Resistance Mutations Research Articles

COMPARISON OF NANOPORE AND CLASSICAL SANGER SEQUENCING TO IDENTIFY MOSQUITO BLOODMEAL HOSTS.

The tools available to vector control districts (VCDs) to collect mosquito surveillance data are constantly evolving. As more VCDs obtain real-time polymerase chain reaction (PCR) instruments and the costs associated with computing power and next-generation sequencing continue to decrease, the option of generating useful molecular data in-house becomes more viable. Measures such as arbovirus testing and genotyping for insecticide resistance mutations using RT-qPCR, and identifying species used for mosquito bloodmeals with next-generation sequencing or Sanger sequencing are examples. In this study we identify mosquito host bloodmeal species using Nanopore sequencing from Oxford Nanopore Technologies. We used MinION and Flongle flow cells and a Mk1C device to sequence 96 barcoded amplicon samples in a single sequencing run, and share details of data analysis using the free-to-use Galaxy bioinformatics platform. After sequencing the same samples with Sanger sequencing, we conclude that Nanopore sequencing is better at identifying species in mixed bloodmeals. This work demonstrates a potential use of nanopore sequencing by VCDs with basic biology laboratory and computing equipment.

Evaluation and Isolation of Mungbean Yellow Mosai Virus Resistant Mutants in M4 and M5 Mutant Generations of Blackgram (Vigna mungo)

Background: The pulse known as “Blackgram” is a significant component of the Indian diet since it provides vegetable protein and balances the diet centered on cereals. Mungbean Yellow Mosaic Virus (MYMV) outbreaks on a regular basis completely destroy crops, causing farmers to suffer enormous losses. Mutation breeding can be an effective strategy for developing crops with desirable traits, such as high yield and resistance to diseases like the Mungbean Yellow Mosaic Virus (MYMV) in blackgram. Methods: This experiment was laid out to study the variability pattern of mutant population in M4 and M5 generation of blackgram variety CO 6 treated with the Gamma rays at 200 Gy, 300 Gy and 400Gy and isolation of viable mutants for Mungbean Yellow Mosaic virus resistance. Significant variability was observed among the different treatments in M4 generation and twenty MYMV resistant mutants ((PKT-BG-200Gy-P07; PKT-BG-200Gy-P24; PKT-BG-200Gy-P29; PKT-BG-200Gy-P35; PKT-BG-200Gy-P36; PKT-BG-200Gy-P45; PKT-BG-300Gy-P13; PKT-BG-300Gy-P16; PKT-BG-300Gy-P28; PKT-BG-300Gy-P33; PKT-BG-300Gy-P41; PKT-BG-300Gy-P4; PKT-BG-300Gy-P43; PKT-BG-300Gy-P76; PKT-BG-300Gy-P81; PKT-BG-300Gy-P88; PKT-BG-300Gy-P93; PKT-BG-400Gy-P15; PKT-BG-400Gy-P21; PKT-BG-400Gy-P23) were selected and evaluated in a randomized block design with three replications under natural conditions. Scoring was done by the modified scale of All India Coordinated Research Project on MuLLaRP. Result: Incidence ranged from scale 0.01 (PKT-BG-300Gy-P41and PKT-BG-300Gy-P42) to 5.30 (PKT-BG-300Gy-P28). The mutants PKT-BG-300Gy-P41 and PKT-BG-300Gy-P42 recorded with maximum yield of 1500 kg/ha and 1445kg/ha were found to be observed as high yielding and MYMV resistant lines for summer season in the Cauvery delta region.

Performance of urine Xpert MTB/RIF ultra in a tuberculosis screening strategy in hospitalized patients with advanced HIV disease: Results from an implementation initiative in Brazil.

To evaluate the performance of Xpert MTB/RIF Ultra testing in urine samples as part of a TB screening strategy in patients with advanced HIV disease (AHD). We conducted a multicentre prospective cohort study across three HIV reference hospitals in Brazil, between January and December 2023. The study included adult hospitalized patients with AHD, defined by a CD4 count of <200 cells/μL in the last 3 months or clinical presentation suggestive of opportunistic infection, without effective antiretroviral treatment. Participants underwent systematic tuberculosis (TB) screening using urine Xpert MTB/RIF Ultra and TB lipoarabinomannan (TB-LAM) tests. The diagnosis performance of urine Xpert MTB/RIF Ultra was assessed including sensitivity, specificity, and positive and negative predictive values. Disease characterization was based on the Global Tuberculosis Dictionary. Survival at 30 and 90 days was also evaluated. Urine molecular testing was performed on 133 patients. Xpert MTB/RIF Ultra showed a sensitivity of 20.7% in bacteriologically confirmed TB cases and 21.2% in cases that included both clinically diagnosed and bacteriologically confirmed TB. The addition of urine Xpert MTB/RIF Ultra to TB-LAM led to the detection of three additional cases, representing a 6.3% increase from the 48 cases detected by TB-LAM. Xpert MTB/RIF Ultra had a specificity of 96.9% and no rifampicine resistance mutations were detected. Overall mortality was 16/133 (12.0%) at 30 days and 25/127 (19.7%) at 90 days. There was a high overlap between urine TB-LAM and Xpert MTB/RIF Ultra results, with the addition of Xpert MTB/RIF providing limited additional benefit for TB screening in patients with AHD.

Analysis of Subtype, Molecular Clusters and Drug Resistance in Newly Diagnosed Individuals with HIV-1 Infection in Jiaxing City, Zhejiang Province, China in 2023

The analysis of the molecular epidemiological characteristics of newly diagnosed HIV-infected patients in Jiaxing City is essential for developing effective HIV prevention. Blood samples were collected from newly diagnosed HIV-infected individuals in Jiaxing City from October 2022 to October 2023, and the HIV-1 pol region gene was amplified and sequenced. These sequences were used to construct a molecular transmission network and analyse transmitted drug resistance mutations. We identified 11 subtypes, of which CRF07_BC and CRF01_AE were the most prevalent. The rate of surveillance drug resistance mutation (SDRM) sites in newly diagnosed cases was 9%. A total of 37 molecular transmission clusters were identified, the largest of which was the CRF07_BC-1 cluster (13 nodes). This cluster has five probable high-risk transmitters. Two additional larger clusters in the molecular network were the heterosexual transmission clusters for middle-aged and older males, CRF08_BC-1 (eight nodes) and CRF85_BC-1 (eight nodes). The mean degree of the two clusters was high, and the patients were high-risk transmitters, indicating a higher risk of HIV transmission. The distribution of HIV-1 subtypes in Jiaxing City was widespread, with moderate levels of transmission resistance. Larger molecule clusters carry a high risk of transmission, indicating that we should strengthen monitoring and intervention.

Mode of Action and Mechanisms of Resistance to the Unusual Polyglycosylated Thiopeptide Antibiotic Persiathiacin A.

Persiathiacin A is a novel thiopeptide antibiotic produced by Actinokineospora species UTMC 2448. It has potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. Thiopeptides, including persiathiacin A, exhibit antibacterial activity by inhibiting protein synthesis. In this study, we characterize the mechanism of action of persiathiacin A and investigate how resistance to this antibiotic can emerge. In vitro assays revealed that persiathiacin A inhibits translation elongation, leading to ribosome stalling. Genetic analysis of resistant Bacillus subtilis mutants identified mutations primarily in the rplK gene encoding ribosomal protein L11, which is the binding site for other 26-membered macrocycle-containing thiopeptides. The resistant mutants showed growth impairment and an increased lag time, even in the absence of persiathiacin. Comparative proteomic analysis of a resistant mutant versus the parental strain revealed multiple changes, indicative of negative effects on protein synthesis. Thus, although persiathiacin-resistant mutants can arise readily by the loss of L11 function, it is likely that such mutants would be severely compromised in pathogenesis. Furthermore, bioinformatics analysis identified differences in the key amino acids within the thiopeptide-binding region of L11 in the persiathiacin producer. These probably prevent the antibiotic from associating with its target, providing a mechanism for self-resistance.

StilPCR increases the effective sequencing length of Illumina targeted next-generation sequencing.

Identifying pathogens, resistance-conferring mutations, and strain types through targeted amplicon sequencing is an important tool. However, due to the limitations of short read sequencing, many applications require the division of limited clinical samples. Here, we present stilPCR (single-tube Illumina long read PCR), which allows the generation of hemi-nested amplicons in a single tube, with Illumina indexes and adapters, effectively increasing the Illumina read length without increasing the input requirements of reagents or sample. We have successfully utilized stilPCR on clinical sputum from tuberculosis patients to detect drug resistance mutations.

Abstract PR004: Brain penetrant allosteric EGFR inhibitors for NSCLC

Abstract Approximately 30-50% of Non-Small Cell Lung Cancer (NSCLC) patients already have CNS disease at time of diagnosis or else develop it during treatment, and there remains a need for better outcomes in these patients. The 3rd-generation covalent EGFR inhibitor osimertinib is an effective therapy for NSCLC patients, improving upon 1st-generation inhibitors gefitinib and erlotinib, and achieving enhanced brain penetration relative to those agents. However, the survival benefit relative to the 1st-generation inhibitors is observed primarily in patients with exon19 deletions rather than the L858R mutation. We developed a series of mutant-selective allosteric EGFR inhibitors that specifically target the L858R mutation (and subsequent resistance mutations), with high selectivity over wt EGFR and across the kinome. Early compounds including the isoindolinone JBJ-09-063 demonstrated good in vivo efficacy in mouse tumor models, despite limited bioavailability. Diversification and optimization of the chemistry, involving a scaffold hopping approach and the replacement of the thiazole amide with benzimidazole, delivered a set of compounds with promising potency and rodent PK. A subset of these achieved good brain exposure in mice and efficacy in an intracranial H1975 brain metastasis model. The mutant-selectivity and ability to co-bind with osimertinib makes an allosteric EGFR inhibitor an ideal partner for combination therapy, with the potential to deliver enhanced outcomes in L858R-driven NSCLC, especially for patients with CNS disease. Citation Format: David Scott, Courtney Cullis, Tyler Beyett, Frederic Feru, Thomas Gero, Krista Gipson, Praful Gokhalle, Nathanael Gray, David Heppner, Sampson Huang, Pasi Jänne, Sandeepraj Pusalkar, Michael Eck. Brain penetrant allosteric EGFR inhibitors for NSCLC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr PR004

The Resistance to EGFR-TKIs in Non-Small Cell Lung Cancer

Lung cancer has emerged as a highly aggressive malignancy posing a significant global health hazard, with a particular concern for the rising incidence and mortality of gene-altered non-small cell lung cancer (NSCLC). As the prevalence of NSCLC rises, the investigation and selection of tumor treatment techniques are gaining significance. The treatment of individuals with genetic mutations in advanced NSCLC has emphasized the application of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Early and subsequent generations of EGFR-TKIs have demonstrated clinical efficacy in NSCLC patients carrying select genetic alterations. Osimertinib, a third-generation EGFR-TKI, has been sanctioned as the standard first-line therapy and is also utilized as a second-line option for individuals who develop the T790M resistance mutation following prior EGFR-TKI treatment. Approximately 10 to 30 percent of patients diagnosed with NSCLC exhibit EGFR mutations. This leads to abnormal activation of cancer genes and inactivation of tumor suppressor genes, resulting in poor prognosis for patients. This paper aims to analyze the development and resistance processes of EGFR inhibitors, thereby enhancing the comprehension of the resistance pathways to EGFR-TKIs.

Thymidine Analogue Mutations with M184V Significantly Decrease Phenotypic Susceptibility of HIV-1 Subtype C Reverse Transcriptase to Islatravir.

Islatravir (ISL) is the first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTtI) with novel modes of action. Data on ISL resistance are currently limited, particularly to HIV-1 non-B subtypes. This study aimed to assess prevalent nucleos(t)ide reverse transcriptase inhibitor (NRTI)-resistant mutations in HIV-1 subtype C for their phenotypic resistance to ISL. Prevalent single and combinations of NRTI-resistant mutations were selected from a routine HIV-1 genotypic drug resistance testing database and introduced into HIV-1 subtype C-like pseudoviruses, which were then tested for ISL susceptibility. Single NRTI-resistant mutations were susceptible or showed only a low level of resistance to ISL. This included thymidine analogue mutations (TAMs, i.e., M41L, D67N, K70R, T215FY, and K219EQ) and non-TAMs (i.e., A62V, K65R, K70ET, L74IV, A114S, Y115F, and M184V). Combinations of M184V with one or more additional NRTI-resistant mutations generally displayed reduced ISL susceptibilities. This was more prominent for combinations that included M184V+TAMs, and particularly M184V+TAM-2 mutations. Combinations that included M184V+K65R did not impact significantly on ISL susceptibility. Our study suggests that ISL would be effective in treating people living with HIV (PLWH) failing tenofovir disoproxil fumarate (TDF)/lamivudine (3TC) or TDF/emtricitabine (FTC)-containing regimens, but would be less effective in PLH failing zidovudine (AZT) with 3TC or FTC-containing regimens.

A MALDI-TOF MS-based multiple detection panel of drug resistance-associated multiple single-nucleotide polymorphisms in Candida tropicalis.

Candida tropicalis is one of the main causes of invasive candidiasis. Rapid identification of antifungal resistance is crucial for selection of an appropriate antifungal to improve patient outcomes. Mutations at specific loci are strongly correlated with resistance to antifungal agents. In this study, we developed a multi-single-nucleotide polymorphism (SNP) panel to accurately identify 36 mutation sites across seven genes of C. tropicalis that are associated with resistance to azoles and/or echinocandins. Ten isolates were selected to test repeatability, and another 20 isolates of C. tropicalis were selected to validate consistency. Intra-assay and inter-assay repeatability of the panel was 100%, with the loci accuracy being 99.44% (716 of 720). Furthermore, 109 isolates were examined for clinical research, and the most commonly detected mutations were G751A and A866T of UPC2, A491T of TAC1, and A395T and C461T of ERG11. The G751A and A866T mutations of UPC2 as well as the A395T and C461T mutations of ERG11 co-existed. The SNP panel enables identification of specific mutations at critical sites of drug-resistant strains to facilitate the rapid selection of appropriate antifungal agents and efficient monitoring of the regional epidemiological trends of resistance of C. tropicalis.IMPORTANCEC. tropicalis infections pose a growing global public health challenge, with mortality rates approaching 40%. C. tropicalis is one of the top four Candida spp. responsible for candidiasis, particularly in the Asia-Pacific region and Latin America, notably affecting patients with neutropenia and malignancies. The azole resistance rate of C. tropicalis ranges from 0% to 30%. Between 2009 and 2018, the China Hospital Invasive Fungal Surveillance Network reported an increase in fluconazole and voriconazole resistance from 5.7% to ~30%. Although resistance to echinocandins and amphotericin B remains low, multi-resistance to echinocandins and azoles has been observed. Current methods for detecting drug resistance are limited by the long turnaround time of antifungal susceptibility testing, low throughput of Sanger sequence to target resistance mutations, complex data analysis, and high costs of second-generation sequencing. We developed and validated a rapid, high-throughput, and cost-effective panel to detect and monitor drug-resistance mutations of C. tropicalis.

Targeted dual-receptor phage cocktail against Cronobacter sakazakii: insights into phage-host interactions and resistance mechanisms.

Cronobacter sakazakii is a notorious foodborne pathogen, frequently contaminating powdered infant formula and causing life-threatening diseases in infants. The escalating emergence of antibiotics-resistant mutants has led to increased interest in using bacteriophage as an alternative antimicrobial agent. Two phages, CR8 and S13, were isolated from feces and soil samples and their morphology, physiology, and genomics were characterized. Phage receptor was determined using deletion mutants lacking flgK, rfaC, fhuA, btuB, lamb, or ompC genes, followed by complementation. Phage-resistant mutants were analyzed for phenotypic changes and fitness trade-offs using motility assays and Caco-2 cell invasion models. CR8 and S13 were identified as members of Caudoviricetes. Phage CR8 and phage S13 utilize flagella and LPS, respectively, to adhere to host cells. Bacterial challenge assay demonstrated delayed emergence of the resistant mutant as well as stronger lytic activity of a phage cocktail consisting of CR8 and S13 than the single phage treatment. Phenotypic analysis of the phage cocktail resistant strain, designated as CSR strain, revealed that the resistance resulted from the impaired receptor proteins for phage, such as defects in motility and alteration in LPS structure. CSR strain exhibited significant attenuation in invading human intestinal epithelial Caco-2 cells compared to WT cells. This study demonstrates that the development of the phage cocktail targeting distinct host receptors can serve as a promising antimicrobial strategy to effectively control C. sakazakii.

The association between antimicrobial resistance mutations and treatment outcomes for Mycoplasma genitalium infections from 2018 to 2022: a cross-sectional study from Auckland, New Zealand.

Background New Zealand has among the highest rates of antimicrobial resistance in Mycoplasma genitalium in the world. The aim of this study was to correlate treatment outcomes with 23S rRNA and parC mutations associated with macrolide and fluroquinolone resistance, respectively, in a cohort of sexual health clinic patients. Methods Laboratory and clinical data were collected for patients with M. genitalium infections attending Auckland Sexual Health Service between 1 January 2018 and 31 December 2022, who had a test-of-cure performed within 21-90days of a treatment episode. Treatment outcomes were correlated with the presence or absence of resistance mutations and treatment regimen utilised. Results A total of 95 infections from 93 patients met the study inclusion criteria. Eighty of 93 (86%) infections with available data were macrolide resistant, with 20 of 74 (27%) having both macrolide resistance and parC mutations. Sixteen of 20 (80%) of parC mutations were G248T (S83I), three of 20 (15%) G259T (D87Y) and one of 20 (5%) A247C (S83R). All macrolide-susceptible infections treated with doxycycline and azithromycin were cured (12/12), as were all macrolide-resistant infections without parC mutations treated with doxycycline and moxifloxacin (37/37). Cure rates for macrolide-resistant infections with parC mutations were lower, with variable and often multiple treatment courses; eight of 16 (50%) were cured using one course of sequential doxycycline and moxifloxacin, seven of nine (78%) with one course of minocycline, zero of two (0%) with pristinamycin and one of one (100%) with doxycycline and sitafloxacin. Conclusions Our findings highlight the differing treatment outcomes for infections with and without parC mutations, offering opportunities to refine management of M. genitalium infections.

Mutant prevention concentrations, in vitro resistance evolution dynamics, and mechanisms of resistance to imipenem and imipenem/relebactam in carbapenem-susceptible Klebsiella pneumoniae isolates showing ceftazidime/avibactam resistance.

Klebsiella pneumoniae carbapenemase (KPC) variants selected during ceftazidime/avibactam treatment usually develop susceptibility to carbapenems and carbapenem/β-lactamase inhibitors, such as imipenem and imipenem/relebactam. We analyzed imipenem and imipenem/relebactam single-step mutant frequencies, resistance development trajectories and differentially selected resistance mechanisms using two representative K. pneumoniae isolates that had developed ceftazidime/avibactam resistance during therapy (ST512/KPC-31 and ST258/KPC-35). Mutant frequencies and mutant prevention concentrations were measured in Mueller-Hinton agar plates containing incremental concentrations of imipenem or imipenem/relebactam. Resistance dynamics were determined after incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic or combination, up to 64 times their baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing and whole genome sequencing. The impact of KPC variants identified in resistant mutants on β-lactam resistance was investigated by cloning experiments. Imipenem/relebactam suppressed the emergence of resistant mutants at lower concentrations than imipenem, slowed down resistance development for both strains, and the resulting mutants yielded lower MICs of carbapenems and carbapenem/β-lactamase inhibitors than those selected with imipenem alone. Characterization of resistant mutants revealed that imipenem resistance was mainly caused by inactivation of OmpK36 and mutations in the KPC β-lactamase. Imipenem/relebactam-resistant mutants also maintained OmpK36 alterations, but mutations in KPC were much less frequent compared with those selected with imipenem alone. Genetic and biochemical characterization of the KPC derivatives identified in the resistant mutants confirmed their role in carbapenem resistance. Our data positions imipenem/relebactam as an attractive therapeutic option for combating ceftazidime/avibactam-resistant KPC-producing K. pneumoniae infections.

Mapping kinase domain resistance mechanisms for the MET receptor tyrosine kinase via deep mutational scanning.

Mutations in the kinase and juxtamembrane domains of the MET Receptor Tyrosine Kinase are responsible for oncogenesis in various cancers and can drive resistance to MET-directed treatments. Determining the most effective inhibitor for each mutational profile is a major challenge for MET-driven cancer treatment in precision medicine. Here, we used a deep mutational scan (DMS) of ∼5,764 MET kinase domain variants to profile the growth of each mutation against a panel of 11 inhibitors that are reported to target the MET kinase domain. We validate previously identified resistance mutations, pinpoint common resistance sites across type I, type II, and type I ½ inhibitors, unveil unique resistance and sensitizing mutations for each inhibitor, and verify non-cross-resistant sensitivities for type I and type II inhibitor pairs. We augment a protein language model with biophysical and chemical features to improve the predictive performance for inhibitor-treated datasets. Together, our study demonstrates a pooled experimental pipeline for identifying resistance mutations, provides a reference dictionary for mutations that are sensitized to specific therapies, and offers insights for future drug development.

Mutations on the A and C genomes of Brassica napus confer herbicide resistance to acetohydroxyacid synthase inhibitors

ABSTRACT The point mutations conferring resistance to the sulfonylurea herbicide, chlorsulfuron, are reported for two Brassica napus lines (19c and 30a) developed by seed mutagenesis using ethyl methanesulfonate. Crosses with the herbicide-susceptible wild-type line generated F2 and backcrossed progeny and confirmed that chlorsulfuron resistance was inherited as a dominant mutation at a single locus in both lines. F2 progeny from crossing 19c and 30a segregated in a 15 resistant: 1 susceptible ratio, establishing that the two herbicide-resistant loci are independently inherited and that the mutations for resistance occur at different loci. PCR amplification and DNA sequencing of regions from the acetohydroxyacid synthase gene revealed two separate point mutations at different nucleotides in the same codon. This results in the substitution of proline-197 for leucine in 19c and proline-197 for serine in 30a. The different amino acid substitutions in the acetohydroxyacid synthase enzyme explain the differing responses of 19c and 30a to sulfonylurea and imidazolinone herbicides. Based on attempts at introgression of chlorsulfuron resistance to a wide range of forage brassicas, we conclude that the 19c mutation resides in the Brassica C genome, whereas the 30a mutation is in the Brassica A genome.

Assessing the potential risk of human pathogen resistance to medical antifungal treatments arising from agricultural use of fungicides with the same mode of action

AbstractA mechanistic basis is described for assessment of resistance risk to medical antifungal treatments from agricultural use of fungicides of the same mode of action. The following need to occur in landscape environments for a risk to be posed by dual use: (i) emergence, whereby a resistant strain emerges by mutation and invasion; (ii) selection, whereby a mutation conferring a fitness advantage is selected for in the presence of fungicide; and (iii) exposure of humans to resistant strains from the landscape, potentially resulting in invasive fungal infection. We identify 20 human pathogens for which there is evidence that all three processes above could, in principle, occur. A model is derived to explore what determines resistance emergence and selection in human pathogens in landscape environments. Knowledge gaps are identified in key parameters. The analysis suggests that emergence and selection were particularly affected by fitness cost associated with the resistance mutation(s) and fungicide concentration. Emergence was also determined by the amount of pathogen reproduction (related to pathogen population size). If fungicide resistance is associated with even a small fitness cost, then environments with low fungicide concentrations, such as field soils, may not be conducive to resistance emergence or selection. These general findings were related to a specific case of observational data from the Netherlands for Aspergillus fumigatus. The analysis supports previous work that compost is towards the high‐risk end of the spectrum for this species. Agricultural soils, nonagricultural land and grassland were lower risk.

Tilting the Scales toward EGFR Mutant Selectivity: Expanding the Scope of Bivalent "Type V" Kinase Inhibitors.

Binding multiple sites within proteins with bivalent compounds is a strategy for developing uniquely active agents. A new class of dual-site inhibitors has emerged targeting the epidermal growth factor receptor (EGFR) anchored to both the orthosteric (ATP) and allosteric sites. Despite proof-of-concept successes, enabling selectivity against oncogenic activating mutations has not been achieved and classifying these inhibitors among kinase inhibitors remains underexplored. This study investigates the structure-activity relationships, binding modes, and biological activity of ATP-allosteric bivalent inhibitors (AABIs). We find that AABIs selectively inhibit drug-resistant EGFR mutants (L858R/T790M and L858R/T790M/C797S) by anchoring a methyl isoindolinone moiety along the αC-helix channel of the allosteric site. In contrast, related Type I1/2 inhibitors target wild-type EGFR but are less effective against resistant mutants. This shift in selectivity demonstrates that mutant-selective AABIs classify as "Type V" bivalent inhibitors.

Development and application of species ID and insecticide resistance assays, for monitoring sand fly Leishmania vectors in the Mediterranean basin and in the Middle East.

Development of insecticide resistance (IR) in sand fly populations is an issue of public health concern, threatening leishmaniasis mitigation efforts by insecticide-based vector control. There is a major knowledge gap in the IR status of wild populations worldwide, possibly attributed to the unavailability of specialized tools, such as bioassay protocols, species baseline susceptibility to insecticides and molecular markers, to monitor such phenomena in sand flies. Sand fly populations from (semi-)rural regions of Greece, Turkey and Iran were sampled and identified to species, showing populations' structure in accordance with previously reported data. Genotyping of known pyrethroid resistance-associated loci revealed the occurrence of voltage-gated sodium channel (vgsc) mutations in all surveyed countries. Knock-down resistance (kdr) mutation L1014F was prevalent in Turkish regions and L1014F and L1014S were recorded for the first time in Iran, and in Turkey and Greece, respectively, yet in low frequencies. Moreover, CDC bottle bioassays against pyrethroids in mixed species populations from Greece indicated full susceptibility, using though the mosquito discriminating doses. In parallel, we established a novel individual bioassay protocol and applied it comparatively among distinct Phlebotomus species' populations, to detect any possible divergent species-specific response to insecticides. Indeed, a significantly different knock-down rate between P. simici and P. perfiliewi was observed upon exposure to deltamethrin. IR in sand flies is increasingly reported in leishmaniasis endemic regions, highlighting the necessity to generate additional monitoring tools, that could be implemented in relevant eco-epidemiological settings, in the context of IR management. Our molecular and phenotypic data add to the IR map in an area with otherwise limited data coverage.

Anti-EGFR Rechallenge in Metastatic Colorectal Cancer and the Role of ctDNA: A Systematic Review and Meta-analysis.

Metastatic colorectal cancer (mCRC) remains a significant clinical challenge. While anti-EGFR inhibitors have improved survival rates, their long-term efficacy is limited by disease progression, which is often associated with the development of acquired resistance mutations. However, some patients may regain sensitivity to anti-EGFR agents after alternative therapies, suggesting a potential benefit for rechallenge strategies. Our study aims to conduct a systematic review and meta-analysis to comprehensively evaluate the efficacy and safety of EGFR rechallenge in patients with mCRC. A systematic search of the MEDLINE, EMBASE, and Cochrane databases was conducted between October 28 and December 24, 2023, to identify clinical trials investigating treatment regimens incorporating panitumumab or cetuximab as a rechallenge strategy. Pooled proportions or hazard ratios (HR) were calculated using a random effects model. Inter-study heterogeneity was assessed using the I2. Among the 2105 articles identified through the search, 13 met the predetermined inclusion criteria. Of these, 12 were phase II studies, encompassing 92.3% of the patient population. Cetuximab was administered to 302 patients (75.1%), whereas panitumumab was utilized in 100 patients (24.9%).A pooled analysis of eight studies demonstrated an objective response rate of 20.50% (95% CI 7.94 to 33.07) and a disease control rate of 67.35% (95% CI 58.60 to 76.09). The median progression-free survival was estimated at 3.5months (95% CI 2.68-6.69), with a median OS of 9.8months (95% CI 6.71-12.89). Patients exhibiting RAS wild-type status in circulating tumor DNA (ctDNA) analysis derived enhanced benefits from anti-EGFR rechallenge (HR: 0.41; 95% CI 0.28-0.60, I2 = 60%). Common grade 3 or higher treatment-related adverse events included neutropenia (22.8%) and rash (14.9%). This meta-analysis underscores the efficacy and safety of anti-EGFR rechallenge as a promising therapeutic approach for a subset of patients afflicted with mCRC. The observed correlation between wild-type RAS status, as determined through ctDNA analysis, and improved OS signals the prospect of precision oncology in guiding treatment decisions.

Using Vesicular Stomatitis Virus as a Platform for Directed Protease Evolution.

Antiviral drugs are essential medications to save the lives of infected people. However, they are under constant threat to become ineffective as viruses evolve quickly. Studying the development of resistance is therefore paramount to understand the impact of mutations on pharmacological treatment and to make informed decisions. Yet, such studies are open to scrutiny, as they are considered gain-of-function research, which is especially problematic with viruses of pandemic potential. In this article, we present a protocol that allows for the selection of antiviral resistance mutations safely, without using the actual virus (e.g., SARS-CoV-2, MERS-CoV). Instead, we use vesicular stomatitis virus (VSV) that serves as a surrogate virus; like other RNA viruses, it is prone to mutations due to its polymerase lacking proofreading. By replacing parts of the VSV genome with transgenes from other viruses, VSV becomes dependent on their function. Thus, we can mount a selection pressure with antivirals targeting the transgenes to subsequently sequence selected resistance mutations. This article provides a protocol for this process as well as a sequencing pipeline that we used to collect mutations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using VSV as a platform for directed protease evolution Alternate Protocol: Dose response assay with TCID50 readout Support Protocol 1: A pipeline for high-throughput VSV sequencing Support Protocol 2: Rescue of VSV.