Mechanisms Of Resistance Research Articles

Multifunctional Nano‐Conductive Hydrogels With High Mechanical Strength, Toughness and Fatigue Resistance as Self‐Powered Wearable Sensors and Deep Learning‐Assisted Recognition System

AbstractHigh mechanical strength, toughness, and fatigue resistance are essential to improve the reliability of conductive hydrogels for self‐powered sensing. However, achieving mutually exclusive properties simultaneously remains challenging. Hence, a novel directed interlocking strategy based on topological network structure and mechanical training is proposed to construct tough hydrogels by optimizing the network structure and modulating the orientation of molecular chains. Combining Zn2+ crosslinked cellulose nanofibers (CNFs) and a polyacrylamide‐poly(vinyl alcohol) double‐network, the unique interlocked‐network structure exhibits an enhanced toughening effect due to hydrogen bonding and metal‐ligand interactions. The aligned nanocrystalline domains achieved by training further contribute to an increase in the toughness and fatigue thresholds. This innovative approach synergistically enhances the mechanical properties of the nano‐conductive hydrogel, achieving a maximum tensile strength of 4.98 MPa and a toughness of 48 MJ m−3. Notably, the CNFs template with anchored polyaniline, when oriented through mechanical training, forms a unique directional conductive pathway, which significantly enhances the power output performance. Besides, a motion recognition system based on a self‐powered sensing device is designed with the assistance of deep learning techniques to accurately identify human motion behaviors. This work showcases a potentially transformative flexible electronic material for self‐powered sensing systems and intelligent recognition systems.

Autophagy unrelated transcriptional mechanisms of hydroxychloroquine resistance revealed by integrated multi-omics of evolved cancer cells

ABSTRACT Hydroxychloroquine (HCQ) and chloroquine are repurposed drugs known to disrupt autophagy, a molecular recycling pathway essential for tumor cell survival, chemotherapeutic resistance, and stemness. We pursued a multi-omic strategy in OVCAR3 ovarian cancer and CCL218 colorectal cancer cells. Two genome-scale screens were performed. In the forward genetic screen, cell populations were passaged for 15 drug pulse-chases with HCQ or vehicle control. Evolved cells were collected and processed for bulk RNA-seq, exome-seq, and single-cell RNA-seq (scRNA-seq). In the reverse genetic screen, a pooled CRISPR-Cas9 library was used in cells over three pulse-chases of HCQ or vehicle control treatments. HCQ evolved cells displayed remarkably few mutational differences, but substantial transcriptional differences. Transcriptomes revealed multiple pathways associated with resistance to HCQ, including upregulation of glycolysis, exocytosis, and chromosome condensation/segregation, or downregulation of translation and apoptosis. The Cas9 screen identified only one autophagy gene. Chromosome condensation and segregation were confirmed to be disrupted by HCQ in live cells and organelle-free in vitro extracts. Transcriptional plasticity was the primary mechanism by which cells evolved resistance to HCQ. Neither autophagy nor the lysosome were substantive hits. Our analysis may serve as a model for how to better position repurposed drugs in oncology.

Antimalarial Mechanisms and Resistance Status of Artemisinin and Its Derivatives

Artemisinin is an endoperoxide sesquiterpene lactone isolated from Artemisia annua and is often used to treat malaria. Artemisinin’s peroxide bridge is the key structure behind its antimalarial action. Scientists have created dihydroartemisinin, artemether, artesunate, and other derivatives preserving artemisinin’s peroxide bridge to increase its clinical utility value. Artemisinin compounds exhibit excellent efficacy, quick action, and minimal toxicity in malaria treatment and have greatly contributed to malaria control. With the wide and unreasonable application of artemisinin-based medicines, malaria parasites have developed artemisinin resistance, making malaria prevention and control increasingly challenging. Artemisinin-resistant Plasmodium strains have been found in many countries and regions. The mechanisms of antimalarials and artemisinin resistance are not well understood, making malaria prevention and control a serious challenge. Understanding the antimalarial and resistance mechanisms of artemisinin drugs helps develop novel antimalarials and guides the rational application of antimalarials to avoid the spread of resistance, which is conducive to malaria control and elimination efforts. This review will discuss the antimalarial mechanisms and resistance status of artemisinin and its derivatives, which will provide a reference for avoiding drug resistance and the research and development of new antimalarial drugs.

Single‐Cell RNA‐Sequencing of Soybean Reveals Transcriptional Changes and Antiviral Functions of GmGSTU23 and GmGSTU24 in Response to Soybean Mosaic Virus

ABSTRACTSoybean mosaic virus (SMV) stands as a prominent and widespread threat to soybean (Glycine max L. Merr.), the foremost legume crop globally. Attaining a thorough comprehension of the alterations in the transcriptional network of soybeans in response to SMV infection is imperative for a profound insight into the mechanisms of viral pathogenicity and host resistance. In this investigation, we isolated 50 294 protoplasts from the newly developed leaves of soybean plants subjected to both SMV infection and mock inoculation. Subsequently, we utilized single‐cell RNA sequencing (scRNA‐seq) to construct the transcriptional landscape at a single‐cell resolution. Nineteen distinct cell clusters were identified based on the transcriptomic profiles of scRNA‐seq. The annotation of three cell types—epidermal cells, mesophyll cells, and vascular cells—was established based on the expression of orthologs to reported marker genes in Arabidopsis thaliana. The differentially expressed genes between the SMV‐ and mock‐inoculated samples were analyzed for different cell types. Our investigation delved deeper into the tau class of glutathione S‐transferases (GSTUs), known for their significant contributions to plant responses against abiotic and biotic stress. A total of 57 GSTU genes were identified by a thorough genome‐wide investigation in the soybean genome G. max Wm82.a4.v1. Two specific candidates, GmGSTU23 and GmGSTU24, exhibited distinct upregulation in all three cell types in response to SMV infection, prompting their selection for further research. The transient overexpression of GmGSTU23 or GmGSTU24 in Nicotiana benthamiana resulted in the inhibition of SMV infection, indicating the antiviral function of soybean GSTU proteins.

Rice Varieties Intercropping Induced Soil Metabolic and Microbial Recruiting to Enhance the Rice Blast (Magnaporthe Oryzae) Resistance

[Background] Intercropping is considered an effective approach to defending rice disease. [Objectives/Methods] This study aimed to explore the resistance mechanism of rice intraspecific intercropping by investigating soil metabolites and their regulation on the rhizosphere soil microbial community using metabolomic and microbiome analyses. [Results] The results showed that the panicle blast disease occurrence of the resistant variety Shanyou63 (SY63) and the susceptible variety Huangkenuo (HKN) were both decreased in the intercropping compared to monoculture. Notably, HKN in the intercropping system exhibited significantly decreased disease incidence and increased disease resistance-related enzyme protease activity. KEGG annotation from soil metabolomics analysis revealed that phenylalanine metabolic pathway, phenylalanine, tyrosine, and tryptophan biosynthesis pathway, and fructose and mannose metabolic pathway were the key pathways related to rice disease resistance. Soil microbiome analysis indicated that the bacterial genera Nocardioides, Marmoricola, Luedemannella, and Desulfomonile were significantly enriched in HKN after intercropping, while SY63 experienced a substantial accumulation of Ruminiclostridium and Cellulomonas. Omics-based correlation analysis highlighted that the community assembly of Cellulomonas and Desulfomonile significantly affected the content of the metabolites D-sorbitol, D-mannitol, quinic acid, which further proved that quinic acid had a significantly inhibitory effect on the mycelium growth of Magnaporthe oryzae, and these three metabolites had a significant blast control effect. The optimal rice blast-control efficiency on HKN was 51.72%, and Lijiangxintuanheigu (LTH) was 64.57%. [Conclusions] These findings provide a theoretical basis for rice varieties intercropping and sustainable rice production, emphasizing the novelty of the study in elucidating the underlying mechanisms of intercropping-mediated disease resistance.

Cancer associated variant enrichment CAVE, a gene agnostic approach to identify low burden variants in chronic lymphocytic leukemia

Intratumoral heterogeneity is an important clinical challenge because low burden clones expressing specific genetic alterations drive therapeutic resistance mechanisms. We have developed CAVE (cancer-associated variant enrichment), a gene-agnostic computational tool to identify specific enrichment of low-burden cancer driver variants in next-generation sequencing (NGS) data. For this study, CAVE was applied to TP53 in chronic lymphocytic leukemia (CLL) as a cancer model. Indeed, as TP53 mutations are part of treatment decision-making algorithms and low-burden variants are frequent, there is a need to distinguish true variants from background noise. Recommendations have been published for reliable calling of low-VAF variants of TP53 in CLL and the assessment of the background noise for each platform is essential for the quality of the testing. CAVE is able to detect specific enrichment of low-burden variants starting at variant allele frequencies (VAFs) as low as 0.3%. In silico TP53 dependent and independent analyses confirmed the true driver nature of all these variants. Orthogonal validation using either ddPCR or NGS analyses of follow-up samples confirmed variant identification. CAVE can be easily deployed in any cancer-related NGS workflow to detect the enrichment of low-burden variants of clinical interest.

Generalized pustular psoriasis patient with a heterozygous hypomorphic MPO variant refractory to intravenous spesolimab

AbstractGeneralized pustular psoriasis (GPP) is a recurrent and sometimes life‐threatening sterile pustular disease. Because interleukin (IL)‐36 is the central cytokine in disease formation, spesolimab, which interferes with IL‐36 receptor signaling, is highly effective. Here, we report a patient with GPP with a heterozygous hypomorphic MPO variant refractory to intravenous spesolimab. Although spesolimab showed excellent clinical efficacy in resolving pre‐existing pustules and erythema, it did not suppress the emergence of new pustules and erythema, which did not decrease the peripheral blood neutrophil count, therefore bimekizumab, an anti‐IL‐17A/IL‐17F antibody, was administered after the second spesolimab infusion, which resolved the pustules and erythema. We discuss the possible reasons for the resistance mechanism to spesolimab.

Carbapenem-Resistant Enterobacterales in the Western Balkans: Addressing Gaps in European AMR Surveillance Map

In the context of globa1l efforts to combat antimicrobial resistance (AMR), the importance of comprehensive AMR data is more crucial than ever. AMR surveillance networks, such as the European Antimicrobial Resistance Surveillance Network (EARS-Net) and the Central Asian and European Surveillance of Antimicrobial Resistance (CAESAR), support member states in obtaining high-quality AMR data. Nevertheless, data gaps persist in some countries, including those in the Western Balkans (WBs), a region with high AMR rates. This review analyzed existing research on carbapenem-resistant Enterobacterales (CRE) to better understand the AMR landscape in the WB countries. The most prevalent CRE was Klebsiella pneumoniae, followed by Escherichia coli, Enterobacter cloacae, and Proteus mirabilis, with sporadic cases of Morganella morganii, Providencia spp., Klebsiella oxytoca, and Citrobacter sedlakii. Carbapenemase production was identified as the most common mechanism of carbapenem resistance, but other resistance mechanisms were not investigated. An increasing trend in carbapenem resistance has been observed over the last decade, alongside a shift in carbapenemase epidemiology from the NDM type in 2013–2014 to the OXA-48 type in recent years. Few studies have applied whole-genome sequencing for CRE analysis, which has demonstrated the spread of resistance determinants across different niches and over time, emphasizing the importance of molecular-based research. The overall low number of studies in the WB countries can be attributed to limited resources, highlighting the need for enhanced support in education, training, technology, and equipment to improve data collection and evaluation.

Integrative transcriptome and metabolome analysis reveals the mechanism of fulvic acid alleviating drought stress in oat

Drought stress inhibits oat growth and yield. The application of fulvic acid (FA) can improve the drought resistance of oats, but the corresponding molecular mechanism of FA-mediated drought resistance remains unclear. Here, we studied the effects of FA on the drought tolerance of oat leaves through physiological, transcriptomic, and metabolomics analyses, and identified FA-induced genes and metabolites related to drought tolerance. Physiological analysis showed that under drought stress, FA increased the relative water and chlorophyll contents of oat leaves, enhanced the activity of antioxidant enzymes (SOD, POD, PAL, CAT and 4CL), inhibited the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and dehydroascorbic acid (DHA), reduced the degree of oxidative damage in oat leaves, improved the drought resistance of oats, and promoted the growth of oat plants. Transcriptome and metabolite analyses revealed 652 differentially expressed genes (DEGs) and 571 differentially expressed metabolites (DEMs) in FA-treated oat leaves under drought stress. These DEGs and DEMs are involved in a variety of biological processes, such as phenylspropanoid biosynthesis and glutathione metabolism pathways. Additionally, FA may be involved in regulating the role of DEGs and DEMs in phenylpropanoid biosynthesis and glutathione metabolism under drought stress. In conclusion, our results suggest that FA promotes oat growth under drought stress by attenuating membrane lipid peroxidation and regulating the antioxidant system, phenylpropanoid biosynthesis, and glutathione metabolism pathways in oat leaves. This study provides new insights into the complex mechanisms by which FA improves drought tolerance in crops.

A virulence-associated small RNA MTS1338 activates an ABC transporter CydC for rifampicin efflux in Mycobacterium tuberculosis

The efficacy of the tuberculosis treatment is restricted by innate drug resistance of Mycobacterial tuberculosis and its ability to acquire resistance to all anti-tuberculosis drugs in clinical use. A profound understanding of bacterial ploys that decrease the effectiveness of drugs would identify new mechanisms for drug resistance, which would subsequently lead to the development of more potent TB therapies. In the current study, we identified a virulence-associated small RNA (sRNA) MTS1338-driven drug efflux mechanism in M. tuberculosis. The treatment of a frontline antitubercular drug rifampicin upregulated MTS1338 by >4-fold. Higher intrabacterial abundance of MTS1338 increased the growth rate of cells in rifampicin-treated conditions. This fact was attributed by the upregulation of an efflux protein CydC by MTS1338. Gel-shift assay identified a stable interaction of MTS1338 with the coding region of cydC mRNA thereby potentially stabilizing it at the posttranscriptional level. The drug efflux measurement assays revealed that cells with higher MTS1338 abundance accumulate less drug in the cells. This study identified a new regulatory mechanism of drug efflux controlled by an infection-induced sRNA in M. tuberculosis.

The p-MYH9/USP22/HIF-1α axis promotes lenvatinib resistance and cancer stemness in hepatocellular carcinoma

Lenvatinib is a targeted drug used for first-line treatment of hepatocellular carcinoma (HCC). A deeper insight into the resistance mechanism of HCC against lenvatinib is urgently needed. In this study, we aimed to dissect the underlying mechanism of lenvatinib resistance (LR) and provide effective treatment strategies. We established an HCC model of acquired LR. Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were used to detect the stemness of HCC cells. Molecular and biochemical strategies such as RNA-sequencing, immunoprecipitation, mass spectrometry and ubiquitination assays were used to explore the underlying mechanisms. Patient-derived HCC models and HCC samples from patients were used to demonstrate clinical significance. We identified that increased cancer stemness driven by the hypoxia-inducible factor-1α (HIF-1α) pathway activation is responsible for acquired LR in HCC. Phosphorylated non-muscle myosin heavy chain 9 (MYH9) at Ser1943, p-MYH9 (Ser1943), could recruit ubiquitin-specific protease 22 (USP22) to deubiquitinate and stabilize HIF-1α in lenvatinib-resistant HCC. Clinically, p-MYH9 (Ser1943) expression was upregulated in HCC samples, which predicted poor prognosis and LR. A casein kinase-2 (CK2) inhibitor and a USP22 inhibitor effectively reversed LR in vivo and in vitro. Therefore, the p-MYH9 (Ser1943)/USP22/HIF-1α axis is critical for LR and cancer stemness. For the diagnosis and treatment of LR in HCC, p-MYH9 (Ser1943), USP22, and HIF-1α might be valuable as novel biomarkers and targets.

Treatment resistance to melanoma therapeutics on a single cell level

Therapy targeting the BRAF-MEK cascade created a treatment revolution for patients with BRAF mutant advanced melanoma. Unfortunately, 80% patients treated will progress by 5 years follow-up. Thus, it is imperative we study mechanisms of melanoma progression and therapeutic resistance. We created a scRNA (single cell RNA) atlas of 128,230 cells from 18 tumors across the treatment spectrum, discovering melanoma cells clustered strongly by transcriptome profiles of patients of origins. Our cell-level investigation revealed gains of 1q and 7q as likely early clonal events in metastatic melanomas. By comparing patient tumors and their derivative cell lines, we observed that PD1 responsive tumor fraction disappears when cells are propagated in vitro. We further established three anti-BRAF-MEK treatment resistant cell lines using three BRAF mutant tumors. ALDOA and PGK1 were found to be highly expressed in treatment resistant cell populations and metformin was effective in targeting the resistant cells. Our study suggests that the investigation of patient tumors and their derivative lines is essential for understanding disease progression, treatment response and resistance.

Damage monitoring of glass/epoxy-curved laminates with different stacking sequences using acoustic emission

Delamination is a major concern in the curvature area of a composite laminate due to the flexural stresses occurring between the laminates. This research focuses on the respective merits of different stacking sequences obtained by layering the glass fiber reinforcement at various orientations in an epoxy resin to manufacture a curved laminate. In particular, laminates with stacking sequences, such as Unidirectional [0°]24, Cross-ply [0°/90°]6S, Angle ply [±45°]6S, and Quasi-isotropic laminates [0°/±45°/90°]3S, were tested by performing four-point bending tests. Acoustic emission (AE) monitoring and experimental analysis characterized damage modes, including delamination. AE parameters, such as amplitude, energy, duration, cumulative count, and peak frequency, were utilized to characterize the damage mechanisms of the various stacking sequences. Experimental AE data confirmed that fiber orientation influences delamination resistance and failure mechanisms. Furthermore, compared to other layup sequences, the peak load of cross-ply (Layup B) was found to be greater by 48.7%, 32.87%, and 50.14%. Cross-ply curved laminates also have higher interlaminar tensile strength and curved beam strength than other sequences. They also failed less often due to delamination than other sequences. Finally, scanning electron microscope characterization verified and validated all damage processes found in the AE data collection. As a result, these findings play a significant role in predicting the lifetime, delamination failure, and strength of curved composite laminates.

Automated Workflow for Phase‐Field Simulations: Unveiling the Impact of Heat‐Treatment Parameters on Bainitic Microstructure in Steel

Bainitic steels are extensively utilized across various sectors, such as the automotive and railway industries, owing to their impressive mechanical properties, including strength, hardness, and fatigue resistance. However, the pursuit of achieving the desired optimal mechanical properties presents considerable challenges due to the intricate bainitic microstructures consisting of multiple phases. To tackle these challenges, an automated workflow is used for extracting 2D and 3D microstructural features. The proposed method allows for a detailed examination of the correlations between microstructure characteristics and the processing parameters, specifically the holding temperature during transformation. In these findings, it is revealed that as the holding temperature decreases, there is a notable reduction in microstructural element size and carbon partitioning. Some of the observations are microstructural features such as area, perimeter, and thickness of the bainitic ferrite grains under two different holding temperatures. Phase‐field simulations results show that the microstructures at lower holding temperatures have finer grains. The distributions of grain areas and perimeters are uniform, with smaller grains dominating at low and high isothermal holding temperatures. While the grain thickness measurements from simulations and experiments at high temperature are qualitatively aligned, data from low temperatures show discrepancies.

Experimental Study on the Frost Resistance of Basalt Fiber Reinforced Concrete

In this paper, the effect of basalt fiber (BF) on the frost resistance of concrete under different curing conditions was investigated, and its frost resistance mechanism was analyzed. Three different curing conditions (normal curing, short-term curing, and seawater curing) were adopted, and concrete with different BF volume contents was designed. Freeze-thaw (FT) tests were carried out using the rapid freezing method to test the frost resistance of basalt fiber reinforced concrete (BFRC). Additionally, the mass loss rate (MLR), relative dynamic modulus of elasticity (RDME) change, and compressive strength reduction of specimens during the freeze-thaw cycles (FTCs) were evaluated. The results show that when the BF content is 0.15%, under normal curing, short-term curing, and seawater curing conditions, the residual compressive strength of BFRC after FTCs was increased by 5.4%, 28.1%, and 30.9%, respectively, compared to plain concrete. By incorporating BF into concrete, the development of microcracks can be effectively retarded, and damage generation during FTCs can be reduced. In addition, the microscopic morphological characteristics and pore structure characteristics of concrete further elucidate the frost resistance mechanism of BFRC from a microscopic perspective.

Profiling the lncRNA–miRNA–mRNA interaction network in the cold-resistant exercise period of grape (Vitis amurensis Rupr.)

BackgroundGrape is a plant that is sensitive to low temperature and vulnerable to low-temperature damage. However, little is known about the roles of lncRNAs, miRNAs and mRNAs in regulating the hypothermia response mechanism in Vitis amurensis Rupr.MethodsIn this study, the expression and regulatory network of low-temperature response genes were studied in the phloem of grape under different low-temperature stress.ResultsHere, we performed analyses related to RNA-seq and miRNA-seq on grape phloem tissues from five periods of cold resistance campaigns. Three RNAs (lncRNAs, miRNAs and mRNAs) obtained by KEGG and GO analyses were used to identify starch and sucrose metabolism associated with cold resistance, and specific changes in BP, CC, and MF were identified in four comparisons. Venn diagrams, thermograms and pathway maps were used to analyze the differentially expressed genes (DEGs), and their specific gene expression during the cold exercise were obtained. The six DEGs finally selected were used for qRT-PCR to verify the RNA-seq data. In addition, we found that the regulatory networks of miRNAs and lncRNAs correspond to the six DEGs. This study will contribute to further experimental studies to elucidate the cold resistance mechanism of Vitis amurensis Rupr.ConclusionsThe low-temperature response genes of grape are mainly enriched in the starch and sucrose metabolism, and they are regulated by miRNAs and lncRNAs. The conclusions will provide basic information for further understanding of the cold resistance mechanism of grape in the future.Graphical

Inhibition of hTERT/telomerase/telomere mediates therapeutic efficacy of osimertinib in EGFR mutant lung cancer

The inevitable acquired resistance to osimertinib (AZD9291), an FDA-approved third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating or T790M resistant mutations, limits its long-term clinical benefit. Telomere maintenance via telomerase reactivation is linked to uncontrolled cell growth and is a cancer hallmark and an attractive cancer therapeutic target. Our effort toward understanding the action mechanisms, including resistance mechanisms, of osimertinib has led to the identification of a novel and critical role in maintaining c-Myc–dependent downregulation of hTERT, a catalytic subunit of telomerase, and subsequent inhibition of telomerase/telomere and induction of telomere dysfunction in mediating therapeutic efficacy of osimertinib. Consequently, osimertinib combined with the telomere inhibitor, 6-Thio-dG, which is currently tested in a phase II trial, effectively inhibited the growth of osimertinib-resistant tumors, regressed EGFRm NSCLC patient-derived xenografts, and delayed the emergence of acquired resistance to osimertinib, warranting clinical validation of this strategy to manage osimertinib acquired resistance.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

Investigation on the mechanisms of carbapenem resistance among the non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae

BackgroundIn Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates.MethodsAll four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses.ResultsMulti-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates.ConclusionsThis study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

Gut microbiota-derived butyrate selectively interferes with growth of carbapenem-resistant Escherichia coli based on their resistance mechanism

ABSTRACT We investigated consequences of resistance acquisition in Escherichia coli clinical isolates during anaerobic (continuous culture) growth and examined their sensitivity to butyrate, a hallmark metabolite of healthy gut microbiota. Strains were stratified based on carrying either a carbapenemase (CARB) or displaying porin malfunctioning (POR). POR displayed markedly altered growth efficiencies, lower membrane stability and increased sensitivity to butyrate compared with CARB. Major differences in global gene expression between the two groups during anaerobic growth were revealed involving increased expression of alternative substrate influx routes, the stringent response and iron acquisition together with lower expression of various stress response systems in POR. Longitudinal analyses during butyrate wash-in showed common responses for all strains as well as specific features of POR that displayed strong initial “overshoot” reactions affecting various stress responses that balanced out over time. Results were partly reproduced in a mutant strain verifying porin deficiencies as the major underlying mechanism for results observed in clinical isolates. Furthermore, direct competition experiments confirmed butyrate as key for amplifying fitness disadvantages based on porin malfunctioning. Results provide new (molecular) insights into ecological consequences of resistance acquisition and can assist in developing measures to prevent colonization and infection based on the underlying resistance mechanism.