Multiple Point Mutations Research Articles

Predicting tigecycline susceptibility in multidrug-resistant Klebsiella species and Escherichia coli strains of environmental origin.

Tigecycline (TGC) is an important antimicrobial agent used as a last resort for difficult-to-treat infections mainly caused by carbapenem-resistant Enterobacteriaceae, but TGC-resistant strains are emerging, raising concerns. In this study, 33 whole-genome characterized multidrug-resistant (MDR) strains (Klebsiella species and Escherichia coli) positive mainly to mcr-1, bla, and/or qnr from the environment were investigated for TGC susceptibility and mutations in TGC resistance determinants, predicting a genotype-phenotype relationship. TGC minimum inhibitory concentrations (MICs) of Klebsiella species and E. coli ranged from 0.25 to 8 and 0.125 to 0.5mg/L, respectively. In this context, KPC-2-producing Klebsiella pneumoniae ST11 and Klebsiella quasipneumoniae subsp. quasipneumoniae ST4417 strains were resistant to TGC, while some E. coli strains of ST10 clonal complex positive for mcr-1 and/or blaCTX-M exhibited reduced susceptibility to this antimicrobial. Overall, neutral and deleterious mutations were shared among TGC-susceptible and TGC-resistant strains. A new frameshift mutation (Q16stop) in RamR was found in a K. quasipneumoniae strain and was associated with TGC resistance. Deleterious mutations in OqxR were identified in Klebsiella species and appear to be associated with decreased susceptibility to TGC. All E. coli strains were determined as susceptible, but multiple point mutations were identified, highlighting deleterious mutations in ErmY, WaaQ, EptB, and RfaE in strains exhibiting decreased susceptibility to TGC. These findings demonstrate that resistance to TGC is not widespread in environmental MDR strains and provide genomic insights about resistance and decreased susceptibility to TGC. From a One Health perspective, the monitoring of TGC susceptibility should be constant, improving the genotype-phenotype relationship and genetic basis.

DDMut: predicting effects of mutations on protein stability using deep learning.

Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.

Efficient Generation of Multiple Seamless Point Mutations Conferring Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus.

Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis.

The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.

Abstract 1685: Overcoming acquired resistance to PROTAC degraders

Abstract Background: Proteolysis-targeting chimera (PROTAC) technology has been widely investigated for cancer treatment and there have been several PROTAC degrader-based drugs used for clinical trials in the past few years. PROTAC degraders are able to target crucial proteins traditionally thought to be “undruggable”, such as transcriptional factors. Hence, there is increasing interest in these drugs due to their highly efficacy and low off-target toxicity. However, upon further investigation, it has been found that after a period of treatment with PROTAC degraders, cancer cells can acquire resistance to these compounds. Thus, it is of increasing importance to figure out the mechanisms of the resistance and to overcome the acquired resistance to these PROTAC degraders. In our study, we classified resistance mechanisms of prostate cancer cells to PROTAC degraders into two classes and identified factors that can induce them to preferentially develop specific resistance mechanisms. Methods and Results: AU-15330 is a PROTAC that specifically degrades the subunits (BRG1, PBRM1, BRM) of SWI/SNF complex. We treated 22rv1, a prostate cancer cell line, with different AU-15330 concentrations over a month to establish AU-15330-resistant cell lines. Whole exome sequencing (WES) analysis of these resistant cell lines found that the resistant cell lines that were developed through high-dose (1uM) AU-15330 treatment acquired multiple point mutations within or adjacent to the AU-15330 targeting bromodomain of BRG1. However, BRG1 point mutations were absent in the resistant cell lines that developed through lower concentration of AU-15330 treatment (100nM). Instead, RNA-seq analysis revealed that ABCB1 (also known as Multidrug Resistance Protein 1) expression was upregulated in cells that developed resistance to AU-15330 at 100nM. This was further confirmed by Western blot and qPCR. Next, we overexpressed ABCB1 in LNCaP and 22RV1, and found that overexpression of ABCB1 indeed made prostate cancer cells resistant to AU15330. In addition, we tested with several different potent PROTAC degraders (ARD616, ZBC260, etc) in ABCB1-expressing AU-15530 resistant cells, and found the ABCB1 expression drove the cells to become resistant to all tested PROATC degraders compared to the parental cells. Lastly, we demonstrated that Zosuquidar, an ABCB1 inhibitor, can overcome the ABCB1-mediated resistance to AU-15330 and other PROTAC degraders, suggesting that inhibiting ABCB1 might be a direct and effective strategy to restore PROTAC degrader’s potency when ABCB1-mediated drug resistance is developed. Conclusion: Our study found that the mechanisms of resistance of PROTAC degraders can vary and may be dependent on drug concentrations. Such concepts may inform future clinical decision-making regarding drug dosing. Citation Format: Tongchen He, Caleb Cheng, Abhijit Parolia, Alex Hopkins, Yuanyuan Qiao, Lanbo Xiao, Arul Chinnaiyan. Overcoming acquired resistance to PROTAC degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1685.

Draft Genome Sequences of 14 Fluoroquinolone-Resistant Escherichia coli Isolates from Imported Shrimp.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA, parC, and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Analysis of receptor binding domain for possible mutations in S gene region of SARS-CoV-2

Humanity has historically been affected by remarkable epidemics and pandemics, including the plague, cholera, influenza, SARS-CoV, and MERS-CoV. A novel coronavirus pandemic known as SARS-CoV-2 is rapidly sweeping the globe. Over the period, the genome of the novel coronavirus has been mutated as it passes through its primary host. The world is reporting multiple point mutations. So the objective of this study was to observe modifications in the partial region of SARS-CoV-2 for the surveillance. This cross sectional study was carried out to detect the modifications in the SARS-CoV-2. For this purpose initial screening of COVID-19 was done to collect the strain of SARS-CoV-2 using RT-PCR. Then, primers were created in order to amplify the area using the S gene of the SARS-CoV-2. The amplified product was sent for the sequencing and bioinformatic tools were used to observed the mutations and data was compared with the Wild strain of the Virus. During the analysis, one of the most important point mutation was D614G caused by the amino acid substitution of aspartic acid with the glycine. Addition to this point mutation, other important mutations have also been observed.

Structure-Based Affinity Maturation of Antibody Based on Double-Point Mutations.

Structure-based site-directed affinity maturation of antibodies can be expanded by multiple-point mutations to obtain various mutants. However, selecting the appropriate number of promising mutants for experimental evaluation from the vast number of combinations of multiple-point mutations is challenging. In this report, we describe how to narrow candidate mutants using the so-called weak interaction analysis such as CH-π and CH-O in addition to widely recognized interactions such as hydrogen bonds.

Multiple point mutations in cytochrome b gene of Babesia gibsoni – A possible cause for buparvaquone resistance

The development and rise in drug-resistant Babesia gibsoni strain is a serious concern among veterinary practitioners. Of several therapeutic strategies followed, buparvaquone-azithromycin combination therapy is widely used to treat small Babesia infections in Asia. The present study focused on buparvaquone-induced mutations in B. gibsoni by analyzing its cytochrome b gene sequence. Among the 12 dogs that were administered with buparvaquone-azithromycin combination therapy, 8 of them were unresponsive to drug-resistant B. gibsoni infection. Hematological parameters were recorded before and after 10 day treatment plan and an improvement in these values was observed. Eight samples with persistent parasitemia after 10 day treatment plan was subjected to cytochrome b gene amplification and analyzed for mutations. On analysis, out of the 25 mutations only 9 were non-synonymous in nature; T15N, S48T, P152L, V167I, A217T, F258Y, M311I, S336G, A337S. Mutation P152L was seen near to Q01 (130–148) binding region and F258Y within the drug binding region Q02 (244–266).

IndelsRNAmute: predicting deleterious multiple point substitutions and indels mutations

BackgroundRNA deleterious point mutation prediction was previously addressed with programs such as RNAmute and MultiRNAmute. The purpose of these programs is to predict a global conformational rearrangement of the secondary structure of a functional RNA molecule, thereby disrupting its function. RNAmute was designed to deal with only single point mutations in a brute force manner, while in MultiRNAmute an efficient approach to deal with multiple point mutations was developed. The approach used in MultiRNAmute is based on the stabilization of the suboptimal RNA folding prediction solutions and/or destabilization of the optimal folding prediction solution of the wild type RNA molecule. The MultiRNAmute algorithm is significantly more efficient than the brute force approach in RNAmute, but in the case of long sequences and large m-point mutation sets the MultiRNAmute becomes exponential in examining all possible stabilizing and destabilizing mutations.ResultsAn inherent limitation in the RNAmute and MultiRNAmute programs is their ability to predict only substitution mutations, as these programs were not designed to work with deletion or insertion mutations. To address this limitation we herein develop a very fast algorithm, based on suboptimal folding solutions, to predict a predefined number of multiple point deleterious mutations as specified by the user. Depending on the user’s choice, each such set of mutations may contain combinations of deletions, insertions and substitution mutations. Additionally, we prove the hardness of predicting the most deleterious set of point mutations in structural RNAs.ConclusionsWe developed a method that extends our previous MultiRNAmute method to predict insertion and deletion mutations in addition to substitutions. The additional advantage of the new method is its efficiency to find a predefined number of deleterious mutations. Our new method may be exploited by biologists and virologists prior to site-directed mutagenesis experiments, which involve indel mutations along with substitutions. For example, our method may help to investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary structure.

Detection of Novel gyrB Mutation in Fluoroquinolone-Resistant Salmonella and Escherichia coli using PCR-RFLP

Abstract Background Emergence of fluoroquinolone resistance in gut pathogens is a cause of concern. Resistant to quinolone is mainly due to the point mutations at the quinolone-resistance determining regions (QRDR). The aim of the study was to develop polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) to detect QRDR mutations in gyrA and gyrB regions in enteric pathogens. Methodology PCR-RFLP was done for gyrA 83 region using HinfI and for gyrB 447 using AcuI for fluoroquinolone resistant and susceptible gut pathogens. The products were also sequenced to confirm the presence of restriction sites. Results In this study, a PCR-RFLP technique was developed to detect gyrA 83 mutations in Salmonella typhi and Escherichia coli. A first of its kind PCR-RFLP was also developed to detect gyrB 447 mutation using a restriction enzyme AcuI. Restriction digestion of gyrA using HinfI resulted in three bands for resistant S. typhi isolates due to the presence of mutation at gyrA 83 and four bands were seen for sensitive S. typhi isolates, while two bands for resistant and three bands were seen in sensitive E. coli isolates. Similarly, restriction digestion of gyrB using AcuI resulted in no digestion for resistant S. typhi isolates and two bands for resistant E. coli isolates. This suggest that there is mutation at gyrB 447 region ofE. coli, while no mutation was found in S. typhi isolates. Conclusion The PCR-RFLP developed in the present study could successfully detect gyrA 83 and gyrB 447 mutations in fluoroquinolone-resistant S. typhi and E. coli. The technique can be efficiently used in epidemiological studies instead of a cost-intensive sequencing method to detect the status of multiple point mutations in gut pathogens.

Molecular characterization of canine and feline kobuvirus infections in Iran.

Kobuviruses are viral pathogens with broad host range presented in human gastroenteritis cases; but, the pathogenesis of these viruses in companion animals is not well described. In the present study, the presence of canine (CaKVs) and feline kobuviruses (FeKVs) was detected in the 100 fecal samples of diarrhoeic and healthy companion dogs and cats by polymerase chain reaction in Tehran, Iran. The prevalence of infection was estimated as 8.00% and 4.00% in dogs and cats, respectively. All positive samples were belonged to non-diarrhoeic animals except for a feline sample being co-infected with panleukopenia. Sequence analysis showed multiple point mutations in canine and feline Iranian strains and new feline strain was detected in the present study. This is the first detection of CaKVs and FeKVs in Iran; but, the exact role of these enteric viral pathogens and their zoonotic risks are better to be clarified in all endemic regions.

Tolerance of Senecavirus A to Mutations in Its Kissing-Loop or Pseudoknot Structure Computationally Predicted in 3' Untranslated Region.

Senecavirus A (SVA) is an emerging virus that belongs to the genus Senecavirus in the family Picornaviridae. Its genome is a positive-sense and single-stranded RNA, containing two untranslated regions (UTRs). The 68-nt-long 3′ UTR is computationally predicted to possess two higher-order RNA structures: a kissing-loop interaction and an H-type-like pseudoknot, both of which, however, cannot coexist in the 3′ UTR. In this study, we constructed 17 full-length SVA cDNA clones (cD-1 to -17): the cD-1 to -7 contained different point mutations in a kissing-loop-forming motif (KLFM); the cD-8 to -17 harbored one single or multiple point mutations in a pseudoknot-forming motif (PFM). These 17 mutated cDNA clones were independently transfected into BSR-T7/5 cells for rescuing recombinant SVAs (rSVAs), named rSVA-1 to −17, corresponding to cD-1 to −17. The results showed that the rSVA-1, -2, -3, -4, -5, -6, -7, -9, -13, and -15 were successfully rescued from their individual cDNA clones. Moreover, all mutated motifs were genetically stable during 10 viral passages in vitro. This study unveiled viral abilities of tolerating mutations in the computationally predicted KLFM or PFMs. It can be concluded that the putative kissing-loop structure, even if present in the 3′ UTR, is unnecessary for SVA replication. Alternatively, if the pseudoknot formation potentially occurs in the 3′ UTR, its deformation would have a lethal effect on SVA propagation.

Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate

ABSTRACT The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus “chicken embryo lethal orphan” (CELO). RESEARCH HIGHLIGHTS AGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing. Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1). Multiple point mutations, site insertions and deletions identified compared to CELO. Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms—multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates—were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.

One-step base editing in multiple genes by direct embryo injection for pig trait improvement.

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

Genome sequence of a multidrug-resistant Campylobacter coli strain isolated from a newborn with severe diarrhea in Lebanon.

A multidrug-resistant (MDR) Campylobacter coli (C. coli) strain was isolated from a 2-month-old newborn who suffered from severe diarrhea in Lebanon. Here, whole-genome sequencing (WGS) analysis was deployed to determine the genetic basis of antimicrobial resistance and virulence in the C. coli isolate and to identify its epidemiological background (sequence type). The identity of the isolate was confirmed using API® Campy, MALDI-TOF, and 16S rRNA gene sequencing analysis. The antimicrobial susceptibility phenotype was determined using the disk diffusion assay. Our analysis showed that resistance to macrolide and quinolone was potentially associated with the presence of multiple point mutations in antibiotic targets on the chromosomal DNA. Furthermore, tetracycline and aminoglycoside resistance were encoded by genes on a pTet plasmid. The blaOXA-61, which is associated with beta-lactam resistance, was also detected in the C. coli genome. A set of 30 genes associated with the virulence in C. coli was detected using WGS analysis. MLST analysis classified the isolate as belonging to a new sequence type (ST-9588), a member of ST-828 complex which is mainly associated with humans and chickens. Taking together, this study provides the first WGS analysis of Campylobacter isolated from Lebanon. The detection of a variety of AMR and virulence determinants strongly emphasizes the need for studying the burden of Campylobacter in Lebanon and the Middle East and North Africa (MENA) region, where information on campylobacteriosis is scant.

Origin of the tight binding mode to ACE2 triggered by multi-point mutations in the omicron variant: a dynamic insight.

The continuous spread of the newly emerged SARS-CoV-2 Omicron variant (B.1.1.529) has become an important reason for the surge in COVID-19 infections. Its numerous mutated residues containing key sites on the receptor-binding domain (RBD) undoubtedly pose new challenges for epidemic control. Although the preventive measures are becoming more sophisticated, the effects of mutations on the binding of the virus to the receptor protein remain to be elucidated. Here, we used molecular dynamics (MD) simulations to investigate the differences in the binding mode between the Omicron variant and the angiotensin-converting enzyme 2 (ACE2) compared to the wild-type strain (WT). Multi-point mutations in the Omicron variant RBD could cause the conformation shift in the large Loop (where T478K and E484A are located), which makes it easier to wrap the N-terminal helix of ACE2 and form tighter contacts. The stronger electrostatic interaction was the main reason for its enhanced binding affinity as compared to WT. This was due to the large number of positively charged patches (N440K, T478K, Q493R, Q498R, and Y505H) formed by the substitution of neutral amino acids at multiple sites. The appearance of these highly polar hydrophilic amino acids may cause local perturbations and affect the electrostatic complementarity of RBD with the ACE2, and further mediate conformational changes. Thus, a more extensive interaction network was found in the mutation system and the complex interaction cluster was formed near E37@ACE2, which was essential for the stable binding of the two. In addition, we speculated that these mutations may affect the electrostatic complementarity with the four potential antibodies to reduce the sensitivity of the virus to antibodies. This study reveals the key details of the Omicron variant binding to ACE2 and provides important theoretical views for the enhanced infectivity of this variant. We hope that these observations can provide timely molecular insights for responding to the Omicron variant pandemic.

Mutagenic Activity of AID/APOBEC Deaminases in Antiviral Defense and Carcinogenesis

Proteins of the AID/APOBEC family are capable of cytidine deamination in nucleic acids forming uracil. These enzymes are involved in mRNA editing, protection against viruses, the introduction of point mutations into DNA during somatic hypermutation, and antibody isotype switching. Since these deaminases, especially AID, are potent mutagens, their expression, activity, and specificity are regulated by several intra-cellular mechanisms. In this review, we discuss the mechanisms of impaired expression and activation of AID/APOBEC proteins in human tumors and their role in carcinogenesis and tumor progression. Also, the diagnostic and potential therapeutic value of increased expression of AID/APOBEC in different types of tumors is analyzed. We assume that in the case of solid tumors, increased expression of endogenous deaminases can serve as a marker of response to immunotherapy since multiple point mutations in host DNA could lead to amino acid substitutions in tumor proteins and thereby increase the frequency of neoepitopes.

Mutagenic Activity of AID/APOBEC Deaminases in Antiviral Defense and Carcinogenesis.

Proteins of the AID/APOBEC family are capable of cytidine deamination in nucleic acids forming uracil. These enzymes are involved in mRNA editing, protection against viruses, the introduction of point mutations into DNA during somatic hypermutation, and antibody isotype switching. Since these deaminases, especially AID, are potent mutagens, their expression, activity, and specificity are regulated by several intracellular mechanisms. In this review, we discuss the mechanisms of impaired expression and activation of AID/APOBEC proteins in human tumors and their role in carcinogenesis and tumor progression. Also, the diagnostic and potential therapeutic value of increased expression of AID/APOBEC in different types of tumors is analyzed. We assume that in the case of solid tumors, increased expression of endogenous deaminases can serve as a marker of response to immunotherapy since multiple point mutations in host DNA could lead to amino acid substitutions in tumor proteins and thereby increase the frequency of neoepitopes.