacements by similar amino acids yield consistent phenotypes, while almost all substitutions of D200 increase thermostability. Thermostability of mutants in the absence of a template is low. The presence of a template increases it, as mutants show a higher affinity to template and processivity. The effect of mutations on thermostability, affinity to a template, and processivity is additive. Thus, the pentuple mutant L139P/D200N/T330P/L603W/E607K demonstrates a 10-, 50-, and 64-fold increase of these parameters, respectively, synthesizes a long 7.5 kb cDNA at ≤59 °C, and shows the fidelity of the wild-type M-MuLV RT. However, the reaction rate correlates negatively with the addition of mutations and the affinity to a template. All mutants retain RNAse H activity.Residue D200 is close to the polymerase active site as the catalytic D224 and D225 are only 7–9 Å away from the D200. Therefore, substitutions of D200 may cause structural rearrangements of the catalytic and neighboring residues that may alter the substrate-binding affinity and the rate of catalysis.Residue L139 is located at the core of the palm domain. It forms a hydrophobic cluster with residues from several distinct sequence regions. The largest and polar substitutions of the L139 drastically diminish the thermostability. However, mutation L139P is advantageous in terms of thermostability, affinity to a template, and processivity. Presumably, P139 stabilizes the hydrophobic cluster .Residue T330 is located at the terminus of a small helix in the thumb domain, presumably close to a template; therefore, T330P could stabilize the DNA-contacting helical motif.Residue L603 is positioned at the helix-unstructured loop boundary at the end of the C-helix in the RNase H domain. All sizeable hydrophobic/aromatic substitutions position 603 improve thermostability, possibly by stabilizing a hydrophobic Y598-L603-I617 cluster at the same loop as the putative phosphate backbone-interacting K609 and K612. Residue E607 is located in a putative DNA-interacting loop, and an additional positive charge E607K could enhance interaction with DNA de novo, while 17 have been identified previously . The majority of selected mutants harbor more than one advantageous mutation (up to 5–6) accompanied by other random substitutions. More than half of sequenced M-MuLV mutants possess substitutions D524G/A/N, D528N/G/A, D653N/D/A/H/V, which are located in RNAse H active site and render RNAse H activity simultaneously with increasing thermostability. Frequent substitutions D200/N/A/G are located close to the polymerase active site. Mutation H204R leads to an increased thermostability and decreased terminal deoxynucleotidyl transferase activity. Mutation H638G results in a hyperactive enzyme R. Skirgaila et al. used 5 rounds of compartmentalized ribosome display to select thermostable mutants of M-MuLV RT. Among 55 selected clones, 39 reveal RT activity. Out of 28 advantageous mutations, 11 mutations are found − M-MuLV RT 6-fold in the presence of the template. The pentuple mutant (M5) shows a broader temperature spectrum (25–70 °C), with maximum activity at 40–55 °C. Calculated transition temperatures (first derivative of melting spectra) are 68 °C (M-MuLV RT) and 75.5 °C (M5) in the presence of the template. M5 binds template-primer with significantly higher (10-fold) affinity compared to wild-type M-MuLV RT. Both H− and H+ M5 M-MuLV RTs can synthesize long cDNA and overcome complex template secondary structures up to 55 °C.After random mutagenesis, B. Arezi and H. Hogrefe demonstrated that mutations E69K, E302R, W313F, L435G, and N454K increase the thermal stability of M-MuLV RT − M-MuLV RT were more susceptible than the wild-type enzyme Later, the M5 M-MuLV RT was shown to be 2–4 folds less sensitive to common reverse transcription inhibitors  than M-MuLV RT, while HSubstitution E69K is thought to provide a secondary binding site in the fingers domain. Residues F303 and G305 are suggested to be facing away from the surface (E302-T306-F309-W313) interacting with the minor groove of the template-primer duplex. Positively charged  or polar (F309N) side chains could improve template-primer binding and thermal resistance by increasing the number of hydrogen bonds formed with a minor groove base or phosphate backbone. Hydrophobic mutation (W313F) remains a difficult case to explain. Unlike tryptophan, phenylalanine was found to be frequently involved in van der Waals contacts with the sugar-phosphate backbone or with DNA bases The connection domain (amino acids 362–474) provides conformational flexibility between the RNA-/DNA-dependent polymerase and the C-terminal RNase H domain. Mutation L435G/M is located in the C-end of β19 and N454K/R – in a large unstructured loop that connects to the RNase H domain. L435 is a solvent-exposed residue residing in a stretch of hydrophobic amino acids (432–436: LVILA) that were independently replaced by lysine to increase solubility (34). It was suggested that local perturbations in β19  could change template-primer interactions. Unlike L435K, L435G/M mutations show the same heat-sensitivity and denaturation/aggregation behavior as wild-type M-MuLV RT in the absence of the template. Residue N454, located distal to L435 in the connection domain, has not been previously implicated in template-primer interactions in M-MuLV RT Y. Katano et al. performed site-saturation mutagenesis of 8 regions  using 8 separate libraries. The most thermostable clone harbors D200C mutation in the palm domain. Among 13 mutants with increased thermostability, 9  are located in the palm domain, 4  – in the fingers domain. Mutation L218L is found in 3 clones, T186C, D200C, E201M, L202M – in 2 clones, making these mutations highly involved in maintaining M-MuLV RT stability at elevated temperatures. Thus, the priority of domains for increasing thermal stability is palm > fingers > thumb, connection. D200C mutant can synthesize cDNA at 4 °C higher temperature than the WT enzyme and is 3-folds more thermostable after the heating at 51 °C T. Yano et al. fused M-MuLV RT with 8xHisTag and Streptavidin Tag on either N- or C-ends and expressed the fusion proteins in silkworm larvae. Both chimeric enzymes retain around 50% of polymerase activity and synthesize long cDNA at 55 °C, while the wild-type enzyme lost this ability 3.6.4As it was shown in many studies, affinity to a template is closely related to the thermal stability of M-MuLV RT. Consequently, in many cases increase of M-MuLV RT thermal stability led to an enhanced ability to bind with a template. The same reasoning could be applied to processivity, as this parameter depends on the tightness of enzyme-template interactions. For that reason, increased affinity to template or processivity of M-MuLV RT was demonstrated in only a few studies.A. Konishi et al. found that among E286R, E302K, L435R, D524A, and quadruple mutants, substitutions E302K, L435R have 1.5-fold higher affinity to RNA template, and E302K, L435R, D524 – 1.5-fold higher affinity to the DNA template, all enzymes bind preferentially with RNA template. E302 mutant is less prone to misincorporation of dATP. All mutants are RNAse H defective, probably, caused by the prevention of proper binding of the active site with the substrate, while residue D524 participates in catalysis Pyrococcus abyssi or DNA-binding Sto7d protein from Sulfolobus tokodaii) with either N- or C-ends of H− M-MuLV RT. Sto7d was also fused with the triple mutant L139P/D200N/T330P H− M-MuLV RT, previously reported as more thermostable. The RNAse activity of Sto7d was disabled by K12L mutation.I. Oscorbin et al. fused DNA-binding domains . The presence of template increases at 2–4-fold thermostability of mutated enzymes. Sto7d at C-end increases processivity 1.5–3-fold for RDDP, 3–4-fold for DDDP, and increases 3–4-times optimal concentration of mono- and divalent ions. Fused Sto7d also improves 2–3 times tolerance to inhibitors . Additional domains do not influence terminal transferase activity. Sto7d at C-end improves several times the efficacy of cDNA synthesis. The observed results are similar to what has been reported for fusions of DNA polymerases. They could be explained as consequences of strengthening binding to a template by the additional domain on the C-end of the enzyme, acting independently from the M-MuLV RT The temperature optimum of all fusion enzymes remains the same with the parent H3.6.5Being one of the core enzymes in modern biotechnology, M-MuLV RT has drawn much attention from numerous companies, manufacturing solutions for scientific research and healthcare. Such curiosity resulted in a vast number of patents describing different ways to improve the performance of M-MuLV RT. While a rigorous survey is out of the present review’s scope, several examples of the achievements in the field are given below to mark the progress made in the past 30 years in the development of commercially available reverse transcriptases.−4 and 32 × 10−4, respectively). It was observed that mutants Y64W, R116M, K152R, T197A are similar to wild-type M-MuLV RT, while Q190F and V223H are more accurate, V223F – more erroneous. The SuperscriptII is also more prone to extend primers on an RNA template with a biased nucleotide pool. An introduction of F309N and V223H mutation increased the fidelity of SuperscriptII on both DNA- and RNA-templates; the mutants were more accurate than the wild-type enzyme. Mutations H204R and T306K were claimed to enhance the thermal stability of M-MuLV RT. Mutants F309N, T197E, and Y133A demonstrate reduced terminal transferase activity. Thus, improved M-MuLV RT, Superscript III One of the most popular reverse transcriptases on the market, SuperscriptII, was described by Invitrogen in 1988 In 2006, a combination of mutations E69K, E302R, W313F, L435G, N454K, and D524N was introduced by Stratagene In 2008, Applied Biosystems presented a novel M-MuLV RT mutant with F155Y, R301L, F309A, D524E/N, E562D, Y586A, D653N, H638G substitutions In 2008, Fermentas claimed a broad set of mutations: E5K, M39V/L, I49V/T, M66L, Q91R/L, P130S, L139P, I179T/V, D200N/A/G, Q221R, Q237R, T287A, A307V, T330P, L333Q, Y344H, A502V, D524A, L528I, H594R/K/Q, L603W/M, E607K/G/A, H634Y, A644V/T, N649S, D653G/A/H/V, K658R/Q, and L671P Life Technologies patented Superscript IV in 2014, harboring mutations P51L, S67R, E69K, T197A, H204R, E302K, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P New England Biolabs claimed in 2016 a set of mutations: H8Y, S56A, T246E, N249D, Q291I, M320L, T330E, and altered N-terminus that is more efficient than Superscript IV in elongation and template switching, particularly on RNA templates with GC-content more than 50% − variants In 2016, Bio-Rad described chimeric reverse transcriptases, combinations of M-MuLV RT and feline leukemia virus reverse transcriptase (FLV RT), and their RNAse H4Among all enzymes used nowadays, M-MuLV RT is one of the most frequently applied. Presumably, only a few DNA polymerases, i.e., Taq- and Pfu-polymerase, are more common in modern biological laboratories. It would not be an overstatement to say that almost all RNA-related studies and clinical protocols rely on the reverse transcriptase discovered 50 years ago. The coronavirus outbreak began in 2019, once again underlined the crucial significance of M-MuLV RT, as this enzyme, together with Taq-polymerase, became a basis for countless test systems for SARS-CoV-2 detection. However, compared to numerous papers describing alteration of Taq-polymerase, the amount of similar studies dedicated to improving M-MuLV is limited. It should also be noted that the high practical importance of M-MuLV RT led to a great number of patents related to the improvement of the enzyme’s properties.A direct relation of known structural features with the beneficial mutations is given in m*dNTPAmino acid residues involved in the polymerase catalysis appear to be crucial for the proper functioning of the enzyme, as VXDD motif and residues interacting with the incoming dNTP are usually invariant. Most mutations in these important positions result in the decrease of polymerase activity and fidelity. A rare exception is V223, as V223A/M mutations increase processivity and decrease KAs it was mentioned earlier, most studies to improve M-MuLV RT concentrated on increasing the M-MuLV RT thermostability. At the same time, other features remain in the shadow of the heavy influence that reaction temperature has on the synthesis of cDNA. Processivity is an exception, as the thermostability of M-MuLV RT is closely related to binding with a template. Thus, a change of template-interacting amino-acids residues could increase the affinity to a template; increased binding of template leads to enhanced thermal stability. Simultaneously, tethering of template binding results in higher processivity and the ability to produce longer cDNA fragments. Elimination of RNAse H activity by site-directed mutagenesis also increases thermostability, while the presence of RNAse H domain itself, specifically its C-helix, is advantageous for a processive synthesis.It is worth pointing that various studies have identified a significant number of mutations beneficial for thermal stability of M-MuLV RT. several of these works deployed high-throughput methodologies such as random mutagenesis, site-saturation mutagenesis, and compartmentalized ribosome display. It would be a legitimate assumption that mutations the most beneficial for thermostability have already been found and described for the past 30 years. For that reason, further development of M-MuLV RT mutants seems to be dedicated to improving other M-MuLV RT characteristics. For instance, another crucial parameter, the fidelity of synthesis by M-MuLV, being extensively studied, remains almost untouched in terms of alterations. However, in the last decades, methods of single-cell analysis and somatic-mutation testing have been booming. These techniques require RTs with higher fidelity than M-MuLV RT, as higher fidelity reduces the number of errors in the sequencing results. In addition, the need for higher accuracy and growing demand for template switching, RNAse H activity, strand displacement stimulate the interest for respective M-MuLV RT mutants.ad hoc; the same could be noted for the studies that relied on various random mutagenesis, direct evolution techniques, and protein chimerization. Therefore, applying in silico prediction methods is another possible direction for future studies. Modern artificial intelligence programs, such as AlfaFold 2 rational.A rational design using software predicting 3D-structure and interaction with a template could facilitate the development of novel improved M-MuLV RT mutants. However, to date, virtually all studies on the field are empirical, either applying only knowledge about wild-type M-MuLV RT spatial structure or relying on a trial and error approach. Thus, reported so far, the rational design to generate novel M-MuLV RT variants has been performed without predicting 3D-structure alterations caused by the introduction of mutations. Explanations of mutations effect are usually made To sum up, we provide in the present review a brief description of M-MuLV RT discovery, structure, thermostability, processivity, fidelity. In addition, we also focused on the attempts to improve M-MuLV RT characteristics. We believe this review will be helpful for the future design of more advantageous M-MuLV RT variants.Igor P. Oscorbin: Writing – original draft. Maxim L. Filipenko: Conceptualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Radix Ophiopogon Japonicus has long been used as traditional Chinese medicine to treat pulmonary disease, and Ophiopogonin B (OP-B) as a bioactive component of it has also been verified to inhibit cell proliferation of various non-small cell lung cancer (NSCLC) cells in vivo and in vitro. Therefore, we wonder whether OP-B is also effective to drug resistant lung cancer cells. Firstly, Cell Counting Kit-8 (CCK8) assay was used to compare the sensitivity of OP-B on NCI-H460, A549, cisplatin resistant A549 (A549/DDP) and paclitaxel resistant A549 (A549/PTX) cells, and A549/DDP cells were shown to be more sensitive to OP-B than other three cell lines, the results were further verified in orthotopic tumor nude mice model and zebrafish tumor model. Moreover, observation of cell morphological feature, mitochondrial membrane potential, LDH release rate, and production of IL-1β all suggested that OP-B induced pyroptosis in A549/DDP cells more significantly than that in A549 cells. Meanwhile, transcriptomic sequencing results between OP-B treated and the Mock A549/DDP group also suggested that OP-B induced more significant Caspase-1/GSDMD dependent pyroptosis in A549/DDP group, which was further verified by VX-765, the inhibitor of Caspase-1. Together, the experimental results suggested that OP-B alleviated DDP resistance of A549 cells through inducing more significant Caspase-1/GSDMD-dependent pyroptosis.Drug resistance has become the main reason for the failure of tumor chemotherapy.  In recent years, lung cancer has become the third leading cause of cancer-related diseases et al. found that GSDMD-N itself can directly induce pyroptosis, which indicates that GSDMD cleavage is really an important index to detect whether pyroptosis occurs or not The mechanism of drug resistance in tumors are complex, which mainly includes drug concentration reduction in tumor cells, abnormal drug metabolic pathway, DNA damage repair dysfunction, regulation of autophagy, blocking of apoptotic pathways, and self-renewal or proliferation of tumor stem cells et al. combined BI2536 (an inhibitor of PLK1) with Cis-Dichlorodiammine platinum (DDP) to treat ESCC cells and found the combination of two drugs had a synergistic effect on tumor inhibition and resulted in pyroptotic death of cancer cells by Caspase-3/GSDME pathway. Their further research revealed that BI2536 can reverse DDP resistance of ESCC by inhibiting DNA damage repair and inducing pyroptosis Currently, some studies found that there is any connection between tumor resistance and pyroptosis Radix Ophiopogon Japonicus, a traditional Chinese medicine often used in pulmonary disease treatment. In our previous studies, OP-B had been verified to have significant inhibition effects on various NSCLC cells Traditional Chinese medicine has been used for thousands of years in China and has unique advantages in cancer treatment. Ophiopogonin B (OP-B) is a bioactive component extracted from OP-B (CAS: 38971-41-4) and OP-D (CAS: 41753-55-3) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd . OP-B and OP-D were dissolved in DMSO as a 10 mmol/l stock solution and stored at 4 °C. Caspase-1 inhibitor VX765 (Belnacasan) (CAS: 273404-37-8) were purchased from Med Chem Express. Polo-188 (CAS:9003-11-6) was purchased from Beijing solarbio science & technology co., Ltd.2.A549 and NCI-H460 cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences . A549/DDP and A549/PTX cells were kind gifts from Professor Zhigang Guo . A549, NCI-H460 cells were cultured in DMEM/F12 medium , and A549/DDP, A549/PTX cells were cultured in RPMI 1640 medium  with 10% fetal bovine serum , supplemented with 1% penicillin/streptomycin solution . All of the cells were maintained at 37 °C in humidified atmosphere of 5% COThe inhibition of OP-B on A549, NCI-H460, A549/PTX, A549/DDP cells was estimated by Cell Counting Kit-8  as described previously The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer  containing 1% phenylmethanesulfonyl fluoride (PMSF) before suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer , then separated on 12% SDS-PAGE  and transferred onto polyvinylidene fluoride (PVDF) membranes . After the membranes were blocked with 5% non-fat milk, they were incubated at 4 °C overnight with primary antibodies against Caspase-1 , GSDMD , GSDMD-N , NLRP3 , and β-actin . After incubation with horseradish peroxidase (HRP)-linked anti-rabbit  or anti-mouse  secondary antibodies for 2 h at room temperature, the bands were detected with a Gel Doc™ XR+ Gel Documentation System  with enhanced chemiluminescence (ECL) reagents .Cells were pelleted, and RNA samples were isolated and sent to Huada  for constructing RNA-seq library. Briefly, total RNAs were isolated, and mRNA was enriched and then were pooled together for cDNA synthesis and sequencing. The transcriptomic RNA sequencing was performed on an Illumina NovaSeq 6000 platform, to create pair-end reads with a length of 150 bp (PE150). The differentially expressed genes (DEGs) were identified using edgeR (|logFC | >1 & FDR < 0.05), and these DEGs were undergone Gene ontology and KEGG pathway analysis.-△△Ct method was used for quantification and fold change for target genes was normalized by internal control.Total RNA was extracted from A549 or A549/DDP cells using TRIzol reagent (Sangon Biotech) according to the manufacturer's protocol. Then, the RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed using cDNA primers specific for mRNA. The gene GAPDH was used as an internal control. All the real-time PCR reactions were performed using Takara′s SYBR Premix Ex Taq™ II (Tli RNaseH Plus) in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The 24 cells/well were seeded into 96-well plates. After exposure to the indicated treatments, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, then blocked with 5% goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. After that, the cells were incubated with a primary antibody against Cox-2 , IL-1β  in antibody dilution buffer /0.3% Triton X-100) overnight at 4 °C. Then, the cells were incubated with a fluorochrome-conjugated anti-rabbit secondary antibody  in ADB for 2 h at room temperature in the dark. Subsequently, the cells were stained with DAPI  for 5 min. Images were obtained under a fluorescence microscope .Cells at a density of 1×10When frozen sections were used for immunofluorescence, the sections were first blocked with goat serum; the rest of the procedure was the same as that for the goat serum-blocked cells.The activity of LDH released into cell culture supernatants was detected using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit  according to the manufacturer's protocol for analyzing pyroptosis.7 in 0.2ml medium of a 1:1 mixture of RPMI 1640 and Matrigel 354,248) were injected into right lung parenchyma of the mice (n=30), and the volume of tumors were monitored by luciferase imaging of live animals using an IVIS Spectrum bioluminescence imaging system  after intraperitoneal injection of 200 μl D-Luciferin substrate . And the mice were mainly used to test the toxicity and pharmacological activity of OP-B on A549 or A549/DDP xenograft mice. The mice for toxicity-detection were divided into 5 groups (6 in each group), including polo-188 group (62.5 mg/ml Poloxamer), OP-B groups (1.5 or 3 mg/Kg OP-B), and the Normal or Mock group . And the mice for pharmacological activity detection were divided into 9 groups, including the Normal group , A549 or A549/DDP Mock groups , A549 OP-B-treatment groups (1.5 or 3 mg/Kg OP-B), A549/DDP OP-B-treatment groups (1.5 or 3 mg/Kg OP-B), and A549 or A549/DDP cyclophosphamide groups (20 mg/Kg cyclophosphamide). All of the mice were treated with intraperitoneal injection . The polo-188 was formulated with 0.9% NaCl, and OP-B was formulated with polo-188. 25 days later, all mice' hearts, livers, lungs and kidneys were harvested, then half of the tissues were used for GPT, GOT and CRE detectinon with microplate test kit , and the rest of them were used for haematoxylin-eosin (H&E) staining, Transmission Electron Microscope (TEM) observation, or immunohistochemistry and immunofluorescence observation.The BALB/c nude mice (4 weeks old) were maintained under specific pathogen‑free (SPF) conditions. Animal welfare and experimental procedures were performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals, and all protocols were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine. To establish the orthotopic xenograft lung cancer model, the luciferase-expressing A549 or A549/DDP cell line with lentivirus was established, then A549 or A549/DDP cells  and resuspended in HBSS were injected into the yolk sac of zebrafish embryos (200 cells/embryo n=100). Then, embryos were incubated at 34 °C. At 72 h post injection, the proliferation of tumor cells was evaluated through a fluorescence stereomicroscope . The software Image J was used to quantify the proliferation rate of tumor cells.The model and OP-B groups were selected for TEM analysis to observe the morphology of A549 cells and A549/DDP cells. The mice were sacrificed, 1/3 of tissue was removed from lung and fixed with 3% glutaraldehyde for more than 2 hours, then immobilized with 1% osmium acid for 2 hours. The tissues were dehydrated step by step with ethanol and acetone and then soaked overnight and embedding agent. After the tissues were embedded, polymerized, repaired, sliced and double stained with uranium acetate and lead citrate, they were observed by TEM (JEM-1011).t-test on two experimental conditions or two-way ANOVA when repeated measures were compared, with p < 0.05 considered statistically significant. All graphs show mean values ± SEM.Data entry and all analyses were performed in a blinded fashion. All statistical analyses were performed using Graph Pad Prism8.0 software. Statistical significance was calculated using two-tailed unpaired 50 values of A549 and A549/PTX cells were 16.47 μM and 229.4 μM, respectively. Under the treatment of DDP, the IC50 values of A549 and A549/DDP cells were 83.92 μM and 2568 μM, respectively. According to the IC50 value of each cell, the resistance index (RI) of A549/PTX and A549/DDP cells was 13.93 and 30.6 respectively  =IC50(A549/PTX)/IC50(A549), RI(A549/DDP) =IC50(A549/DDP)/IC50(A549)).First of all, we used CCK-8 assay to test cell viability of A549, A549/PTX or A549/DDP cells under different doses of PTX and DDP for 24h to prove the drug resistance of A549/DDP and A549/PTX cells Fig. B. Under Next, we compared the inhibitory effects of OP-B on NCI-H460, A549, A549/PTX and A549/DDP cells, with a concentration range of 1.25~20 μM for 24 or 48 h treatment , and observed tumor growth by bioluminescence image. After the models being successfully built, 3 mg/kg of OP-B was administered to the mice consecutively for 14 days, meanwhile, bioluminescence images were taken on 0 and 14th days  by microinjection technology, after 3 days treatment of 5 μM OP-B, the fluorescence intensity of zebrafish in each group was measured by fluorescent stereo-microscope Fig. E. Statisin vivo and in vitro.Taken together, OP-B has more significant inhibitory effect on DDP-resistant A549 tumors In order to further explore the reason why A549/DDP cells are more sensitive to OP-B than A549 cells, we took the lung tissues from the mice mentioned in Fig. Next, we used JC-1 mitochondrial membrane potential (MMP) probe to label the above cells. After 24 hours treatment with OP-B (5 μM), flow cytometry was used to detect the changes in the number of red and green fluorescence cells to quantify the MMP. The results showed that the level of MMP in A549 and A549/DDP cells decreased after OP-B treatment, while it decreased more significantly in A549/DDP cells Fig. B-D.Since the occurrence of pyroptosis is accompanied by the release of a large amount of inflammatory substance and decrease of mitochondrial membrane potential, we then tested LDH release rate and the changes of mitochondrial membrane potential (MMP) in A549 and A549/DDP cells. The results showed that after OP-B (5 μM and 10 μM) treatment for 6, 12 and 24 hours, the release of LDH in both cell lines increased in dose-dependent manner, and it reached peak at 12 h Fig. E and F. in vivo  for 24h, the immunofluorescence results showed that the expression levels of Cox2 and IL-1β in both of the cell lines were significantly increased, and the expression level of the two proteins was higher at 10 μM of OP-B than that at 5 μM  to screen differentially expressed genes (DEGs) between OP-B treated and the Mock A549/DDP cells. In a comparison between A549/DDP cells treated with or without OP-B, the transcriptome analysis found 1149 downregulated genes and 690 upregulated genes  in vivo and in vitroRadix Ophiopogonis is commonly used in traditional Chinese medicine to treat lung diseases. In various chemical compositions of ophiopogonis, saponin is the key to quality control of it. OP-B, as a saponin compound, had been proved to have obvious anti-lung cancer effect in vivo and in vitro also verified the hypothesis of pyroptosis, and further investigation of Caspase-1/GSDMD pathway by qRT-PCR and western blot both proved that OP-B induced more significant pyroptosis in A549/DDP cells than that in A549 cells.Further observation of the tumor tissues under electron microscope found that the morphology of the tumor cells in A549/DDP orthotopic tumor models showed more significant feature of pyroptotic cell death as bubbles swelling from the plasma membrane and causing lysis and massive release of cellular contents. As occurrence of pyroptosis is always followed by releasing of many proinflammatory factors, including IL-1β, IL-18, ATP, and HMGB1 23, 24. In these two works, Zhang et al. found that in the pyroptosis-activated immune microenvironment, CD8+ T cells and NK cells reciprocally induced pyroptosis in tumor cells via granzyme B, thereby establishing a positive feedback loop et al. revealed that pyroptosis by their bioorthogonal system in less than 15% of tumor cells was sufficient to clear an entire tumor graft operating in live animals As tumor cells always show innate resistance to apoptosis, the development of new strategies to induce pyroptosis may provide more efficient cancer therapy options and improve patient survival In summary, our study demonstrated that OP-B suppressed A549/DDP cells growth more significantly than A549 cells. The mechanism of it may involve that OP-B induced A549/DDP cells produced more significantly pyroptosis than A549 cells by Caspase-1/GSDMD pyroptosis pathway Fig. . Our fin


Nontargeted analysis can be used for the rapid screening and confirmatory analysis of veterinary drugs and their metabolites, which are important for the comprehensive safety evaluation of animal-derived foods. Here, a novel nontargeted screening approach based on liquid chromatography coupled with electrospray ionization–high-resolution mass spectrometry (LC/ESI–HR-MS) was developed to determine erythromycin, clarithromycin, and their metabolites in chicken liver microsomes. Erythromycin and clarithromycin were incubated in vitro in the presence of NADPH for 60 min to generate metabolites in chicken liver microsomes. After the incubation, the supernatant was extracted using ultrasonic shaking, orbital shaking, and centrifugation before analysis using LC/ESI-HR-MS in positive ion mode on an Agilent Eclipse Plus C18 column  with 0.1 percent formic acid-water and acetonitrile as the mobile phases for gradient elution at 0.4 mL/min. The results show that erythromycin can produce N-desmethyl-erythromycin A in chicken liver microsomes, but clarithromycin cannot produce N-desmethyl-clarithromycin in chicken liver microsomes. The N-desmethyl-erythromycin A and N-desmethyl-clarithromycin were tentatively identified in chicken liver microsomes using the established quick analytic method, which will provide a theoretical foundation for future research on pharmacokinetics and drug elimination in poultry. Legionella, Mycoplasma, and Chlamydia. Soluble erythromycin thiocyanate powder has been used as a veterinary medicine to treat chickens artificially infected with chronic respiratory diseases [Erythromycin (ERY) and clarithromycin (CLA) are macrolide antibiotics (MACs) with broad-spectrum antibacterial activity and have effects on both Gram-positive and Gram-negative bacteria. The antibacterial mechanism of ERY and CLA involves irreversible binding to the 50S subunit of the bacterial ribosome and selective inhibition of protein synthesis by blocking transpeptidation and mRNA displacement, which is the same as the antibacterial mechanism of chloramphenicol ,2. MACs diseases . Althougdiseases ,5,6. Chidiseases ,8,9,10.Aframomum melegueta, Denniettia, and Millettia aboensis extracts on CYP3A activity in intestinal and liver microsomes. The cytochrome P450 enzymes in liver microsomes of poultry  that are orthologous to cytochrome P450 enzymes in human and mouse liver microsomes are CYP1A1/2, CYP2A6, and CYP3A4 [3)2 branches. In the present work, chicken liver microsomes containing CYP3A4 orthologues, the liver toxicity of MACs, and medicinal chemical structural factors were considered and ERY and CLA in chicken liver microsomes were incubated with N-demethylation metabolites.The monitoring of veterinary drug metabolites in animal-derived foods has not received enough attention. However, studying such metabolite residues is extremely important for the safety of animal-derived foods. Phase I and phase II reactions are the two stages of drug metabolism in the liver. The first oxidation and reduction reactions in phase I are catalysed by monooxidases (mixed-function oxidases) in the liver. This group of enzymes constitute a complex microsomal system with haemoglobin cytochrome P-450 (CYP) as the core enzyme . Studiesd CYP3A4 ,17,18,19d CYP3A4  reportedThe separation and detection characteristics of liquid chromatography tandem mass spectrometry (LC-MS/MS) make it ideal for the investigation of veterinary medication residues in complicated food matrices . Many taThe purpose of this study was to verify whether ERY and CLA can produce N-desmethyl-erythromycin A and N-desmethyl-clarithromycin in chicken liver microsomes. Buspirone (BUS) can be metabolized by the CYP3A4 enzyme and thus was added as a positive control in this experiment. An in vitro incubation test was carried out in chicken liver microsomes, and a cofactor group and no cofactor (control) group were set up to verify the N-desmethyl-erythromycin A and N-desmethyl-clarithromycin. A nontargeted screening method based on LC-TOF-MS/MS was established to determine BUS, ERY, CLA, and their metabolites in chicken liver microsomes. This study will provide a scientific basis for the pharmacokinetics and drug elimination of ERY, CLA, and their metabolites in poultry and for the monitoring of MACs residues in animal-derived foods.Erythromycin A dihydrate , CLA (≥95% standard), buspirone hydrochloride, dimethylsulfoxide , and the reduced form of β-nicotinamide dinucleotide phosphate  were purchased from Sigma-Aldrich . N-Desmethyl-erythromycin A and N-desmethyl clarithromycin were purchased from Santa Cruz Biotechnology . Potassium phosphate buffer  was purchased from Biosesang . HPLC-grade methanol and acetonitrile (ACN) were supplied by J.T. Baker Chemical . Chicken liver microsomes (PL-MIC-201) were purchased from PRIMACYT GmbH  and stored at −80 °C before use. All chemicals were of the highest purity available.Standard 20 mM stock solutions of erythromycin A dihydrate, buspirone hydrochloride, N-desmethyl-erythromycin A, and N-desmethyl-clarithromycin were prepared in pure methanol. A standard 20 mM stock solution of CLA was prepared in DMSO. These standard stock solutions were stored at −20 °C. Standard working solutions of erythromycin A dihydrate, CLA, buspirone hydrochloride, N-desmethyl-erythromycin A, and N-desmethyl-clarithromycin at 20 μM were prepared by diluting the stock solutions with 0.1 M potassium phosphate buffer (pH 7.4). The standard working solutions were prepared for immediate use through gradual dilution. A 10 mM NADPH solution was prepared in 0.1 M potassium phosphate buffer (pH 7.4). An internal standard (IS) solution of 1 μg/mL CLA and an IS solution of 1 μg/mL erythromycin A dihydrate were prepared in pure ACN and stored at −20 °C.The incubation system (100 μL) contained 65 μL of potassium phosphate buffer , 20 μL of chicken liver microsomes (5 mg/mL), 5 μL of a compound (20 μM) and 10 μL of NADPH (10 mM). After a 5 min preincubation period at 37 °C, the reaction was started by introducing 10 μL of NADPH (10 mM) as the cofactor solution. The final study groups were divided into cofactor and no cofactor (control) groups. After preincubation for 5 min, 10 μL of potassium phosphate buffer  was added to the control group. At 0 and 60 min, the reaction was quenched with 100 μL of ice-cold ACN with IS . For 0 min samples, the reaction was quenched immediately after the 5-min preincubation, followed by the addition of 10 μL of NADPH (10 mM) and 10 μL of potassium phosphate buffer  to the cofactor and control groups, respectively. Following brief sonication (5 min) and vortexing (5 min), the mixtures were centrifuged for 30 min at 3000 rpm and 4 °C. The supernatant was transferred to an Agilent 96-well plate and then analysed on an Agilent 6530 Q-TOF LC-MS/MS instrument.v/v) and the above-mentioned column temperature and flow rate conditions. HR-MS detection was carried out in positive ionization mode with the following ion source parameters: capillary voltage, 4000 V; drying gas, 12 L/min and 325 °C; nebulizer gas, 35 psi; and sheath gas, 10 L/min and 350 °C. Mass spectra were acquired in positive ion auto MS/MS mode  with a full scan range of m/z (100–1000) and a mass scan rate of 5 spectra/s. The mass spectrometer was run in an extended dynamic range mode with a mass range of m/z 50–3200. The mass resolving power was greater than 12,000 FWHM at m/z 1552. Liquid chromatographic separation and mass spectrometric detection were performed on an Agilent 1200 series HPLC system and an Agilent 6530 Q-TOF LC-MS/MS system equipped with a dual AJS ESI ion source. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column  by gradient elution with a mobile phase consisting of 0.1 vol.% formic acid in water (A) and ACN (B) at 40 °C: 0–2 min, 5 vol.% B; 2–3 min, 50 vol.% B; 3–4 min, 75 vol.% B; and 4-5 min, 5 vol.% B. The injection volume was 5 μL with a flow rate of 0.4 mL/min. Before sample injection, the instrument was equilibrated under the initial gradient conditions  and Agilent MassHunter Metabolite ID software (version B.04.00) . The remaining content was evaluated by dividing the ratio of the response of the target compound to the response of the IS at 60 min of incubation by the ratio of the response of the target compound to the response of the IS at 0 min of incubation in the cofactor group or control group.+ at m/z 386.2577, 734.4741, 748.4898, 720.4581, and 734.4756 . Midazolam was used as the CYP3A4 substrate to evaluate the strength of drug interactions mediated by CLA and ERY. CLA and ERY caused strong and moderate interactions, respectively, possibly because the strengths of their inhibition of the main drug-metabolizing enzyme CYP3A4 differ but they do not inhibit the activity of the drug transporter P-GP ,38. ChicBased on previous studies by Zhou et al.  and Lee Compared with targeted and quantitative approaches, a nontargeted screening method based on matching fragmentation characteristics can confirm and analyse unknown substances and collect as much substance information as possible, with wide coverage. Thus, this study used a nontargeted screening method based on LC-HR-MS to detect BUS, erythromycin A, CLA, and their metabolites in chicken liver microsomes. We extracted the ion chromatograms of BUS, CLA (IS), and N-desmethyl-erythromycin A in chicken liver microsomes at m/z 386.2551, 748.4842, and 720.4529 , respectively. In the extracted ion chromatograms, the retention times of BUS, IS, and N-desmethyl-erythromycin A are 4.70, 5.22, and 4.77 min, respectively. The extracted ion chromatograms of erythromycin A , N-desmethyl-erythromycin A, and IS from the cofactor and control groups at 60 min and 0 min incubation are shown in The in vitro incubation test of CLA in chicken liver microsomes was carried out based on erythromycin A; erythromycin A was used as an IS and detected by LC-HR-MS. We extracted the ion chromatograms of BUS, IS, and N-desmethyl-clarithromycin  in chicken liver microsomes, and the retention times of these compounds were 4.71, 4.80, and 5.12 min, respectively. Residues of erythromycin A, N-desmethyl-erythromycin A, and CLA in animal-derived foods can pose human health risks due to toxicity. Therefore, drug metabolism residues in animal-derived foods should be considered. This study evaluated the levels of erythromycin A and CLA metabolism remaining in chicken liver microsomes, as shown in In this study, whether erythromycin A and CLA produce N-desmethyl-erythromycin A and N-desmethyl-clarithromycin in chicken liver microsomes was tentatively identified by LC-HR-MS. Through comparison of the extracted ion chromatograms with standard chromatogram, it was found that erythromycin A can produce N-desmethyl-erythromycin A in chicken liver microsomes, but CLA cannot produce N-desmethyl-clarithromycin. Nontargeted screening based on the LC-HR-MS method is fast, efficient, and sensitive and is suitable for the detection of veterinary drugs and their metabolites in animal-derived foods. This study will provide a scientific basis and detection methods for the monitoring of erythromycin A, CLA, and N-desmethyl-erythromycin A in poultry.


During public health crises like the COVID-19 pandemic, ultraviolet-C (UV-C) decontamination of N95 respirators for emergency reuse has been implemented to mitigate shortages. Pathogen photoinactivation efficacy depends critically on UV-C dose, which is distance- and angle-dependent and thus varies substantially across N95 surfaces within a decontamination system. Due to nonuniform and system-dependent UV-C dose distributions, characterizing UV-C dose and resulting pathogen inactivation with sufficient spatial resolution on-N95 is key to designing and validating UV-C decontamination protocols. However, robust quantification of UV-C dose across N95 facepieces presents challenges, as few UV-C measurement tools have sufficient (1) small, flexible form factor, and (2) angular response. To address this gap, we combine optical modeling and quantitative photochromic indicator (PCI) dosimetry with viral inactivation assays to generate high-resolution maps of “on-N95” UV-C dose and concomitant SARS-CoV-2 viral inactivation across N95 facepieces within a commercial decontamination chamber. Using modeling to rapidly identify on-N95 locations of interest, in-situ measurements report a 17.4 ± 5.0-fold dose difference across N95 facepieces in the chamber, yielding 2.9 ± 0.2-log variation in SARS-CoV-2 inactivation. UV-C dose at several on-N95 locations was lower than the lowest-dose locations on the chamber floor, highlighting the importance of on-N95 dose validation. Overall, we integrate optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations, establishing a versatile approach to characterize UV-C photoinactivation of pathogens contaminating complex substrates such as N95s. With established applications in water8, air11, and non-porous surface13 disinfection, ultraviolet-C (UV-C) germicidal (200–280 nm) irradiation was identified as one of the most promising and accessible methods for N95 decontamination15. UV-C decontamination of N95s has been reported for multiple types of pathogens21, including SARS-CoV-2 (the virus that causes COVID-19)22. Upon sufficient absorption by nucleic acids, UV-C inactivates pathogens by damaging their genetic material12; thus, UV-C decontamination efficacy is critically dependent on total received dose (integrated irradiance over time). While the level of decontamination necessary for N95 reuse may depend on institutional or federal guidelines, ≥ 3-log10 (≥ 99.9%) viral inactivation has previously been estimated to be a suitable decontamination threshold based on typical on-N95 viral load23 (“log10” subsequently referred to as “log”).In response to global shortages of N95 respirators during the COVID-19 pandemic, a broad range of crisis-capacity strategies for decontamination and reuse of these complex, multilayered, made-for-single-use protective textiles have been proposed and investigated in a growing body of literature21, the applied surface dose required to decontaminate all N95 layers is orders of magnitude higher than the dose required on non-porous surfaces24, and varies between N95 models18 due to substantial differences in material composition. UV-C decontamination protocols must ensure that all N95 surfaces receive sufficient dose for pathogen inactivation, while also not exceeding the exposure threshold for material degradation, as high cumulative doses of UV-C degrade N95 material25. Additionally, UV-C dose is nonuniformly distributed across the complex, 3D N95 surface due to Lambert’s cosine law26 and self-shadowing3. Thus, UV-C distribution across N95 surfaces is highly dependent on N95 morphology, as well as the decontamination system and N95 positioning. Accurate UV-C dose measurements are critical to understanding pathogen photoinactivation for a variety of decontamination applications28. However, the unique considerations involved in N95 decontamination complicate determination of the UV-C dose applied to all N95 surfaces in a decontamination system, impacting both research and implementation30.N95s present distinct challenges for UV-C decontamination: due to limited UV-C penetration through N95 material layers6, assess UV-C dose and viral inactivation on flat coupons of N95 material. N95 coupon studies determine the UV-C dose required for viral inactivation throughout the porous N95 material layers, but fail to capture the impact of the 3D facepiece shape on the received UV-C dose across the N95 surface. Other approaches use optical modeling to estimate the UV-C distribution across N95 surfaces from the UV-C dose measured in a single location, in order to relate approximate UV-C dose to SARS-CoV-2 inactivation22. Optical modeling is an attractive approach to study UV-C distribution, as it can recapitulate nearly any UV-C system to provide a high-resolution map of irradiance distribution33 via entirely user-defined system parameters. However, optical models alone cannot capture non-idealities such as irradiance fluctuations, bulb-to-bulb differences in power output, and environmental and material changes over time35. Additionally, while the modularity of optical modeling is advantageous for broad applicability, the model accuracy depends on both the optics expertise of the user and the accuracy of user-defined parameters such as the reflective and scattering properties of all materials. Thus, the high resolution and rapid iteration capabilities of optical simulations would be most valuable when coupled with in-situ validation measurements.Effective implementation requires translation of robust research studies linking on-N95 surface dose to viral inactivation for a given UV-C source emission spectrum and N95 model. However, coincident on-N95 dose and viral inactivation measurements are infeasible as the measurement sensor would shadow the pathogen. Furthermore, most UV-C dosimetry tools lack sufficient spatial resolution, throughput, and angular response for on-N95 measurements. As a result, UV-C dose for N95 decontamination is typically characterized indirectly. For example, to circumvent challenges associated with making UV-C dose measurements on non-planar surfaces, many studies, including a recent study of SARS-CoV-229. PCIs complement simulation results by providing absolute dose measurements and empirical validation. Planar, paper-like dosimeters similar to PCIs have been shown to have ideal angular detection response36, though the angular response of UV-C PCIs has not been quantified. The low cost and small, flexible form factor of PCIs supports quantitative, spatially resolved and high-throughput on-N95 PCI dosimetry in the same exposure and in nearly the same on-N95 location as inoculated pathogens, minimizing confounding factors such as temporal or spatial variation29 and angular dependence of UV-C irradiance26. Thus, PCIs may comprise a cornerstone to better inform safe and effective UV-C decontamination, especially when corroborated by further study to confirm PCI angular response and suitability for readout by diverse, lower-cost color readers.To this end, a promising in-situ method has recently been developed to quantify on-N95 dose using UV-C photochromic indicators (PCIs)Here, to investigate the impact of UV-C dose variation on SARS-CoV-2 inactivation on N95s, we introduce a method to simultaneously map UV-C dose and SARS-CoV-2 viral inactivation across N95 respirator facepieces. We integrate two approaches for high-spatial-resolution on-N95 dosimetry: PCI quantification and optical modeling. We develop an optical modeling workflow to characterize UV-C dose distribution across N95s within a decontamination chamber to rapidly iterate on experimental design, and simultaneously inform and validate this model using in-situ PCI dose quantification. From the high-resolution simulated N95 dose maps, we identify pairs of proximal measurement sites receiving equivalent UV-C dose in order to measure UV-C dose at SARS-CoV-2 inoculation sites within the same UV-C exposure. For the first time, we apply quantitative in-situ PCI dosimetry to simultaneously quantify UV-C dose and SARS-CoV-2 inactivation across a model N95 facepiece (intra-N95) at multiple locations (intra-chamber), providing new, practical insight into how N95 facepiece shape impacts decontamination efficacy.All UV-C decontamination experiments were performed with Spectronics XL-1000 UV-C chambers with BLE-8T254 low-pressure amalgam bulbs. Irradiance was measured using calibrated, NIST-traceable ILT 1254/TD UV-C radiometers  and corresponding ILT DataLight III meter software. A custom notch in the UV-C chamber doors allowed a cable to pass through for in-situ radiometer measurements. One chamber and radiometer were used exclusively in a biosafety level 3 (BSL-3) laboratory for SARS-CoV-2 inactivation experiments, while another set was used for all experiments outside BSL-3. The UV-C irradiance over time and space within the two chambers were concordant  was < 10%. To measure doses beyond the PCI dynamic range on planar surfaces, a 1.1 mm-thick Borofloat glass attenuator  with 12.4% ± 0.4% UV-C transmittance (measured in a UV-C chamber) was placed over the PCI. We generated calibration curves specific to the PCI batch, attenuator, and colorimeter to quantify UV-C dose from PCI color change (CIEDE2000 ΔE) with respect to an unexposed reference  were cut into quarters prior to use. D65/10° L*a*b* PCI color was measured using an RM200QC spectrocolorimeter  and/or Color Muse colorimeter . The Color Muse was aligned over the PCI using a template , other than cases where PCI measurements are normalized to a maximum PCI reading, we report total error: the root sum square of standard deviations associated with both replicate variation and propagated uncertainty in PCI dose estimation. All other error values report the standard deviation of replicate measurements.31, and positioned and/or duplicated to match in-situ chamber locations. All simulations were performed with “Use Polarization”, “Scatter NSC Rays”, “Split NSC Rays” and “Ignore Errors” engaged. Detector data were exported and analyzed using custom MATLAB scripts. Because the optical model may not accurately predict absolute dose due to environmental fluctuations, simulation results were normalized to the maximum value within the analyzed domain ).To create a model of the respirator compatible with the optical modeling software, a 3M 1860 N95 with straps removed was scanned using a Creaform Go!SCAN 3D. After additional pre-processing, the scan of the N95 was positioned within a CAD model of the UV-C chamber . Simulated UV-C dose at each location was extracted from the optical model using custom MATLAB scripts.UV-C dose distribution across the chamber floor was characterized in situ at 15 evenly spaced locations , (2) highest point height if not corner height (lowest point height if corner is highest) (h), (3) rotation along the N95 surface  corner height (d) was then defined as |C-h|. The angle of rotation toward the N95 surface  of SARS-CoV-2 stock at 8 × 107 TCID50/mL were loaded per N95 inoculation site and dried for 3.5 h at room temperature in a biosafety cabinet. After UV-C exposure, inoculation sites were excised with a 12 mm-diameter biopsy punch (MedexSupply ACD-P1250), incubated in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin for ≥ 30 min, and viable SARS-CoV-2 virus was quantified by a TCID50 assay. All study procedures were approved by the UC Berkeley Committee for Laboratory and Environmental Biosafety and conducted in agreement with BSL-3 requirements.For all viral inactivation experiments, SARS-CoV-2 preparation, stock titration, inoculation, and TCIDThe UV-C dose response of SARS-CoV-2 was assessed by measuring viral inactivation on 3M 1860 N95 coupons inoculated with SARS-CoV-2 and exposed to different UV-C doses. By mapping UV-C irradiance across the chamber floor, we identified 5 locations of equivalent irradiance at which to place a radiometer, PCI, and 3 inoculated N95 coupons . To account for output degradation29  were cut from the edge of N95s to include the raised, sealed seam to minimize layer separation. The seam did not prevent the coupons from lying flat during UV-C exposure. Both a radiometer and PCI (with Borofloat attenuator for doses beyond the PCI upper limit of quantification) were used to quantify in-situ UV-C dose applied during each exposure. Exposure time was estimated by dividing the target dose by the irradiance at the coupon platform  was compared. The inoculated heating control coupon was placed on a platform of the same height as the coupon and PCI platforms, and in a location receiving approximately the same irradiance. A custom acrylic cover, verified to block all UV-C, was then placed on top of the heating control coupon, so that the coupon would be exposed to the temperature rise in the chamber but not to UV-C. A PCI was placed near the heating control coupon, under the acrylic cover, to verify that the heating control coupon is not irradiated with UV-C.50 assay was performed to assess SARS-CoV-2 viability (Note S2).After each exposure, PCI(s) were measured with both the RM200QC and the Color Muse. Biopsies were excised from all irradiated coupons, as well as one unexposed control coupon stored at room temperature outside the UV-C chamber. A TCIDcorrcoef function in MATLAB. The linear regression and confidence intervals on the linear fit parameters were determined with the MATLAB functions fit (with the ‘poly1’ model) and confint, respectively. The 95% prediction intervals on the linear regression function were determined using the MATLAB function predint with simultaneous functional bounds.To simultaneously measure UV-C dose and SARS-CoV-2 inactivation on 3M 1860 N95 facepieces, PCIs (without attenuator) were affixed to N95s at each chosen dose measurement site, and accompanying SARS-CoV-2 inoculation sites outlined in advance to facilitate accurate viral deposition. SARS-CoV-2 was inoculated at each paired inoculation site. After drying, two N95s (‘corner’ and ‘front’ N95s) were placed in a UV-C chamber after bulb warm-up. To monitor dose during each exposure, a radiometer and PCI were also placed at their respective positions near the two corners of the chamber floor , expired 3M 1860 N95s were used to preserve non-expired N95s for healthcare workers.29, we introduce simultaneous measurement of SARS-CoV-2 inactivation and UV-C dose on N95 facepieces. We extend characterization of the quantitative PCI dosimetry method29 in two ways. First, we measured the angular response of PCIs and verified a near-ideal response  and non-expired coupons (N = 2 coupons per condition), we observed minimal impact of expiration status on UV-C decontamination efficacy among the batches of 3M 1860 N95s tested  have been shown to yield > 3-log reduction of SARS-CoV-239. Droplet imbibition into porous matrices is a complex process that depends on properties of the fluid and substrate40, differences in inoculation volume and solution, and N95 material, all of which may influence the proportion of virus which penetrates into inner N95 layers. Thus, the system scrutinized here is an idealized model system and the SARS-CoV-2 dose response behavior observed is not anticipated to represent SARS-CoV-2 inactivation in clinical settings, where different N95 models and soiling are expected to substantially increase the necessary UV-C dose. Given the results and precision of the TCID50 assay .We next assessed an applied UV-C dose range of 50–500 mJ/cm50. The tandem approach allowed simultaneous measurement at multiple locations on intact N95 facepieces: a measurement not feasible with radiometers or viral inactivation measurements alone.Having established the UV-C dose response of SARS-CoV-2 on flat N95 coupons, we next investigated the magnitude of N95 shape-induced UV-C dose variation, as received UV-C is dependent on incident angle and distance from the UV-C source. Concomitantly, we sought to understand how the nonuniform on-N95 UV-C dose translated to SARS-CoV-2 viral inactivation efficacy. We aimed to map SARS-CoV-2 inactivation differences and UV-C dose nonuniformity across the N95 facepiece by simultaneously quantifying on-N95 dose with in-situ PCI measurements and SARS-CoV-2 inactivation via TCIDlocations to make on-N95 measurements that sample the range of delivered UV-C doses, and (2) measurement sites within each location receiving the same dose. Each location must be large enough to house two proximal measurement sites each ~ 13 mm in diameter.Because a PCI placed at the SARS-CoV-2 inoculation site would shadow the virus inoculum, we used optical simulation to identify pairs of adjacent on-N95 measurement sites which receive equal dose. With paired measurement sites, the UV-C dose received by a SARS-CoV-2 inoculation site can be monitored using a PCI placed at the proximal equivalent-dose site. Optical simulation rapidly reports the irradiance distribution across easily tunable N95 configurations with high spatial resolution, facilitating identification of: (1) 43, but care must be taken to ensure all N95s receive sufficient dose. Additionally, N95s must be separated to prevent cross-contamination. In the studied decontamination system, three N95s can be staggered within the chamber . Given the lateral symmetry in dose distribution within the chamber  location, compared to a relative uncertainty of ~ 5% at all other locations when PCIs are measured with the RM200QC  due to the irregular N95 facepiece geometry and the off-center positioning of the N95s in the chamber. We observed that for locations receiving normalized UV-C doses > 0.34 , the doses across each proximal pair of PCI and inoculation sites were within 6.0% of each other, both in simulation and when measured in situ. For normalized UV-C doses ≤ 0.34 (at more steeply sloped and/or shadowed locations), the simulated doses at each pair of measurement sites were within 11.1% of each other and the doses measured in situ were within 11.8% ± 6.0% of each other   estimated from a linear regression on the dose–response curve (Pearson correlation coefficient r = − 0.8842) is ~ 19 mJ/cm2, higher than the D90 of ~ 1.4 mJ/cm2 for dried SARS-CoV-2 on a nonporous surface45, as expected  variability in the slope of the coupon surface caused by separation of N95 layers along the three sides without a seam . The observed 2.9 ± 0.2-log difference in SARS-CoV-2 survival across N95s within one chamber is substantial, given that effective N95 decontamination has previously been defined in some studies as ≥3-log reduction of pathogens. To our knowledge, this is the first study to rigorously quantify both UV-C dose and viral inactivation at paired locations on intact N95s, to understand how UV-C dose distribution and resulting decontamination efficacy depend on N95 facepiece shape.Similar to the coupon study, we observe varying SARS-CoV-2 inactivation among replicate inoculation sites receiving similar UV-C dose (1.1 ± 0.8-log difference in inactivation between replicates). We hypothesize that replicate-to-replicate variation in measured SARS-CoV-2 inactivation level in either the on-N95 and/or coupon studies may be due to: (1) the quantal nature of the TCID50, we also investigated whether dose monitoring at low-irradiance locations on the chamber floor could directly indicate the lowest on-N95 UV-C dose (Note S4). The doses in the corners of the chamber floor were 49.5% ± 1.6% (radiometer location) and 44.0 ± 0.7% (floor PCI location) of the maximum on-N95 dose, whereas the lowest on-N95 dose measured was 6.0% ± 1.6% of the maximum on-N95 dose  receives the highest dose while the more steeply sloped regions near the base of the sides of the N95 (location b) receive some of the lowest doses that can be measured on the front N95 with our approach, given the footprint of the PCI and SARS-CoV-2 inoculation site. Across the locations sampled on the front N95, simulation predicted a 3.0-fold difference in UV-C dose, and we measured a 2.8 ± 0.4-fold difference in UV-C dose using PCIs in situ. This variation in UV-C dose yielded a 2.8 ± 1.5-fold difference in SARS-CoV-2 log reduction (from 1.6 ± 1.2-log reduction at location b to 3.4 ± 0.4-log reduction at location a). While placing the N95 directly in the center of the UV-C chamber rather than offset toward the door would increase UV-C dose uniformity, throughput may be reduced, as the number of N95s that could fit in the chamber without contacting each other would be reduced from three to one.Across the 3 measured locations on the facepiece of the corner N95, simulation predicted an 8.1-fold difference in UV-C dose and we measured a 10.2 ± 3.3-fold difference in dose. This variation in UV-C dose yielded a 4.9 ± 1.3-fold difference in SARS-CoV-2 log reduction . However, because the measurement locations were chosen to evenly sample the range of UV-C doses applied across both (front and corner) N95s, the measured locations on the corner N95 did not capture the maximum corner N95 dose near the apex  in UV-C dose and SARS-CoV-2 log reduction, the difference in UV-C dose (10.2-fold) was greater than the difference in SARS-CoV-2 log reduction (4.9-fold) on the corner N95. We hypothesize that the corner N95 receives UV-C doses which may be in the shoulder of the SARS-CoV-2 survival curve, where SARS-CoV-2 inactivation is not fully log-linear with dose25. The simulation and in-situ dose measurement workflows we demonstrate here help inform N95 positioning within decontamination systems to optimize decontamination cycle time, pathogen inactivation, and the maximum number of safe reuses.Characterization of UV-C dose distribution across N95s within a decontamination system is valuable for informing decontamination protocols and evaluating throughput. In our system, we observed substantially lower variation in UV-C dose and SARS-CoV-2 inactivation across a single N95, as compared to across both N95s in the chamber, which suggests that approaches to increase decontamination throughput should be carefully considered. Including more N95s in the chamber may not necessarily increase throughput as compared to a single N95 in the center of the chamber, as multiple N95s likely have more nonuniform on-N95 dose because they are more spread out and can shadow each other. Greater UV-C dose nonuniformity increases the exposure time needed for all N95 surfaces to reach the minimally acceptable UV-C dose, which in turn affects the total number of safe reprocessing cycles prior to N95 material degradation Fig. A25. The 29 may extend the dynamic range of PCIs measured with lower-cost color readers.In summary, we have demonstrated that the N95 facepiece shape and position within a UV-C decontamination system have substantial influence on the on-N95 UV-C dose distribution and concomitant decontamination efficacy. We introduce a workflow to combine optical modeling and in-situ quantitative PCI dosimetry to characterize on-N95 UV-C dose with high spatial resolution, high throughput, and near-ideal angular response. For the first time, we combined simultaneous and robust quantitative UV-C dose measurements with SARS-CoV-2 inactivation measurements at specific locations on N95 respirators to probe the relationship between on-N95 dose and pathogen inactivation within each UV-C exposure. The substantial variation in on-N95 UV-C dose and SARS-CoV-2 inactivation we observed in a single decontamination chamber highlights how nonuniform UV-C dose distribution impacts pathogen inactivation and total UV-C exposure . We further demonstrated that a lower-cost colorimeter accurately quantifies dose from PCIs, making the PCI quantification workflow more accessible. Additional investigation into alternative color metrics29 using a camera, flatbed scanner, or other imaging modalities can analyze smaller PCI regions for finer in-situ dose measurement resolution. Additionally, PCI flexibility facilitates measurement on a variety of nonplanar N95 surfaces, though measurements of irregular topologies to which one large PCI cannot conform may be infeasible or may require an impractical number of measurements with small PCIs on each individual face. Optical simulations are also applicable to any N95 shape that can be accurately captured using a 3D scanner. However, for N95 models with greater N95-to-N95 variability in shape, small differences in N95 shape  may substantially impact the resulting UV-C dose distribution. For example, small changes in the pleat configuration and/or opening angle of duckbill and pleated N95s may yield large differences in dose distribution due to shadowing and angle-dependent UV-C dose. The rapid iterations possible with optical simulation can provide preliminary evaluations of UV-C dose distribution sensitivity to N95 shape variability, highlighting cases in which in-situ dose measurements with PCIs may be particularly valuable. Likewise, pathogen inactivation can be measured on a variety of N95 models22, as long as the pathogen sample can be accurately applied and dried on the N95 location of interest (some hydrophobic and/or steeply sloped N95 regions may be difficult to inoculate without sample migration from the region of interest).The approaches introduced here for characterizing UV-C dose and pathogen inactivation with cup-shaped 3M 1860 N95s are versatile and widely applicable to other N95 models or other substrates. The location and spatial resolution of in-situ UV-C dose measurements are limited only by PCI size and readout resolution. In this study, PCI size was limited by the spectrocolorimeter aperture size; however, alternative color readout methods2 UV-C dose on-N95. Overall, the on-N95 UV-C dosimetry approach here facilitates characterization of decontamination protocols of any UV-C system, supporting system-specific validation that is critical to ensuring safe and effective N95 decontamination.Future studies are needed to characterize SARS-CoV-2 dose response in more clinically relevant conditions, such as with the addition of soiling agents and on varying N95 models. Extending the dynamic range of PCIs, while maintaining a near-ideal angular response, is also critical for measurement of ⪆260 mJ/cmSupplementary Information.


Neutralizing antibodies (nAbs) elicited against the receptor-binding site (RBS) of the spike protein of wild-type SARS-CoV-2 are generally less effective against recent variants of concern. RBS residues E484, K417 and N501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on ACE2 binding and K417N and E484K mutations on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternate binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies. The COVID-19 pandemic has already lasted for over a year, but new infections are still escalating throughout the world. While several different COVID-19 vaccines have been deployed globally, a major concern is the emergence of antigenically distinct SARS-CoV-2 variants of concern (VOCs). In particular, the B.1.1.7 lineage that arose in the UK . We therD 3.3 nM vs 7.0 nM), whereas K417N substantially reduced ACE2 binding (41.6 nM). E484K slightly reduced binding (11.3 nM). Importantly, N501Y could rescue binding of K417N (9.0 nM), and the triple mutant K417N/E484K/N501Y (as in B.1.351) had similar binding (6.5 nM) to wild type  . Twenty H Y33 and Y52 make hydrophobic interactions with the aliphatic moiety of K417, and its ε-amino group interacts with CDR H3 through a salt bridge (D97 or E97), hydrogen bond (H-bond), or cation-π interaction (F99) (IGHV3-53/3-66 RBD antibodies can adopt two different binding modes (on (F99) . K417N/Ton (F99) . ConsistH26GYTFTG(Y)Y33, 50W(I)N/S(P)XSXGTX58, 73TS(I)S/T76 motifs are important for RBD binding . Although only a small part of the epitope interacts with the light chains of 2-4, S2M11, and C121, VL 32 and 91  play an important role in forming a hydrophobic pocket together with VH residues for binding RBD-F486, which is another key binding residue in such classes of antibodies  and S54 are involved in polar interactions with the RBD-E484 side chain that would be altered by substitution with Lys  and a salt bridge (VL R91)  from a COVID-19 patient  and make(VL R91)  that exp(VL R91)  compared(VL R91) . Thus, I(VL R91) .A further group of antibodies target the back side of the RBS ridge (RBS-C) , mainly Two other non-RBS sites that are distant from K417 and E484 have been repeatedly shown to be neutralizing sites on the SARS-CoV-2 RBD, namely the CR3022 cryptic site and S309 proteoglycan site . AntibodThe most potent neutralizing antibodies to SARS-CoV-2 generally tend to target the RBS (table S3), as they directly compete with receptor binding. Such RBS antibodies often interact with K417, E484, or N501, which are located in the RBS, and are therefore sensitive to RBS mutations in the VOCs. On the other hand, antibodies targeting the CR3022 and S309 sites are often less potent, but are less affected by the VOCs, as their epitopes do not contain mutated residues. In fact, recent studies have shown that sera from convalescent or vaccinated individuals can retain neutralization activity, albeit reduced, against the mutated variants (As SARS-CoV-2 continues to circulate in humans and increasing numbers of COVID-19 vaccines are administered, herd immunity to SARS-CoV-2 should be approached locally and globally. However, as with other RNA viruses, such as influenza and HIV (