Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance.

Rifampicin (RIF), the frontline drug against M. tuberculosis, is completely ineffective against M. abscessus, partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF. Using RNA-seq, we show that exposure of M. abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M. tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes in vitro. Finally, we show that the tip of the PCh-loop of Mab_helR, present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP.

The intrinsic macrolide resistome of Escherichia coli.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

Insights into gemcitabine resistance in pancreatic cancer: association with metabolic reprogramming and TP53 pathogenicity in patient derived xenografts.

BACKGROUND: With poor prognosis and high mortality, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Standard of care therapies for PDAC have included gemcitabine for the past three decades, although resistance often develops within weeks of chemotherapy initiation through an array of possible mechanisms. METHODS: We reanalyzed publicly available RNA-seq gene expression profiles of 28 PDAC patient-derived xenograft (PDX) models before and after a 21-day gemcitabine treatment using our validated analysis pipeline to identify molecular markers of intrinsic and acquired resistance. RESULTS: Using normalized RNA-seq quantification measurements, we first identified oxidative phosphorylation and interferon alpha pathways as the two most enriched cancer hallmark gene sets in the baseline gene expression profile associated with intrinsic gemcitabine resistance and sensitivity, respectively. Furthermore, we discovered strong correlations between drug-induced expression changes in glycolysis and oxidative phosphorylation genes and response to gemcitabine, which suggests that these pathways may be associated with acquired gemcitabine resistance mechanisms. Thus, we developed prediction models using baseline gene expression profiles in those pathways and validated them in another dataset of 12 PDAC models from Novartis. We also developed prediction models based on drug-induced expression changes in genes from the Molecular Signatures Database (MSigDB)'s curated 50 cancer hallmark gene sets. Finally, pathogenic TP53 mutations correlated with treatment resistance. CONCLUSION: Our results demonstrate that concurrent upregulation of both glycolysis and oxidative phosphorylation pathways occurs in vivo in PDAC PDXs following gemcitabine treatment and that pathogenic TP53 status had association with gemcitabine resistance in these models. Our findings may elucidate the molecular basis for gemcitabine resistance and provide insights for effective drug combination in PDAC chemotherapy.

Aberrant m5C hypermethylation mediates intrinsic resistance to gefitinib through NSUN2/YBX1/QSOX1 axis in EGFR-mutant non-small-cell lung cancer.

BACKGROUND: RNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear. METHODS: The correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. RESULTS: RNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1). CONCLUSIONS: Our study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

Intrinsic Resistance to Colistin in the Genus Hafnia.

A bacterial species is considered to be intrinsically resistant to an antimicrobial when nearly all of the wild-type isolates (i.e., those without acquired resistance) exhibit minimum inhibitory concentration (MIC) values that are sufficiently high such that susceptibility testing is unnecessary, and that the antimicrobial should not be considered for therapy. Accordingly, knowledge of intrinsic resistance influences both the selection of treatment regimens and the approach to susceptibility testing in the clinical laboratory, where unexpected results also facilitate the recognition of microbial identification or susceptibility testing errors. Previously, limited data have suggested that Hafnia spp. may be intrinsically resistant to colistin. We evaluated the in vitro activity of colistin against 119 Hafniaceae that were isolated from human samples: 75 (63%) from routine clinical cultures and 44 (37%) from stool samples of travelers undergoing screening for antimicrobial resistant organisms. Broth microdilution colistin MICs were ≥4 µg/mL for 117 of 119 (98%) isolates. Whole-genome sequencing of 96 of the isolates demonstrated that the colistin-resistant phenotype was not lineage-specific. 2 of the 96 (2%) isolates harbored mobile colistin resistance genes. Compared to whole-genome sequencing, VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and VITEK 2 GN ID failed to consistently distinguish between Hafnia alvei, Hafnia paralvei, and Obesumbacterium proteus. In conclusion, using a reference antimicrobial susceptibility testing method and a genetically diverse collection of isolates, we found Hafnia spp. to be intrinsically resistant to colistin. The recognition of this phenotype will help inform rational approaches by which to perform antimicrobial susceptibility testing and therapy for patients with infections that are caused by Hafnia spp.

The intrinsic resistance of Fusarium solani to the Fusarium-specific fungicide phenamacril is attributed to the natural variation of both T218S and K376M in myosin5.

Fusarium solani is responsible for causing root rot in various crops, resulting in wilting and eventual demise. Phenamacril, a specific inhibitor of myosin5 protein, has gained recognition as an effective fungicide against a broad spectrum of Fusarium species. It has been officially registered for controlling Fusarium diseases through spray application, root irrigation, and seed dipping. In this study, phenamacril was observed to exhibit negligible inhibitory effects on F. solani causing crop root rot, despite the absence of prior exposure to phenamacril. Considering the high selectivity of phenamacril, this phenomenon was attributed to intrinsic resistance and further investigated for its underlying mechanism. Sequence alignment analysis of myosin5 proteins across different Fusarium species revealed significant differences at positions 218 and 376. Subsequent homology modeling and molecular docking results indicated that substitutions T218S, K376M, and T218S&K376M impaired the binding affinity between phenamacril and myosin5 in F. solani. Mutants carrying these substitutions were generated via site-directed mutagenesis. A phenamacril-sensitivity test showed that the EC50 values of mutants carrying T218S, K376M, and T218S&K376M were reduced by at least 6.13-fold, 9.66-fold, and 761.90-fold respectively compared to the wild-type strain. Fitness testing indicated that mutants carrying K376M or T218S&K376M had reduced sporulation compared to the wild-type strain. Additionally, mutants carrying T218S exhibited an enhanced virulence compared to the wild-type strain. However, there were no significant differences observed in mycelial growth rates between the mutants and the wild-type strain. Thus, the intrinsic differences observed at positions 218 and 376 in myosin5 between F. solani and other Fusarium species are specifically associated with phenamacril resistance. The identification of these resistance-associated positions in myosin5 of F. solani has significantly contributed to the understanding of phenamacril resistance mechanisms, thereby discouraging the use of phenamacril for controlling F. solani.

A Phase II Exploratory Study to Identify Biomarkers Predictive of Clinical Response to Regorafenib in Patients with Metastatic Colorectal Cancer Who Have Failed First-Line Therapy.

Single-agent regorafenib is approved in Canada for metastatic colorectal cancer (mCRC) patients who have failed previous lines of therapy. Identifying prognostic biomarkers is key to optimizing therapeutic strategies for these patients. In this clinical study (NCT01949194), we evaluated the safety and efficacy of single-agent regorafenib as a second-line therapy for mCRC patients who received it after failing first-line therapy with an oxaliplatin or irinotecan regimen with or without bevacizumab. Using various omics approaches, we also investigated putative biomarkers of response and resistance to regorafenib in metastatic lesions and blood samples in the same cohort. Overall, the safety profile of regorafenib seemed similar to the CORRECT trial, where regorafenib was administered as ≥ 2 lines of therapy. While the mutational landscape showed typical mutation rates for the top five driver genes (APC, KRAS, BRAF, PIK3CA, and TP53), KRAS mutations were enriched in intrinsically resistant lesions. Additional exploration of genomic-phenotype associations revealed several biomarker candidates linked to unfavorable prognoses in patients with mCRC using various approaches, including pathway analysis, cfDNA profiling, and copy number analysis. However, further research endeavors are necessary to validate the potential utility of these promising genes in understanding patients' responses to regorafenib treatment.

Antimicrobial resistance: new insights and therapeutic implications.

Antimicrobial resistance has not been a new phenomenon. Still, the number of resistant organisms, the geographic areas affected by emerging drug resistance, and the magnitude of resistance in a single organism are enormous and mounting. Disease and disease-causing agents formerly thought to be contained by antibiotics are now returning in new forms resistant to existing therapies. Antimicrobial resistance is one of the most severe and complicated health issues globally, driven by interrelated dynamics in humans, animals, and environmental health sectors. Coupled with various epidemiological factors and a limited pipeline for new antimicrobials, all these misappropriations allow the transmission of drug-resistant organisms. The problem is likely to worsen soon. Antimicrobial resistance in general and antibiotic resistance in particular is a shared global problem. Actions taken by any single country can adversely or positively affect the other country. Targeted coordination and prevention strategies are critical in stopping the spread of antibiotic-resistant organisms and hence its overall management. This article has provided in-depth knowledge about various methods that can help mitigate the emergence and spread of antimicrobial resistance globally. KEY POINTS: • Overview of antimicrobial resistance as a global challenge and explain various reasons for its rapid progression. • Brief about the intrinsic and acquired resistance to antimicrobials and development of antibiotic resistance in bacteria. • Systematically organized information is provided on different strategies for tackling antimicrobial resistance for the welfare of human health.

Successful treatment of aortic valve endocarditis caused by Enterococcus casseliflavus: a case report.

BACKGROUND: Enterococcus casseliflavus is rarely isolated from human specimens. To the best of our knowledge, there are no reports on its detailed treatment course and prognosis. Here, we present the first known case of E. casseliflavus endocarditis with a detailed treatment course. CASE PRESENTATION: An 86-year-old Japanese woman was transferred to the emergency department with dyspnoea, wheezing, and lumbago. Her medical history included hypertension, chronic kidney disease, idiopathic interstitial pneumonia, and rectal carcinoma. Physical examination revealed expiratory wheezes and a diastolic murmur (Levine 2/6) at the 4th right sternal border. Chest radiography revealed bilateral interstitial opacities and slight cardiac dilatation. Transthoracic echocardiography demonstrated the presence of mobile vegetation with perforation, prolapse, and regurgitation of the aortic valve. With a suspicion of infective endocarditis, we started administering intravenous ampicillin/sulbactam. Thereafter, blood cultures identified E. casseliflavus through matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The antimicrobial treatment was then switched to ampicillin plus gentamicin. The patient underwent aortic valve replacement on the thirteenth hospital day. She was administered intravenous ampicillin and gentamicin for 6 weeks. The patient was discharged 8 weeks after admission. CONCLUSIONS: Our case demonstrated that E. casseliflavus could cause infective endocarditis, which can be successfully treated with a 6-week regimen of ampicillin and gentamicin in combination with proper surgical treatment.

Intrinsic and acquired resistance to CDK4/6 inhibitors and potential overcoming strategies.

Abnormal activation of the cyclin-dependent kinases (CDKs), which result in aberrant cell proliferation, is one of the inherent characteristics of tumor. Thus targeting the activity of CDKs represents a promising tumor therapeutic strategy. Currently, the specific inhibitors that target CDK4 and CDK6 have been approved for the treatment of estrogen receptor positive, human epidermal growth factor receptor 2 negative (ER+ HER2-) breast cancer in combination with endocrine therapy; other combination strategies are being tested in a number of clinical trials. However, the acquired resistance to CDK4/6 inhibitors has emerged. As the cell cycle is orchestrated by a series of biological events, the alterations of other molecular events that regulate the cell cycle progression may be involved in intrinsic resistance to CDK4/6 inhibitors. In this review we mainly discuss the mechanisms underlying intrinsic resistance and acquired resistance to CDK4/6 inhibitors as well as combination strategies with other signal pathway inhibitors being tested in clinical and pre-clinical studies, to extend the use of CDK4/6 inhibitors in tumor treatment.

Acibenzolar-S-Methyl and Resistance Quantitative Trait Loci Complement Each Other to Control Apple Scab and Fire Blight.

Diversifying disease control methods is a key strategy to sustainably reduce pesticides. Plant genetic resistance has long been used to create resistant varieties. Plant resistance inducers (PRI) are also considered to promote crop health, but their effectiveness is partial and can vary according to the environment and the plant genotype. We investigated the putative interaction between intrinsic (genetic) and PRI-induced resistance in apple when affected by scab and fire blight diseases. A large F1 mapping population was challenged by each disease after a pre-treatment with acibenzolar-S-methyl (ASM) and compared with the water control. Apple scab and fire blight resistance quantitative trait loci (QTLs) were detected in both conditions and compared. ASM exhibited a strong effectiveness in reducing both diseases. When combined, QTL-controlled and ASM-induced resistance acted complementarily to reduce the symptoms from 85 to 100%, depending on the disease. In our conditions, resistance QTLs were only slightly or rarely affected by ASM treatment, despite their probable implication in various stages of the resistance buildup. Implications of these results are discussed considering already known results, the underlying mechanisms, cross protection of both types of resistance against pathogen adaptation, and practical application in orchard conditions.

Coming from the Wild: Multidrug Resistant Opportunistic Pathogens Presenting a Primary, Not Human-Linked, Environmental Habitat.

The use and misuse of antibiotics have made antibiotic-resistant bacteria widespread nowadays, constituting one of the most relevant challenges for human health at present. Among these bacteria, opportunistic pathogens with an environmental, non-clinical, primary habitat stand as an increasing matter of concern at hospitals. These organisms usually present low susceptibility to antibiotics currently used for therapy. They are also proficient in acquiring increased resistance levels, a situation that limits the therapeutic options for treating the infections they cause. In this article, we analyse the most predominant opportunistic pathogens with an environmental origin, focusing on the mechanisms of antibiotic resistance they present. Further, we discuss the functions, beyond antibiotic resistance, that these determinants may have in the natural ecosystems that these bacteria usually colonize. Given the capacity of these organisms for colonizing different habitats, from clinical settings to natural environments, and for infecting different hosts, from plants to humans, deciphering their population structure, their mechanisms of resistance and the role that these mechanisms may play in natural ecosystems is of relevance for understanding the dissemination of antibiotic resistance under a One-Health point of view.

Disparate outer membrane exclusionary properties underlie intrinsic resistance to hydrophobic substances in Pseudomonas spp. isolated from surface waters under triclosan selection.

Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.

A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

Mutation Variants and Co-Mutations as Genomic Modifiers of Response to Afatinib in HER2-Mutant Lung Adenocarcinoma.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-mutant lung cancer remains an orphan of specific targeted therapy. The variable responses to anti-HER2 therapies in these patients prompt us to examine impact of HER2 variants and co-mutations on responses to anti-HER2 treatments in lung cancer. PATIENTS AND METHODS: Patients with stage IV/recurrent HER2-mutant lung cancers identified through next-generation sequencings were recruited from seven hospitals. The study comprised a cohort A to establish the patterns of HER2 variants and co-mutations in lung cancer and a cohort B to assess associations between HER2 variants, co-mutations, and clinical outcomes. RESULTS: The study included 118 patients (cohort A, n = 86; cohort B, n = 32). Thirty-one HER2 variants and 35 co-mutations were detected. Predominant variants were A775_G776insYVMA (49/118, 42%), G778_P780dup (11/118, 9%), and G776delinsVC (9/118, 8%). TP53 was the most common co-mutation (61/118, 52%). In cohort B, objective response rates with afatinib were 0% (0/14, 95% confidence interval [CI], 0%-26.8%), 40% (4/10, 14.7%-72.6%), and 13% (1/8, 0.7%-53.3%) in group 1 (A775_G776insYVMA, n = 14), group 2 (G778_P780dup, G776delinsVC, n = 10), and group 3 (missense mutation, n = 8), respectively (p = .018). Median progression-free survival in group 1 (1.2 months; 95% CI, 0-2.4) was shorter than those in group 2 (7.6 months, 4.9-10.4; hazard ratio [HR], 0.009; 95% CI, 0.001-0.079; p < .001) and group 3 (3.6 months, 2.6-4.5; HR, 0.184; 95% CI, 0.062-0.552; p = .003). TP53 co-mutations (6.317; 95% CI, 2.180-18.302; p = .001) and PI3K/AKT/mTOR pathway activations (19.422; 95% CI, 4.098-92.039; p < .001) conferred additional resistance to afatinib. CONCLUSION: G778_P780dup and G776delinsVC derived the greatest benefits from afatinib among HER2 variants. Co-mutation patterns were additional response modifiers. Refining patient population based on patterns of HER2 variants and co-mutations may help improve the efficacy of anti-HER2 treatment in lung cancer. IMPLICATIONS FOR PRACTICE: Human epidermal growth factor receptor 2 (HER2)-mutant lung cancers are a group of heterogenous diseases with up to 31 different variants and 35 concomitant genomic aberrations. Different HER2 variants exhibit divergent sensitivities to anti-HER2 treatments. Certain variants, G778_P780dup and G776delinsVC, derive sustained clinical benefits from afatinib, whereas the predominant variant, A775_G776insYVMA, is resistant to most anti-HER2 treatments. TP53 is the most common co-mutation in HER2-mutant lung cancers. Co-mutations in TP53 and the PI3K/AKT/mTOR pathway confer additional resistance to anti-HER2 treatments in lung cancer. The present data suggest that different HER2 mutations in lung cancer, like its sibling epidermal growth factor receptor, should be analyzed independently in future studies.

Inhibition of Akt/mTOR pathway overcomes intrinsic resistance to dasatinib in triple-negative breast cancer.

Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.

Cell permeability, ß-lactamase activity, and transport contribute to high level of resistance to ampicillin in Lysobacter enzymogenes.

Discovery of multidrug resistance (MDR) in environmental microorganisms provides unique resources for uncovering antibiotic resistomes, which could be vital to predict future emergence of MDR pathogens. Our previous studies indicated that Lysobacter sp. conferred intrinsic resistance to multiple antibiotics at high levels, especially ampicillin, the first broad-spectrum ß-lactam antibiotics against both Gram-positive and Gram-negative bacteria. However, the underlying molecular mechanisms for resistance to ampicillin in Lysobacter enzymogenes strain C3 (LeC3) remain unknown. In this study, screening a Tn5 transposon mutant library of LeC3 recovered 12 mutants with decreased ampicillin resistance, and three mutants (i.e., tatC, lebla, and lpp) were selected for further characterization. Our results revealed that genes encoding ß-lactamase (lebla) and twin-arginine translocation (tatC) system for ß-lactamase transport played a pivotal role in conferring ampicillin resistance in L. enzymogenes. It was also demonstrated that the lpp gene was not only involved in resistance against ß-lactams but also conferred resistance to multiple antibiotics in L. enzymogenes. Permeability assay results indicated that decreased MDR in the lpp mutant was in part due to its higher cellular permeability. Furthermore, our results showed that the difference of LeC3 and L. antibioticus strain LaATCC29479 in ampicillin susceptibility was partly due to their differences in cellular permeability, but not due to ß-lactamase activities.

Role of epaQ, a Previously Uncharacterized Enterococcus faecalis Gene, in Biofilm Development and Antimicrobial Resistance.

Enterococcus faecalis is a commensal of the human gastrointestinal tract; it is also an opportunistic pathogen and one of the leading causes of hospital-acquired infections. E. faecalis produces biofilms that are highly resistant to antibiotics, and it has been previously reported that certain genes of the epa operon contribute to biofilm-associated antibiotic resistance. Despite several studies examining the epa operon, many gene products of this operon remain annotated as hypothetical proteins. Here, we further explore the epa operon; we identified epaQ, currently annotated as encoding a hypothetical membrane protein, as being important for biofilm formation in the presence of the antibiotic daptomycin. Mutants with disruptions of epaQ were more susceptible to daptomycin relative to the wild type, suggesting its importance in biofilm-associated antibiotic resistance. Furthermore, the ΔepaQ mutant exhibited an altered biofilm architectural arrangement and formed small aggregates in liquid cultures. Our cumulative data show that epa mutations result in altered polysaccharide content, increased cell surface hydrophobicity, and decreased membrane potential. Surprisingly, several epa mutations significantly increased resistance to the antibiotic ceftriaxone, indicating that the way in which the epa operon impacts antibiotic resistance is antibiotic dependent. These results further define the key role of epa in antibiotic resistance in biofilms and in biofilm architecture.IMPORTANCEE. faecalis is a common cause of nosocomial infection, has a high level of antibiotic resistance, and forms robust biofilms. Biofilm formation is associated with increased antibiotic resistance. Therefore, a thorough understanding of biofilm-associated antibiotic resistance is important for combating resistance. Several genes from the epa operon have previously been implicated in biofilm-associated antibiotic resistance, pathogenesis, and competitive fitness in the GI tract, but most genes in this locus remain uncharacterized. Here, we examine epaQ, which has not been characterized functionally. We show that the ΔepaQ mutant exhibits reduced biofilm formation in the presence of daptomycin, altered biofilm architecture, and increased resistance to ceftriaxone, further expanding our understanding of the contribution of this operon to intrinsic enterococcal antibiotic resistance and biofilm growth.

Cellular sensing of viral DNA and viral evasion mechanisms.

Mammalian cells detect foreign DNA introduced as free DNA or as a result of microbial infection, leading to the induction of innate immune responses that block microbial replication and the activation of mechanisms that epigenetically silence the genes encoded by the foreign DNA. A number of DNA sensors localized to a variety of sites within the cell have been identified, and this review focuses on the mechanisms that detect viral DNA and how the resulting responses affect viral infections. Viruses have evolved mechanisms that inhibit these host sensors and signaling pathways, and the study of these antagonistic viral strategies has provided insight into the mechanisms of these host responses. The field of cellular sensing of foreign DNA is in its infancy, but our currently limited knowledge has raised a number of important questions for study.

Evidences Suggesting that Distinct Immunological and Cellular Responses to Light Damage Distinguishes Juvenile and Adult Rat Retinas.

To unravel the mechanisms behind the higher resistance to light damage of juvenile (JR) versus adult (AR) rats, Sprague Dawley rats were exposed to a bright luminous environment of 10, 000 lux. The light-induced retinopathy (LIR) was assessed with histology, electroretinography and immunohistochemistry (IHC). In JR, 2 days of exposure induced the typical LIR, while >3 days added little LIR. IHC revealed a subtle migration of microglia (Iba1 marker) from the inner to the outer retina after 3 days of exposure in JR contrasting with the stronger reaction seen after 1 day in AR. Similarly, in JR, the Müller cells expressed less intense GFAP, CNTF and FGF2 staining compared to AR. Our results suggest that in JR the degree of retinal damage is not proportional to the duration of light exposure (i.e., dose-independent retinopathy), contrasting with the dose-dependent LIR reported in AR. The immature immune system in JR may explain the delayed and/or weaker inflammatory response compared to AR, a finding that would also point to the devastating contribution of the immune system in generating the LIR phenotype, a claim also advanced to explain the pathophysiology of other retinal degenerative disorders such as Age-related Macular Degeneration, Diabetic Retinopathy and Retinitis Pigmentosa.