Selection Of Mutants Research Articles

Synergistic Combination of Linezolid and Fosfomycin Closing Each Other's Mutant Selection Window to Prevent Enterococcal Resistance.

Enterococci, the main pathogens associated with nosocomial infections, are resistant to many common antibacterial drugs including β-lactams, aminoglycosides, etc. Combination therapy is considered an effective way to prevent bacterial resistance. Preliminary studies in our group have shown that linezolid combined with fosfomycin has synergistic or additive antibacterial activity against enterococci, while the ability of the combination to prevent resistance remains unknown. In this study, we determined mutant prevention concentration (MPC) and mutant selection window (MSW) of linezolid, fosfomycin alone and in combination including different proportions for five clinical isolates of Enterococcus and characterized the resistance mechanism for resistant mutants. The results indicated that different proportions of linezolid combined with fosfomycin had presented different MPCs and MSWs. Compared with linezolid or fosfomycin alone, the combination can restrict the enrichment of resistant mutants at a lower concentration. A rough positive correlation between the selection index (SI) of the two agents in combination and the fractional inhibitory concentration index (FICI) of the combination displayed that the smaller FICI of linezolid and fosfomycin, the more probable their MSWs were to close each other. Mutations in ribosomal proteins (L3 and L4) were the mechanisms for linezolid resistant mutants. Among the fosfomycin-resistant mutants, only two strains have detected the MurA gene mutation related to fosfomycin resistance. In conclusion, the synergistic combination of linezolid and fosfomycin closing each other’s MSW could effectively suppress the selection of enterococcus resistant mutants, suggesting that the combination may be an alternative for preventing enterococcal resistance. In this study, the resistance mechanism of fosfomycin remains to be further studied.

A Leucyl-tRNA Synthetase Inhibitor with Broad-Spectrum Anti-Mycobacterial Activity.

Global infections by non-tuberculous mycobacteria (NTM) are steadily rising. New drugs are needed to treat NTM infections, but the NTM drug pipeline remains poorly populated and focused on repurposing or reformulating approved antibiotics. We sought to accelerate de novo NTM drug discovery by testing advanced compounds with established activity against Mycobacterium tuberculosis 3-aminomethyl 4-halogen benzoxaboroles, a novel class of leucyl-tRNA synthetase inhibitors, were recently discovered as active against M. tuberculosis Here, we report that the benzoxaborole EC/11770 is not only a potent anti-tubercular agent but is active against the M. abscessus and M. avium complexes. Focusing on M. abscessus, which causes the most difficult-to-cure NTM disease, we show that EC/11770 retained potency against drug-tolerant biofilms in vitro and was effective in a mouse lung infection model. Resistant mutant selection experiments showed a low frequency of resistance and confirmed leucyl-tRNA synthetase as the target. This work establishes the benzoxaborole EC/11770 as a novel preclinical candidate for the treatment of NTM lung disease and tuberculosis and validates leucyl-tRNA synthetase as an attractive target for the development of broad-spectrum anti-mycobacterials.

Identification of 16S rRNA mutations in Mycoplasma genitalium potentially associated with tetracycline resistance in vivo but not selected in vitro in M. genitalium and Chlamydia trachomatis.

Tetracyclines are widely used for the treatment of bacterial sexually transmitted infections (STIs) and recently have been used successfully for post-exposure prophylaxis of STIs in MSM. We investigated the in vitro and in vivo development of tetracycline resistance in Chlamydia trachomatis and Mycoplasma genitalium and evaluated 16S rRNA mutations associated with acquired resistance in other bacteria. In vitro selection of resistant mutants of reference strains of C. trachomatis and M. genitalium was undertaken by serial passage in medium containing subinhibitory concentrations of tetracycline or doxycycline, respectively. The 16S rRNA gene of the two microorganisms was amplified and sequenced at different passages, as were those of 43 C. trachomatis- and 106 M. genitalium-positive specimens collected in France from 2013 to 2019. No tetracycline- or doxycycline-resistant strains of C. trachomatis and M. genitalium, respectively, were obtained after 30 serial passages. The tetracycline and doxycycline MICs were unchanged and analysis of the 16S rRNA gene, the molecular target of tetracyclines, of C. trachomatis and M. genitalium revealed no mutation. No mutation in the 16S rRNA gene was detected in C. trachomatis-positive specimens. However, six M. genitalium-positive specimens harboured a mutation potentially associated with tetracycline resistance without known prior tetracycline treatment for patients. Tetracyclines did not select in vitro-resistant mutants of C. trachomatis or M. genitalium. However, 16S rRNA mutations either responsible for or associated with tetracycline resistance in other bacteria, including mycoplasma species, were identified in several M. genitalium-positive specimens.

Enhanced production of clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain.

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3% (5.21 ± 0.26 g/l) in a flask culture and 17.4% (6.11 ± 0.36 g/l) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C

Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.

Genetic improvement of pyrethrum (Tanacetum cinerariifolium Sch. Bip.) through gamma radiation and selection of high yield stable mutants through seven post-radiation generations

Purpose To increase the size of the flowers for easy plucking, flower yield, pyrethrins content (%), and elite mutant selection in pyrethrum. Materials and methods To increase pyrethrum production and acclimatize in north Indian plain condition, a genetic improvement program was undertaken to widen the range of variations for size and yield of flowers and pyrethrins content (%) in pyrethrum crop. Pyrethrum seeds of the variety Avadh were irradiated with gamma rays at 20 to 300 Gy doses in Gamma chamber 5000 (cobalt-60 research irradiator). Results Observations gathered visually in M1 based on vigor, synchronization of flowering, and flower’s size. Out of 90 M2 families, 20 mutants were raised in M3 along with the check-in preliminary evaluation trial. The four promising mutants, 1 (20 Gy-3), 7 (40 Gy-5), 10 (40 Gy-8), 14 (60 Gy19-10) was grown for four years in a bench-scale trial (randomized block design, replicated thrice) to test the yield performance and selection of high yielding elite mutant (s). It has been found that pyrethrum is sensitive to gamma rays irradiation and produced a high range of qualitative and quantitative variations. After massive screening over four years, two promising mutants for high dry flower yield and pyrethrins content, namely 7 (40 Gy-5), and 10 (40GY-8) were isolated. Conclusions The mutagenesis changed traits mean in positive or negative directions. Pyrethrum plant is highly sensitive to gamma irradiation and produced a high range of variability in the qualitative and quantitative traits. The mutagenesis changed the mean of traits in both positive and negative directions. Due to mutagenic efficacy, two mutants 7 (40 Gy-5), and 10 (40GY-8) were expressed high performance for pyrethrin percent i.e., 87.23 and 59.78% improvement over the check variety ‘Avadh’, with synchronous flowering. These two mutants are in the pipeline for release as a variety for cultivation in the North Indian plains.

Design, Synthesis and Antibacterial Studies of Some New Pyridopyrimidine Derivatives as Biotin Carboxylase Inhibitors

Present study reports the development of novel pyridopyrimidine derivatives as biotin carboxylase inhibitors with potent antibacterial activity. These compounds were designed to avoid possibility of resistance development. Accordingly eighteen compounds were synthesised and characterized on the basis of spectral data. These compounds were tested for their antibacterial potential by the enzyme kinetic assay against the biotin carboxylase. The minimum inhibitory concentration (MIC) and single step resistance studies were also performed. Compound 2-((2-Phenylpyrido[2,3-d]pyrimidin-4-yl)amino)phenol (6o) showed promising activity in biotin carboxylase inhibition with low MIC. It showed molecular docking score of -7.96, this compound showed formation of hydrogen bonds with the active site residues and van Der Walls intractions. The MIC of compounds under investigation was in the rage of 2-5µg/mL over most of the strains studied. It also showed the mutant selection windows of around five which is better than the reference compound rifampin. This compound 6o can be studied further and developed into a potential antibacterial lead molecule.

Bacteriophages in dairy plants.

Bacteriophages represent the main microbiological threat for the manufacture of fermented foods. The dairy industry is the most affected by this problem, as phages are naturally present in raw milk, surfaces, vats, tanks, floors, and distributed by air displacements. Cheese whey may also contain high phage concentrations. Prophages harbored by lysogenic strains could be induced, generating new lytic phages. In this context, where phages cannot be eradicated from dairies, methods of phage monitoring are mandatory. These are mainly based in microbiological features, like classical methods, that are the most used, economic and simple to carry out. Phage DNA detection and quantification by PCR and qPCR, more complex and expensive, are faster, although not able to discern between viable and non-viable virions. Electron microscopy allows direct visualization and characterization of phage morphology, but the apparatus is expensive. Alternative methods based in other phage traits also exist, though less studied and not applicable on a daily basis. Recognition of contamination sources and correct phage monitoring in dairy factories allow a correct application of control measures. These include general measures such as proper factory design, efficient programs of sanitization, good treatment of raw materials, especially milk, and careful handling of by-products. Additionally, the use of starts cultures should be adequate, with application of rotation schemes when possible. Finally, the selection of bacteriophage insensitive mutants (BIM) is essential, and can be achieved simply and empirically, though the study of CRISPR-Cas and other newly discovered mechanisms provide a more rational basis to obtain BIMs with optimized features.

Selection of M5 short-stemmed mutants from 200 gray gamma ray irradiation in Mentik Wangi rice

Mentik Wangi rice is native to Indonesia, it has an oval round shape of rice, yellow brown rice, and fragrant aroma. Mentik Wangi has a weakness that is relatively long harvest time, the plant is too high so it easily fell. This research aims to determine the performance of M5 generation of mutant rice and to select M5 generation of mutant plants resulted from 200 gray gamma irradiation which has short stems with high productivity. The research was carried out by planting 36 genotypes, namely 35 M4 lines of 200 gray gamma ray irradiation and control plant (without irradiation). Observation data were analyzed descriptively and t test to determine differences in M5 generation of Mentik Wangi rice from 200 gray gamma ray irradiation with control plants (without irradiation). Based on the research results that Mentik Wangi rice plants with gamma ray irradiation 200 Gray have better performance compared to control plants (without irradiation) and there are 28 individuals M5 Mentik Wangi rice mutants selected by short-stemmed plants and have high productivity.

Influence of different antibiotic groups on the development of mutational resistance to colistin among Klebsiella pneumoniae

Objective. To determine the concentration of colistin, preventing the selection of colistin-resistant mutants of K. pneumoniae, and to evaluate the effect of antibiotics of different groups on the development of mutational resistance to colistin. Materials and Methods. Minimum inhibitory concentrations (MIC) of colistin were determined for 88 K. pneumoniae strains by the method of serial microdilutions in broth, and carbapenemase genes were detected. The selection of colistin-resistant subpopulations was performed on cation-adjusted MüllerHinton agar (MHA) with the addition of 16 mg/l colistin. Mutant prevention concentration (MPC) of colistin is determined on MHA containing 0, 1, 2, 4, 8, 16, 32, 64 and 128 mg/l of colistin. Also, MPCs of colistin were determined in the presence of a fixed concentration of the second antibiotic: clarithromycin (2 mg/l), azithromycin (2 mg/l), rifampicin (1 mg/l), clindamycin (0.5 mg/l), meropenem (8 mg/l), linezolid (2 mg/l), amikacin (1 mg/l), vancomycin (2 mg/l), doxycycline (2 mg/l). Results. All strains remained susceptible to colistin (colistin MIC 0.06–1.0 mg/l). Resistance to meropenem (MIC > 8 mg/l) was detected in 48 strains (54.5%), 46 of them were carbapenemase producers: KPC – 6 strains (6.8%), OXA-48 – 26 strains (29.5%), NDM – 14 strains (15.9%). Growth of colonies on MHA with 16 mg/l of colistin was found for 96.6% of the strains. The frequency of mutational resistance occurrence ranged from 6 × 10-9 to 10-6 (median: 2 × 10-7). The mutational nature of colistin resistance was confirmed for 36.4% of the strains. The MPC values of colistin were in the range of 16–256 mg/l; (MPC50 32 mg/l, MPC90 256 mg/l) and significantly (32–1024 times) exceeded the MIC values. In the presence of 1 mg/l of rifampicin, the MPC of colistin decreased 4–64 times (MPC50 4 mg/l, MPC90 4 mg/l). In the presence of 2 mg/l of doxycycline, MPC of colistin decreased 2–64 times for all strains (MPC50 8 mg/l, MPC90 16 mg/l). The presence of linezolid (2 mg/l) and vancomycin (2 mg/l) did not significantly change MPC of colistin. Meropenem at a concentration of 8 mg/l had no significant effect on colistin MPC for carbapenemase-producing K. pneumoniae strains. None of the antibiotics lowered the MPC50 of colistin to its clinically achievable serum concentrations. Conclusions. A high frequency of formation of mutational resistance to colistin in K. pneumoniae was revealed. The MPC values of colistin are outside the range of clinically achievable serum concentrations and may decrease in the presence of other antibiotics.

Forward mutations in sars-cov-2 cause waves of covid-19

Time space between waves of Covid-19 is linked to selection of most appropriate virus mutant(s)/variant(s), in the Spike (S) gene, generated during massive and error prone replication of the virus in the human populations that are either naive or partially immune due to infection immunity or vaccinal immunity, having higher transmissibility with immune-escape feature and increased pathogenicity. The mutations in the S gene have been found to be progressive to resist prevalent virus neutralizing antibodies in the hosts. The first mutation of significance was D (Asp) to G (Gly) at the position 614 of the Spike protein, over the original Wuhan strain/virus. Subsequently, with in a short time span of less than 18 months since December 2019, many Variants/mutants of SARS-CoV-2, viz., B.1.1.7, B.1.427, B.1.1.28.2, B.1.429, B.1.617, B.1.351, and P.1 have been found associated with the current waves of the Covid-19 pandemic in different countries. The latest significant S variants identified in the second wave in India and elsewhere are B.1.617 and its Kappa (k), Delta (8) and Delta plus (8+; Lys to Asn at position 417) strains, in the order of appearance. The lambda (X) variant is prevalent in South America. The P.2 variant B.1.1.28.2 was antigenically dominant over B.1 D614G virus. The k and 8 variants of the S gene were partially resistant to neutralization by existing vaccinal antibodies, and the 8 variant has been feared to ignite third wave in many countries of Americas, Asia and Europe. The SARS-CoV-2 possibly has ‘Quasispecies’ structure, like the transboundary animal pathogen Foot and Mouth Disease (FMD) virus, and continuous forward mutations in the S gene has been the cause of the fact that "previous immunity acquired during the course of the natural infection (or vaccination) is not fully competent to protect against subsequent mutants selected (in the nature) that leads to new waves of the disease". The waves of Covid-19 have been guided by emergence of sequential antigenic variants, e.g. B.1 D614G virus ^B.1 8 mutant ^B.1 8+ mutant. Intermittent/inter-wave drop in incidence of the infection between the Covid-19 waves could be attributed to very short term (less than 3 months?) infection/vaccination immunity that is breached by the successive antigenic mutant (s) selected. Vaccination and virus circulation happening simultaneously, can facilitate faster selection of neutralization-escape mutants, which is now evident and may lead to new waves of the disease/infection at short intervals of less than 6 months. All variants described here are antigenically dissimilar, whereas the current Covid-19 vaccines are monovalent. Therefore, it may be essential and beneficial to have multivalent vaccines or with higher antigenic mass in monovalent vaccines. Further, it is required that nasal swabs of a certain percentage of vaccinates be collected at the time of vaccination and preserved for retrospect analysis of their infection status, that in turn will reveal the infection status of the population in villages/cities/districts/State.

1486. Phylogenetic and alpha toxin variant analyses of Staphylococcus aureus strains isolated from patients during the SAATELLITE study

BackgroundSuvratoxumab is a human monoclonal antibody that neutralizes S. aureus (SA) alpha toxin (AT). SAATELLITE, a phase 2 study of the safety and efficacy of suvratoxumab for reducing the incidence of SA pneumonia (NCT02296320), was conducted within the consortium for Combatting Bacterial Resistance in Europe.MethodsA total of 304 SA isolates (baseline, onset and last available isolates from suspected serious bacterial infections, SSBIs) collected from the lower respiratory tract samples from 165 subjects during SAATELLITE were subjected to whole genome sequencing.AT gene (hla) sequences were translated and amino acid variation was identified in comparison to the reference SA USA300 FPR3757. Phylogenetic analysis, genomic annotation and ST analysis were performed.AT expression in SA culture supernatants was performed by ELISA. Representative isolates with novel AT subtypes that had not been identified in previous studies were tested for hemolytic activity and suvratoxumab neutralizing activity.Wilcoxon rank sum test and Fisher’s exact test were performed, respectively: a) to compare difference in baseline AT expression in relation to SA pneumonia incidence; b) to evaluate the association between occurrence of AT stop codons and incidence of SA pneumonia at baseline, as well as the association between occurrence of AT stop codons and treatment arms at post baseline.ResultsWe identified a total of 44 sequence types (STs) and 21 unique AT subtypes, 7 of which have not been described previously. No substitutions were located in the suvratoxumab binding region and all novel AT subtypes displaying lytic activity were neutralized by suvratoxumab.We detected stop codons Q113B and W205B in AT sequences in 53 and 2 SA isolates, respectively. We uncovered no significant associations of: 1) baseline AT expression with SA pneumonia incidence [p=0.967]; 2) occurrence of AT gene stop codon with either SA pneumonia incidence [p >0.999] or suvratoxumab treatment [p=0.103; lower frequency of stop codons in suvratoxumab arm versus placebo].ConclusionOur data indicated that: 1) suvratoxumab target region in (AT) remains conserved; 2) suvratoxumab is active against all AT variants identified to date; 3) suvratoxumab did not exert pressure on SA clinical isolates for selection of escape mutants.DisclosuresDavid E. Tabor, PhD, AstraZeneca (Employee, Shareholder) Andrey Tovchigrechko, PhD, AstraZeneca (Employee, Shareholder)KitePharma, a Gilead company (Employee, Shareholder) Bret R. Sellman, PhD, AstraZeneca (Employee, Shareholder) Michael McCarthy, n/a, AstraZeneca (Employee) Kathryn Shoemaker, MS, AstraZeneca (Employee) Hasan S. Jafri, MD, FAAP, AstraZeneca (Employee) Mark T. Esser, PhD, AstraZeneca (Employee) Alexey Ruzin, PhD, AstraZeneca (Employee, Shareholder)

Revisiting the mode of action of the antimalarial proguanil using the yeast model

Proguanil in combination with its synergistic partner atovaquone has been used for malaria treatment and prophylaxis for decades. However its mode of action is not fully understood. Here we used yeast to investigate its activity. Proguanil inhibits yeast growth, causes cell death and acts in synergy with atovaquone. It was previously proposed that the drug would target the system that maintains the mitochondrial membrane potential when the respiratory chain is inhibited. However our data did not seem to validate that hypothesis. We proposed that proguanil would not have a specific target but accumulate in the mitochondrial to concentrations that impair multiple mitochondrial functions leading to cell death. Selection and study of proguanil resistant mutants pointed towards an unexpected resistance mechanism: the decrease of CoQ level, which possibly alters the mitochondrial membrane properties and lowers proguanil intramitochondrial level.

Nisin Influence on the Antimicrobial Resistance Ability of Canine Oral Enterococci

Periodontal disease (PD) is one of the most common diseases in dogs. Although previous studies have shown the potential of the antimicrobial peptide nisin for PD control, there is no information regarding its influence in the development of antimicrobial resistance or horizontal gene transfer (HGT). Nisin’s mutant prevention concentration (MPC) and selection window (MSW) were determined for a collection of canine oral enterococci. Isolates recovered after the determination of the MPC values were characterized for their antimicrobial profile and its nisin minimum inhibitory and bactericidal concentrations. The potential of vanA HGT between Enterococcus faecium CCGU36804 and nine clinical canine staphylococci and enterococci was evaluated. Nisin MPC values ranged from 400 to more than 600 μg/mL. In comparison with the original enterococci collection, the isolates recovered after the determination of the nisin MPC showed increased resistance towards amoxicillin/clavulanate (5%), vancomycin (5%), enrofloxacin (10%), gentamicin (10%) and imipenem (15%). The HGT of vanA gene was not observed. This work showed that nisin selective pressure may induce changes in the bacteria’s antimicrobial resistance profile but does not influence horizontal transfer of vanA gene. To our knowledge, this is the first report of nisin’s MPC and MSW determination regarding canine enterococci.

Genetically encoded multimode reporter of adaptor complex 3 traffic in budding yeast.

AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway's importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

Methoxy-Substituted Hydroxychalcone Reduces Biofilm Production, Adhesion and Surface Motility of Acinetobacter baumannii by Inhibiting ompA Gene Expression.

An increasing lack of available therapeutic options against Acinetobacter baumannii urged researchers to seek alternative ways to fight this extremely resistant nosocomial pathogen. Targeting its virulence appears to be a promising strategy, as it offers considerably reduced selection of resistant mutants. In this study, we tested antibiofilm potential of four synthetic chalcone derivatives against A. baumannii. Compound that showed the greatest activity was selected for further evaluation of its antivirulence properties. Real-time PCR was used to evaluate mRNA expression of biofilm-associated virulence factor genes (ompA, bap, abaI) in treated A. baumannii strains. Also, we examined virulence properties related to the expression of these genes, such as fibronectin- and collagen-mediated adhesion, surface motility, and quorum-sensing activity. The results revealed that the expression of all tested genes is downregulated together with the reduction of adhesion and motility. The conclusion is that 2'-hydroxy-2-methoxychalcone exhibits antivirulence activity against A. baumannii by inhibiting the expression of ompA and bap genes, which is reflected in reduced biofilm formation, adhesion, and surface motility.

A decoy to neutralize SARS-CoV-2

Coronavirus Many efforts to develop therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the interaction between the spike protein, which decorates the surface of the virus, and its host receptor, human angiotensin-converting enzyme 2 (hACE2). Linsky et al. describe a de novo design strategy that allowed them to engineer decoy proteins that bind to the spike protein by replicating the hACE2 interface. The best decoy, CTC-445, bound with low nanomolar affinity, and selection of viral mutants that decrease binding is unlikely because this would also affect binding to hACE2. A bivalent version of CTC-445 bound even more tightly, neutralized SARS-CoV-2 infection of cells, and protected hamsters from a SARS-CoV-2 challenge. The stable decoy has the potential for respiratory therapeutic delivery. Science , this issue p. [1208][1] [1]: /lookup/doi/10.1126/science.abe0075

Spectral clustering based mutant reduction for mutation testing

Abstract Context: Mutation testing techniques, which attempt to construct a set of so-called mutants by seeding various faults into the software under test, have been widely used to generate test cases as well as to evaluate the effectiveness of a test suite. Its popularity in practice is significantly hindered by its high cost, majorly caused by the large number of mutants generated by the technique. Objective: It is always a challenging task to reduce the number of mutants while preserving the effectiveness of mutation testing. In this paper, we make use of an intelligent technique, namely spectral clustering, to improve the efficacy of mutant reduction. Method: First of all, we give a family of definitions and the method to calculate the distance between mutants according to the weak mutation testing criteria. Then we propose a mutant reduction method based on spectral clustering (SCMT), including the determination method of the number of clusters, spectral clustering of mutants, and selection of representative mutants. Results: The experimental studies based on 12 object programs show that the new approach can significantly reduce the number of mutants without jeopardizing the performance of mutation testing. As compared with other benchmark techniques, the new approach based on weak mutation testing criteria cannot only consistently deliver high effectiveness of mutation testing, but also help significantly reduce the time-cost of mutation testing. Conclusion: It is clearly demonstrated that the use of spectral clustering can help enhance the cost-effectiveness of mutation testing. The research reveals some potential research directions for not only mutation testing but also the broad area of software testing.

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria.

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.

Machine Learning Enables Selection of Epistatic Enzyme Mutants for Stability Against Unfolding and Detrimental Aggregation.

Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov'SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside‐the‐box, predictions not found in other state‐of‐the‐art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness.