Mutations In Target Genes Research Articles

A four-hit mechanism is sufficient for meningioma development.

Meningiomas are central nervous system tumors whose incidence increases with age. Benign meningioma pathogenesis involves germline or somatic mutation of target genes, such as NF2, leading to clonal expansion. We used an established cancer epidemiology model to investigate the number of rate-limiting steps sufficient for benign meningioma development. Incidence data was obtained from the Surveillance, Epidemiology and End Results Program (SEER) for nonmalignant meningioma from 2004 to 2020. Age-adjusted incidence rates per 100,000 person-years were divided into 5-year bands. This was repeated for vestibular schwannomas as a negative control. The Armitage-Doll methodology was applied. Mathematical solutions correcting for volatile tumor microenvironments were applied to fit higher-order models using polynomial regression when appropriate. A 75:25 training:test split was utilized for validation. 222,509 cases of benign meningiomas were identified. We noted strong linear relationships between log-transformed incidence and age across the cohort and multiple subpopulations: male, white, black, Hispanic, Asian/Pacific Islander, and American Indian subpopulations all demonstrated R2 = 0.99. Slopes were between 3.1 and 3.4, suggesting a four-step process for benign meningioma development. Female patients exhibited nonlinear deviations, but the corrected model demonstrated R2 = 0.99 with a four-hit pathway. This model performed robustly on test data with R2 = 0.99. Vestibular schwannomas demonstrated a slope of 2.1 with R2 = 0.99, suggesting a separate three-step process. Four mutations are uniquely required for the development of benign meningiomas. Correcting for volatile tumor microenvironments reliably accounted for nonlinear deviations in behavior. Further studies are warranted to elucidate genomic findings suggestive of key mutations in this pathway.

The landscape of targetable gene mutations in neuroendocrine cervical cancer

The landscape of targetable gene mutations in neuroendocrine cervical cancer

Potential Mechanisms of Hexaconazole Resistance in Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum is a serious fungal disease that can dramatically impact wheat production. At present, disease control is mainly achieved by the use of chemical fungicides. Hexaconazole (IUPAC name: 2(2,4-dichlorophenyl)-1-(1,2,4-triazol-1-yl)hexan-2-ol) is a widely used triazole fungicide, but the sensitivity of F. graminearum to this compound has yet to be established. The current study found that the EC50 values of 83 field isolates of F. graminearum ranged between 0.06 and 4.33 μg/ml, with an average EC50 value of 0.78 μg/ml. Assessment of four hexaconazole-resistant laboratory mutants of F. graminearum revealed that their mycelial growth and pathogenicity were reduced compared with their parental isolates and that asexual reproduction was reduced by resistance to hexaconazole. Meanwhile, the mutants appeared to be more sensitive to abiotic stress associated with SDS and H2O2, while their tolerance to high concentrations of Congo red, and Na+ and K+ increased. Molecular analysis revealed numerous point mutations in the FgCYP51 target genes that resulted in amino acid substitutions, including L92P and N123S in FgCYP51A, as well as M331V, F62L, Q252R, A412V, and V488A in FgCYP51B, and S28L, S256A, V307A, D287G, and R515I in FgCYP51C, three of which (S28L, S256A, and V307A) were conserved in all of the resistant mutants. Furthermore, the expression of the FgCYP51 genes in resistant strains was found to be significantly (P < 0.05) reduced compared with their sensitive parental isolates. Positive cross-resistance was found between hexaconazole and metconazole and flutriafol, as well as with the diarylamine fungicide fluazinam, but not with propiconazole, and the phenylpyrrole fungicide fludioxonil, or with tebuconazole, which actually exhibited negative cross-resistance. These results provide valuable insight into resistant mechanisms to triazole fungicides in F. graminearum, as well as the appropriate selection of fungicide combinations for the control of FHB to ensure optimal wheat production.

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Gene deletion by transconjugation and homologous recombination revealed that Rim101 and Rra1 are required for M. sympodialis growth at higher pH. In addition, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These pH-sensing signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together, these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.IMPORTANCEThe ability to adapt to host pH has been previously associated with microbial virulence in several pathogenic fungal species. Here we demonstrate that a fungal-specific alkaline response pathway is conserved in the human skin commensal fungus Malassezia sympodialis (Ms). This pathway is characterized by the pH-dependent activation of the Rim101/PacC transcription factor that controls cell surface adaptations to changing environmental conditions. By disrupting genes encoding two predicted components of this pathway, we demonstrated that the Rim/Pal pathway is conserved in this fungal species as a facilitator of alkaline pH growth. Moreover, targeted gene mutation and comparative transcriptional analysis support the role of the Ms Rra1 protein as a cell surface pH sensor conserved within the basidiomycete fungi, a group including plant and human pathogens. Using an animal model of atopic dermatitis, we demonstrate the importance of Ms Rim/Pal signaling in this common inflammatory condition characterized by increased skin pH.

Effect on Non-Small Cell Lung Cancer after Combination of Driver Gene Mutations and Anti-PD-1/PD-L1 Immunotherapy as Well as Chemotherapy.

We aimed to reveal the correlation between pathological indicators and PD-L1, between gene mutation status in lung cancer through clinico-pathological data and lung cancer-related gene mutation and PD-L1 expression analysis. The study was conducted in Jinhua Municipal Central Hospital, Zhejiang, China from 2017 to 2022. PD-L1 testing and targeted gene mutations detection were evaluated. The clinical characteristics of these non-small cell lung cancer (NSCLC) samples have been obtained. The groups (LUAD, n=142; LUSC, n=143) were grouped according to clinico-pathological features and PD-L1 expression (Yes/No or High/Low), and the clinico-pathological and genetic and molecular features and correlation with PD-L1 expression were compared across the above groups. Comparisons and analyses were made between different treatment schemes. Lung adenocarcinoma (LUAD, n=142) and lung squamous carcinoma (LUSC, n=143) samples were enrolled (median age: 64 years old). Pleural invasion and M staging were significantly different from PD-L1 alterations (P<0.05). The percentage of patients with PD-L1 tumor proportion score (TPS)≥50% was 36.24% and the percentage of patients with PD-L1 TPS<50% was 29.53%. The percentage of patients with PD-L1 high-expressed and treated by immunotherapy was 75.93% and 63.41% experienced Partial Response/Complete Response. The mutations ratio of EGFR, ALK, KRAS, MET, RET and TP53 were 28.86%, 1.34%, 6.04%, 0.67%, 1.34% and 0.67%, respectively. KRAS mutation was significantly different from PD-L1 alterations (P<0.01). There are individual differences in PD-L1 expression, which can also vary depending on the different clinical features. Specific molecular features correlate with differential PD-L1 expression and may influence the response to therapy.

Genotypic and phenotypic characterization of resistance to fenhexamid, carboxin, and, prochloraz, in Botrytis cinerea isolates collected from cut roses in Colombia.

Gray mold, caused by Botrytis sp., is a significant disease in Colombian rose crops and its control depends primarily on the intensive use of chemically synthesized fungicides. Despite the importance of this pathogen, there is limited information in Colombian floriculture about molecular taxonomy of species, fungicide resistance of populations and their genetic mechanism of resistance. In this study, we analyze 12 isolates of this fungus collected from rose-producing crops in the Department of Cundinamarca and conducted phylogenetic analysis using HSP60, G3PDH, and RPB2 gene sequences. Additionally, we realize phenotypic and genotypic characterization of resistance to the fungicides fenhexamid, carboxin, and prochloraz, evaluating the in vitro EC50 and presence of mutations of target genes of each isolate. All isolates were characterized as Botrytis cinerea in the phylogenetic analysis and presents different levels of resistance to each fungicide. These levels are related to mutations in target genes, with predominancy of L195F and L400F in the ERG27 gene to fenhexamid resistance, H272R/Y in the SDHB gene for carboxin resistance, and Y136F in the CYP51 gene for prochloraz resistance. Finally, these mutations were not related to morphological changes. Collectively, this knowledge, presented for the first time to the Colombian floriculture, contribute to a better understanding of the genetic diversity and population of B. cinerea from rose-producing crops in the department of Cundinamarca, and serve as a valuable tool for making informed decisions regarding disease management, future research, and improving crop management and sustainability in the Colombian floriculture industry.

Phenotypic and genotypic assessment of fluoroquinolones and aminoglycosides resistances in Pseudomonas aeruginosa collected from Minia hospitals, Egypt during COVID-19 pandemic

BackgroundOne of the most prevalent bacteria that cause nosocomial infections is Pseudomonas aeruginosa. Fluoroquinolones (FQ) and aminoglycosides are vital antipseudomonal drugs, but resistance is increasingly prevalent. The study sought to investigate the diverse mechanisms underlying FQ and aminoglycoside resistance in various P. aeruginosa strains particularly during the COVID-19 crisis.MethodsFrom various clinical and environmental samples, 110 P. aeruginosa isolates were identified and their susceptibility to several antibiotic classes was evaluated. Molecular techniques were used to track target gene mutations, the presence of genes encoding for quinolone resistance, modifying enzymes for aminoglycosides and resistance methyltransferase (RMT). Efflux pump role was assessed phenotypically and genotypically. Random amplified polymorphic DNA (RAPD) analysis was used to measure clonal diversity.ResultsQnrS was the most frequently encountered quinolone resistance gene (37.5%) followed by qnrA (31.2%) and qnrD (25%). Among aminoglycoside resistant isolates, 94.1% harbored modifying enzymes genes, while RMT genes were found in 55.9% of isolates. The aac(6')-Ib and rmtB were the most prevalent genes (79.4% and 32.3%, respectively). Most FQ resistant isolates overexpressed mexA (87.5%). RAPD fingerprinting showed 63.2% polymorphism.ConclusionsAminoglycosides and FQ resistance observed in this study was attributed to several mechanisms with the potential for cross-contamination existence so, strict infection control practices are crucial.

Performances of the Idylla GeneFusion Assay: contribution to a rapid diagnosis of targetable gene fusions in tumour samples

AimsWe aimed to evaluate the performances of the Idylla GeneFusion Assay (IGFA) designed to detect, in a single, rapid and fully automated assay, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3...

Malassezia responds to environmental pH signals through the conserved Rim/Pal pathway.

During mammalian colonization and infection, microorganisms must be able to rapidly sense and adapt to changing environmental conditions including alterations in extracellular pH. The fungus-specific Rim/Pal signaling pathway is one process that supports microbial adaptation to alkaline pH. This cascading series of interacting proteins terminates in the proteolytic activation of the highly conserved Rim101/PacC protein, a transcription factor that mediates microbial responses that favor survival in neutral/alkaline pH growth conditions, including many mammalian tissues. We identified the putative Rim pathway proteins Rim101 and Rra1 in the human skin colonizing fungus Malassezia sympodialis. Gene deletion by transconjugation and homologous recombination revealed that Rim101 and Rra1 are required for M. sympodialis growth at higher pH. Additionally, comparative transcriptional analysis of the mutant strains compared to wild-type suggested mechanisms for fungal adaptation to alkaline conditions. These pH-sensing signaling proteins are required for optimal growth in a murine model of atopic dermatitis, a pathological condition associated with increased skin pH. Together these data elucidate both conserved and phylum-specific features of microbial adaptation to extracellular stresses.

Drug resistance in breast cancer

Objective: The obstacle of resistance throughout breast cancer treatment leads to failure of therapy and progress in the disease. Current developments in our knowledge of the molecular pathways behind resistance to therapies in breast cancer will be covered in this brief review. Materials and methods: The presented data in this review is gathered from multiple sources such as clinical observations (information about how patients react to systemic treatments), experimental models (data gathered from in vitro and in vivo drug resistance simulation framework), review of genetic alterations and epigenetic changes that contribute to resistance is done through genomic and epigenetic studies. The drug resistance mechanisms: target mutations, novel signaling pathways, modifications to the tumor microenvironment, emerging biomarkers, genetic and epigenetic modifications (targeted gene mutations, modifications in drug transporter expression, activation of signaling pathways). Conclusion: Ongoing investigations are essential for identifying successful approaches to combat drug resistance in breast cancer. The process of refinement of combining medicines, development of novel targeted treatments, and research into mechanisms that limit drug transport are among the areas of focus.

Conserved Evolutionary Trajectory Can Be Perturbed to Prevent Resistance Evolution under Norfloxacin Pressure by Forcing Mycobacterium smegmatis on Alternate Evolutionary Paths.

Antibiotic resistance is a pressing health issue, with the emergence of resistance in bacteria outcompeting the discovery of novel drug candidates. While many studies have used Adaptive Laboratory Evolution (ALE) to understand the determinants of resistance, the influence of the drug dosing profile on the evolutionary trajectory remains understudied. In this study, we employed ALE on Mycobacterium smegmatis exposed to various concentrations of Norfloxacin using both cyclic constant and stepwise increasing drug dosages to examine their impact on the resistance mechanisms selected. Mutations in an efflux pump regulator, LfrR, were found in all of the evolved populations irrespective of the drug profile and population bottleneck, indicating a conserved efflux-based resistance mechanism. This mutation appeared early in the evolutionary trajectory, providing low-level resistance when present alone, with a further increase in resistance resulting from successive accumulation of other mutations. Notably, drug target mutations, similar to those observed in clinical isolates, were only seen above a threshold of greater than 4× the minimum inhibitory concentration (MIC). A combination of three mutations in the genes, lfrR, MSMEG_1959, and MSMEG_5045, was conserved across multiple lineages, leading to high-level resistance and preceding the appearance of drug target mutations. Interestingly, in populations evolved from parental strains lacking the lfrA efflux pump, the primary target of the lfrR regulator, no lfrR gene mutations are selected. Furthermore, evolutional trajectories originating from the ΔlfrA strain displayed early arrest in some lineages and the absence of target gene mutations in those that evolved, albeit delayed. Thus, blocking or inhibiting the expression of efflux pumps can arrest or delay the fixation of drug target mutations, potentially limiting the maximum attainable resistance levels.

Role of Pharmacotherapy in Treatment of Meningiomas

Meningiomas constitute the most common primary tumors of the central nervous system. Despite maximum treatment, grade 2/3 meningiomas are associated with a high risk of recurrence. Stereotactic radiosurgery is the treatment of choice as adjuvant treatment for grade 2/3 meningiomas. Currently, pharmacotherapies, including molecular targeted therapy for various growth factors, their receptors, and the associated pathways, have shown limited effectiveness for management of refractory or recurrent meningiomas. Therefore, novel systemic treatment approaches are urgently required in such cases. Recent advances in genetics and epigenetics and the identification of specific genetic alterations have led to new classifications of these tumors and the development of therapeutic targets. Identification of targeted gene mutations may lead to precision-based medicine. Other therapeutic approaches such as immune checkpoint inhibitors rarely elicit a significant response in meningiomas with a high tumor mutation burden. Combination therapies that affect these multiple targets are also considered adjuvant therapeutic options. Comprehensive/in-depth research is warranted to investigate the safety and efficacy of other treatment strategies, including chimeric antigen receptor T-cells, oncolytic virus immunotherapy, and gene therapy. In this article, we review the current evidence regarding the efficacy of systemic treatments available in the literature and discuss recent and ongoing trials for meningiomas.

Insecticide resistance and characteristics of mutations related to target site insensitivity of diamondback moths in Taiwan

Diamondback moth (DBM, Plutella xylostella) is the most significant pest of cruciferous vegetables as they rapidly develop high-level resistance to many insecticides. Monitoring DBM susceptibility and target-site mutation frequency is essential for pest control. In this study, 10 insecticides were tested on 11 field populations. Frequencies of target-site mutations (including para, ace1, Rdl1, RyR1, and nAChRα6 genes) were estimated by pyrosequencing. Insecticides registered after 2007 for DBM control in Taiwan, i.e., spinetoram, chlorantraniliprole, chlorfenapyr, metaflumizone, and flubendiamide, showed >80% mortality toward several populations; Bacillus thurigiensis, emamectin benzoate, and chlorfluazuron showed medium to low efficacy in all populations; and tolfenpyrad and mevinphos were highly ineffective. Susceptibility to insecticides varied substantially among populations: eight out of nine populations were highly susceptible to spinetoram, but only one was susceptible to flubendiamide. Target-site mutations related to organophosphates, pyrethroids, fipronil, and diamides were detected in all populations, but there were few spinosad and spinetoram mutations. Our three-year field study demonstrated rapid efficacy loss for all insecticides tested, particularly for more toxic insecticides. Skipped-generation selection of a field DBM strain to emamectin benzoate, metaflumizone, chlorantraniliprole, and flubendiamide revealed that mortality rates dropped from 60 to 80% to <10% after 6 generations. Next-generation sequencing was performed to identify possible target gene mutations. A resistance management program that considers the instability of resistance to some chemicals and pertinent data on resistance mechanisms should be established. Identifying compounds to overcome high-frequency field DBM point mutations could be beneficial for pest control.

Comparative genomic analysis of Brazilian Mesomycoplasma ovipneumoniae strains revealed genomic differences associated with the geographic origin and health status and mutations in the gyrA

Sheep respiratory disease (SRD) is a multifactorial illness commonly affecting sheep. Mesomycoplasma (Mycoplasma) ovipneumoniae is one of the most important etiological agents of SRD and should be better understood, especially in countries where it was recently detected, such as Brazil. Also, the intensive use of quinolones in mycoplasmal infections increases the selective pressure for resistance to this drug class, and no data about antimicrobial resistance in Brazil is available. Therefore, this study aimed to perform a comparative genomic analysis of newly isolated Brazilian M. ovipneumoniae strains, identify point mutations in target genes that may be associated with antibiotic resistance, and perform a phylogenomic analysis of these strains with available genome representatives of M. ovipneumoniae. Glucose-fermenting fried egg-like colonies identified as M. ovipneumoniae were obtained after a culture of tracheobronchial lavage from infected sheep. The genomes were sequenced, de novo assembled and comparatively evaluated. Important putative virulence factors were detected in all isolates: the analysis of the average nucleotide homology of all these genes with the M. ovipneumoniae ATCC 29419 revealed associations between clpB, lgt, tuf, and dnaJ genes and geographic location. In addition, nucleotide substitutions in a few positions of the Quinolone-Resistant Determinant Region of the gyrA gene, including the Ser83Ala, were detected. The phylogenomic analysis showed that the Brazilian isolates belonged to two different clades corresponding to geographic location, and the isolates from São Paulo showed high similarity, which differs from isolates from Rio de Janeiro. This first genomic analysis of the Brazilian M. ovipneumoniae genomes demonstrates strain segregation according to location and health status, reinforcing the importance of continuous surveillance and diagnostics of this bacteria causing sheep respiratory disease in the Brazilian flocks.

Callus-specific CRISPR/Cas9 system to increase heritable gene mutations in maize.

A callus-specific CRISPR/Cas9 (CSC) system with Cas9 gene driven by the promoters of ZmCTA1 and ZmPLTP reduces somatic mutations and improves the production of heritable mutations in maize. The CRISPR/Cas9 system, due to its editing accuracy, provides an excellent tool for crop genetic breeding. Nevertheless, the traditional design utilizing CRISPR/Cas9 with ubiquitous expression leads to an abundance of somatic mutations, thereby complicating the detection of heritable mutations. We constructed a callus-specific CRISPR/Cas9 (CSC) system using callus-specific promoters of maize Chitinase A1 and Phospholipid transferase protein (pZmCTA1 and pZmPLTP) to drive Cas9 expression, and the target gene chosen for this study was the bZIP transcription factor Opaque2 (O2). The CRISPR/Cas9 system driven by the maize Ubiquitin promoter (pZmUbi) was employed as a comparative control. Editing efficiency analysis based on high-throughput tracking of mutations (Hi-TOM) showed that the CSC systems generated more target gene mutations than the ubiquitously expressed CRISPR/Cas9 (UC) system in calli. Transgenic plants were generated for the CSC and UC systems. We found that the CSC systems generated fewer target gene mutations than the UC system in the T0 seedlings but reduced the influence of somatic mutations. Nearly 100% of mutations in the T1 generation generated by the CSC systems were derived from the T0 plants. Only 6.3-16.7% of T1 mutations generated by the UC system were from the T0 generation. Our results demonstrated that the CSC system consistently produced more stable, heritable mutants in the subsequent generation, suggesting its potential application across various crops to facilitate the genetic breeding of desired mutations.

Updated study results of novel FAK/ALK/ROS1 inhibitor APG-2449 in patients (pts) with non-small-cell lung cancer (NSCLC) resistant to second-generation ALK inhibitors.

3124 Background: ALK inhibitors increase FAK pathway gene expression in ALK+ NSCLC cell lines, with the highest induced expression in drug-tolerant persister cells. This suggests that FAK pathway activation is involved in ALK inhibitor-induced early adaptive tolerance in ALK+ NSCLC. Investigational APG-2449 is a novel, orally active FAK inhibitor and a third-generation (3G) ALK/ROS1 tyrosine kinase inhibitor (TKI) with potent activity in preclinical models. APG-2449 is well tolerated at the recommended phase 2 dose (RP2D) and showed preliminary efficacy in pts resistant to 2G ALK TKIs. Here, we provide further safety and efficacy results and demonstrate associations between this therapy and FAK signaling pathways. Methods: After the RP2D was determined as 1,200 mg daily (QD), pts with NSCLC were enrolled into 2 dose expansion cohorts. Cohort 1 included pts who were resistant to 2G ALK TKIs. Cohort 2 included those who were ALK or ROS1 TKI naïve. Results: As of December 9, 2023, 144 pts (median [range] age, 53 [21-78] years; 53.5% female) with NSCLC, mesothelioma, or ovarian cancer were treated with APG-2449 at doses of 150 to 1,500 mg. A total of 129 (89.6%) pts had treatment-related adverse events (TRAEs), the most frequent of which were elevated serum creatinine (49.3%), ALT (42.4%), and AST (36.1%); nausea (28.5%); vomiting (23.6%); diarrhea (22.9%); and decreased leukocyte count (22.2%). In all, 20 (13.9%) TRAEs were grade ≥ 3. Of pts with TKI-naïve NSCLC (n = 36) treated at the RP2D, the overall response (ORR) and disease control rates (DCR = CR + PR + SD) were 68.2% (15/22) and 90.1% (20/22) in ROS1 TKI-naïve pts; and 78.6% (11/14) and 100% (14/14) in ALK TKI-naïve pts. Median progression-free survival (mPFS) in ALK TKI-naïve pts was not reached. Of 22 pts with NSCLC resistant to 2G ALK inhibitors and without targetable bypass gene mutations (e.g., KRAS G12C, BRAF V600E), 9 had PRs (9/22; 40.9%) and the mPFS during APG-2449 treatment at RP2D was 11.86 months. Nine of 12 pts in the RP2D group achieved intracranial PR, resulting in an intracranial ORR of 75.0%. Before APG-2449 administration, 17 tumor tissue samples were collected from pts with NSCLC resistant to 2G ALK inhibitors (in the RP2D group), and IHC was used to assess protein levels of phosphorylated FAK (pFAK). The PFS of pts treated with APG-2449 correlated with pFAK levels in baseline tumor tissue, and pts with higher pFAK levels were more likely to benefit from APG-2449 treatment. Conclusions: APG-2449 demonstrated preliminary efficacy in pts with NSCLC whose disease was TKI naïve and resistant to 2G ALK inhibitors, especially in brain metastases. High pFAK expression levels in baseline tumor tissue correlated with improved APG-2449 treatment responses in pts with NSCLC resistant to 2G ALK inhibitors. Internal study identifier: APG2449XC101. Clinical trial information: NCT03917043 .

Circulating tumor DNA- and cancer tissue-based next-generation sequencing reveals comparable consistency in targeted gene mutations for advanced or metastatic non-small cell lung cancer.

Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy. This study aimed to investigate the consistency of next-generation sequencing (NGS) results between circulating tumor DNA (ctDNA)-based and tissue-based in non-small cell lung cancer (NSCLC) and identify the patient characteristics that favor ctDNA testing. Patients who diagnosed with NSCLC and received both ctDNA- and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center, Sichuan University West China Hospital between December 2017 and August 2022 were enrolled. A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS. The unweighted Cohen's kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6. Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS. A total of 85 patients were enrolled, of which 22.4% (19/85) had stage III disease and 56.5% had stage IV disease. Forty-four patients (51.8%) showed consistent gene mutation types between ctDNA-based and tissue-based NGS, while one patient (1.2%) tested negative in both approaches. Advanced diseases and metastases to other organs would be fit for the ctDNA-based NGS, and the generalized linear model showed that T stage, M stage, and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS. ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS, and it could be considered in advanced or metastatic NSCLC.

Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Whole genome sequencing of uropathogenic E. coli from Ireland reveals diverse resistance mechanisms and strong correlation with phenotypic (EUCAST) susceptibility testing

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes.In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets.Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6′)-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB.These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.

Clinical and Therapeutic Intervention of Hypereosinophilia in the Era of Molecular Diagnosis.

Hypereosinophilia (HE) presents with an elevated peripheral eosinophilic count of >1.5 × 109/L and is composed of a broad spectrum of secondary non-hematologic disorders and a minority of primary hematologic processes with heterogenous clinical presentations, ranging from mild symptoms to potentially lethal outcome secondary to end-organ damage. Following the introduction of advanced molecular diagnostics (genomic studies, RNA sequencing, and targeted gene mutation profile, etc.) in the last 1-2 decades, there have been deep insights into the etiology and molecular mechanisms involved in the development of HE. The classification of HE has been updated and refined following to the discovery of clinically novel markers and targets in the 2022 WHO classification and ICOG-EO 2021 Working Conference on Eosinophil Disorder and Syndromes. However, the diagnosis and management of HE is challenging given its heterogeneity and variable clinical outcome. It is critical to have a diagnostic algorithm for accurate subclassification of HE and hypereosinophilic syndrome (HES) (e.g., reactive, familial, idiopathic, myeloid/lymphoid neoplasm, organ restricted, or with unknown significance) and to follow established treatment guidelines for patients based on its clinical findings and risk stratification.