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1.
Sci Rep ; 14(1): 4163, 2024 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378700

RESUMO

Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.


Assuntos
Aminoglicosídeos , Antibacterianos , Antibacterianos/farmacologia , Aminoglicosídeos/farmacologia , Escherichia coli/genética , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Estreptomicina/farmacologia , Gentamicinas/farmacologia , Neomicina/farmacologia
2.
ACS Infect Dis ; 10(2): 527-540, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38294409

RESUMO

Gram-negative bacterial infections are difficult to manage as many antibiotics are ineffective owing to the presence of impermeable bacterial membranes. Polymicrobial infections pose a serious threat due to the inadequate efficacy of available antibiotics, thereby necessitating the administration of antibiotics at higher doses. Antibiotic adjuvants have emerged as a boon as they can augment the therapeutic potential of available antibiotics. However, the toxicity profile of antibiotic adjuvants is a major hurdle in clinical translation. Here, we report the design, synthesis, and biological activities of xanthone-derived molecules as potential antibiotic adjuvants. Our SAR studies witnessed that the p-dimethylamino pyridine-derivative of xanthone (X8) enhances the efficacy of neomycin (NEO) against Escherichia coli and Pseudomonas aeruginosa and causes a synergistic antimicrobial effect without any toxicity against mammalian cells. Biochemical studies suggest that the combination of X8 and NEO, apart from inhibiting protein synthesis, enhances the membrane permeability by binding to lipopolysaccharide. Notably, the combination of X8 and NEO can disrupt the monomicrobial and polymicrobial biofilms and show promising therapeutic potential against a murine wound infection model. Collectively, our results unveil the combination of X8 and NEO as a suitable adjuvant therapy for the inhibition of the Gram-negative bacterial infections.


Assuntos
Infecções por Bactérias Gram-Negativas , Xantonas , Animais , Camundongos , Antibacterianos/farmacologia , Biofilmes , Escherichia coli , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Mamíferos , Neomicina/farmacologia , Xantonas/farmacologia
3.
J Laryngol Otol ; 138(4): 431-435, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38224038

RESUMO

OBJECTIVE: This review assessed the effectiveness of the nurse-led children's epistaxis clinic in streamlining patient care and avoiding unnecessary general anaesthesia. METHODS: A retrospective case note review was conducted of children attending the nurse-led epistaxis clinic between 2019 and 2021. RESULTS: A total of 718 children were seen over three years. Twelve (1.7 per cent) had a known coagulopathy. Of the children, 590 (82 per cent) had visible vessels and 29 (4 per cent) had mucosal crusting. Silver nitrate cautery was attempted under topical anaesthesia in 481 children, with 463 (96 per cent) successful cauterisations. Fifteen (3 per cent) were cauterised under general anaesthesia. Of the children, 706 (99 per cent) were prescribed nasal antiseptic preparations; this was the sole treatment for 58 (8 per cent). Blood investigations were requested for eight children (1 per cent) and haematology referral for three (0.4 per cent). CONCLUSION: This is the largest published series of children's nosebleeds. Given the short-lived benefit from cautery, it is suggested that general anaesthesia should not be offered routinely. However, improved haematology referral criteria are required to increase underlying diagnosis.


Assuntos
Clorexidina , Epistaxe , Criança , Humanos , Epistaxe/cirurgia , Epistaxe/diagnóstico , Estudos Retrospectivos , Neomicina , Papel do Profissional de Enfermagem , Cauterização
4.
Biol Res ; 57(1): 3, 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-38217055

RESUMO

BACKGROUND: Sensorineural hearing loss (SNHL) poses a major threat to both physical and mental health; however, there is still a lack of effective drugs to treat the disease. Recently, novel biological therapies, such as mesenchymal stem cells (MSCs) and their products, namely, exosomes, are showing promising therapeutic potential due to their low immunogenicity, few ethical concerns, and easy accessibility. Nevertheless, the precise mechanisms underlying the therapeutic effects of MSC-derived exosomes remain unclear. RESULTS: Exosomes derived from MSCs reduced hearing and hair cell loss caused by neomycin-induced damage in models in vivo and in vitro. In addition, MSC-derived exosomes modulated autophagy in hair cells to exert a protective effect. Mechanistically, exogenously administered exosomes were internalized by hair cells and subsequently upregulated endocytic gene expression and endosome formation, ultimately leading to autophagy activation. This increased autophagic activity promoted cell survival, decreased the mitochondrial oxidative stress level and the apoptosis rate in hair cells, and ameliorated neomycin-induced ototoxicity. CONCLUSIONS: In summary, our findings reveal the otoprotective capacity of exogenous exosome-mediated autophagy activation in hair cells in an endocytosis-dependent manner, suggesting possibilities for deafness treatment.


Assuntos
Exossomos , Neomicina , Neomicina/toxicidade , Neomicina/metabolismo , Exossomos/metabolismo , Células Ciliadas Auditivas , Autofagia/fisiologia
5.
Int J Biol Macromol ; 256(Pt 1): 127779, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37981280

RESUMO

Ligand-RNA interaction assay provides the basis for developing new RNA-binding small molecules. In this study, fluorescent copper nanoclusters (CuNCs) were first prepared using two kinds of HIV-1 RNA targets, rev-responsive element (RRE) and transactivator response element (TAR) RNA, as new templates, and it was found that the fluorescence of the single RNA-templated CuNCs was negligible. Using neomycin as a model drug, the fluorescence could be augmented (approximately 6 times) for the neomycin/RNA-templated CuNCs. Thus, a novel method was developed for ligand-RNA interactions by observing the fluorescence changes in CuNCs prepared using RNA before and after the addition of ligands. The preparation parameters of neomycin/RNA-CuNCs were optimized. The as-prepared CuNCs were characterized using UV-vis spectroscopy, fluorescence spectroscopy, and high-resolution transmission electron microscope. Circular dichroism spectral analysis showed that RRE and TAR were inclined to form a double-stranded structure after interaction with neomycin, which was more conducive to the formation of CuNCs. The interactions of neomycin and three test drugs (amikacin, gentamicin, and tobramycin) with RNA were investigated using the proposed method, and the binding constants and number of binding sites were obtained through theoretical calculations. This study provides a novel approach for ligand-RNA interaction assays.


Assuntos
HIV-1 , Nanopartículas Metálicas , RNA , Fluorescência , HIV-1/genética , Cobre/química , Ligantes , Neomicina , Espectrometria de Fluorescência/métodos , Nanopartículas Metálicas/química , Corantes Fluorescentes/química
6.
Biopreserv Biobank ; 22(1): 21-28, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36656160

RESUMO

Aims: Bacterial contamination may occur in feces during collection and processing of semen. Bacteria not only compete for nutrients with spermatozoa but also produce toxic metabolites and endotoxins and affect sperm quality. The aim of the present study was to investigate the effect of antibiotic supplementation on the sperm quality of Indian red jungle fowl, estimation and isolation of bacterial species and their antibiotic sensitivity. Materials and Methods: Semen was collected and initially evaluated, diluted, and divided into six experimental extenders containing gentamicin (2.5 µg/mL), kanamycin (31.2 µg/mL), neomycin (62.5 mg/mL), penicillin (200 U/mL), and streptomycin (250 µg/mL), and a control having no antibiotics were cryopreserved and semen quality was evaluated at post-dilution, post-cooling, post-equilibration, and post-thawing stages (Experiment 1). A total aerobic bacterial count was carried out after culturing bacteria (Experiment 2) and subcultured for antibiotic sensitivity (Experiment 3). Results: It was shown that penicillin-containing extender improved semen quality (sperm motility, plasma membrane integrity, viability, and acrosomal integrity) compared with the control and other extenders having antibiotics. The bacteria isolated from semen were Escherichia coli, Staphylococcus spp., and Bacillus spp. Antibiotic sensitivity results revealed that E. coli shows high sensitivity toward neomycin, kanamycin, and penicillin. Staphylococcus spp. shows high sensitivity toward streptomycin, neomycin, and penicillin. Bacillus spp. shows high sensitivity toward kanamycin and penicillin. Conclusions: It was concluded that antibiotics added to semen extender did not cause any toxicity and maintained semen quality as that of untreated control samples, and penicillin was identified as most effective antibiotic. It is recommended that penicillin can be added to the semen extender for control of bacterial contamination without affecting the semen quality of Indian red jungle fowl.


Assuntos
Antibacterianos , Preservação do Sêmen , Masculino , Humanos , Antibacterianos/farmacologia , Sêmen/microbiologia , Análise do Sêmen , Escherichia coli , Motilidade dos Espermatozoides , Preservação do Sêmen/métodos , Espermatozoides , Penicilinas/farmacologia , Estreptomicina/farmacologia , Neomicina/farmacologia , Bactérias , Canamicina/farmacologia
7.
Hear Res ; 441: 108916, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38103445

RESUMO

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição , Animais , Cobaias , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Ciliadas Auditivas/patologia , Epitélio/metabolismo , Cóclea/metabolismo , Neomicina
8.
Mikrochim Acta ; 191(1): 34, 2023 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-38108923

RESUMO

Magnetic solid phase extraction with the functionalization of protein onto micro- or nano-particles as a probe is favorable for the discovery of new drugs from complicated natural products. Herein, we aimed to develop a rapid method by immobilizing halogenated alkane dehalogenase (Halo)-tagged calcium-sensing receptor (CaSR) directly out of crude cell lysates onto the surface of magnetic microspheres (MM) with no need to purify protein. Thereby we achieved CaSR-functionalized MM for revealing adsorption characteristics of agonist neomycin and screening ligands from herbal medicine Radix Astragali (RA). About 43.87 mg CaSR could be immobilized per 1 g MM within 30 min, and the acquired CaSR-functionalized MM showed good stability and activity for 4 weeks. The maximum adsorption capacity of neomycin on CaSR-functionalized MM was determined as 4.70 × 10-4 ~ 3.96 × 10-4 mol/g within 277 ~ 310 K, and its adsorption isotherm characteristics described best by the Temkin model were further validated using isothermal titration calorimetry. It was inferred that CaSR's affinity for neomycin was driven by electrostatic forces in a spontaneous process when the system reached an equilibrium state. Moreover, the ligands from the RA extract were screened, three of which were assigned as astragaloside IV, ononin, and calycosin based on HPLC-MS. Our findings demonstrated that the functionalization of a receptor onto magnetic materials designed as an affinity probe has the capability to recognize its agonist and capture the ligands selectively from complex matrices like herbs.


Assuntos
Neomicina , Receptores de Detecção de Cálcio , Microesferas , Adsorção , Ligantes , Fenômenos Magnéticos
9.
Front Immunol ; 14: 1264060, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38130726

RESUMO

Sialic acids are terminal sugars of the cellular glycocalyx and are highly abundant in the nervous tissue. Sialylation is sensed by the innate immune system and acts as an inhibitory immune checkpoint. Aminoglycoside antibiotics such as neomycin have been shown to activate tissue macrophages and induce ototoxicity. In this study, we investigated the systemic subcutaneous application of the human milk oligosaccharide 6'-sialyllactose (6SL) as a potential therapy for neomycin-induced ototoxicity in postnatal mice. Repeated systemic treatment of mice with 6SL ameliorated neomycin-induced hearing loss and attenuated neomycin-triggered macrophage activation in the cochlear spiral ganglion. In addition, 6SL reversed the neomycin-mediated increase in gene transcription of the pro-inflammatory cytokine interleukin-1ß (Il-1b) and the apoptotic/inflammatory kinase Pik3cd in the inner ear. Interestingly, neomycin application also increased the transcription of desialylating enzyme neuraminidase 3 (Neu3) in the inner ear. In vitro, we confirmed that treatment with 6SL had anti-inflammatory, anti-phagocytic, and neuroprotective effects on cultured lipopolysaccharide-challenged human THP1-macrophages. Thus, our data demonstrated that treatment with 6SL has anti-inflammatory and protective effects against neomycin-mediated macrophage activation and ototoxicity.


Assuntos
Neomicina , Ototoxicidade , Camundongos , Animais , Humanos , Neomicina/toxicidade , Antibacterianos/efeitos adversos , Aminoglicosídeos , Anti-Inflamatórios/farmacologia
10.
Neurosci Lett ; 817: 137518, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37844727

RESUMO

In mammals, aminoglycoside antibiotic-induced injury to hair cells (HCs) and associated spiral ganglion neurons (SGNs) is irreversible and eventually leads to permanent hearing loss. Efforts have been directed towards the advancement of efficacious therapeutic treatments to protect hearing loss, but the ideal substance for treating the damaged cochlear sensory epithelium has yet to be identified. Berberine (BBR), a quaternary ammonium hydroxide extracted from Coptis chinensis, has been found to display potential anti-oxidant and neuroprotective properties. However, its involvement in aminoglycoside antibiotic-induced ototoxicity has yet to be explored or assessed. In the present study, we explored the possible anti-oxidative properties of BBR in mitigating neomycin-triggered ototoxicity. An improved survival of HCs and SGN nerve fibers (NFs) in organ of Corti (OC) explants after neomycin with BBR co-treatment was observed, and BBR treatment attenuated reactive oxygen species (ROS) generation and reduced cleaved caspase-3 signaling by activating six phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling relative subtypes, and the addition of PI3K/AKT suppressor LY294002 resulted in a decrease in the protective effect. The protective effect of BBR against ototoxicity was also evident in a neomycin-injured animal model, as evidenced by the preservation of HC and SGN in mice administered subcutaneous BBR for 7 days. In summary, all results suggest that BBR has potential as a new and effective otoprotective agent, operating via the PI3K/AKT signaling pathway.


Assuntos
Berberina , Perda Auditiva , Ototoxicidade , Animais , Camundongos , Antibacterianos/toxicidade , Apoptose , Berberina/farmacologia , Berberina/uso terapêutico , Perda Auditiva/induzido quimicamente , Perda Auditiva/prevenção & controle , Neomicina/toxicidade , Ototoxicidade/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
Appl Environ Microbiol ; 89(10): e0055923, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37787538

RESUMO

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.


Assuntos
Enterite , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Animais , Suínos , Neomicina/farmacologia , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/epidemiologia , Fazendas , Antibacterianos/farmacologia , Plasmídeos/genética , Escherichia coli Enterotoxigênica/genética , Patrimônio Genético , Dinamarca , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana
12.
PeerJ ; 11: e15562, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37701833

RESUMO

Background: Aminoglycoside antibiotics are used for treating certain acute infections. However, these drugs cause ototoxicity by inducing inner ear hair cell death. Aims/Objectives: We investigated the protective effect of a nanoemulsion of the carotenoid astaxanthin on mammalian inner ear hair cells against neomycin-induced ototoxicity. Material and Methods: Dose-response relationship, quantification of hair cell loss, and reactive oxygen species production were assayed in response to neomycin with and without astaxanthin in cultured utricles of CBA/N mice. In addition, auditory brain response (ABR) and hair cell loss after exposure to the nanoformulation and loud noise were examined in vivo in guinea pigs. Results: Astaxanthin suppressed neomycin-induced reduction of hair cells by reducing the production of hydroxy radicals. Furthermore, hair cell loss in the second rotation of the cochlea was significantly lower in the astaxanthin group than in the noise-only group. Conclusions and Significance: The blood-labyrinth barrier limits the successful delivery of drugs for inner ear complications. However, in the nanoemulsion form, astaxanthin can penetrate the round window (fenestra ovale) membrane, enabling topical administration. Thus, astaxanthin nanoemulsion could be useful in treating ototoxicity in individuals with inner ear complications.


Assuntos
Orelha Interna , Ototoxicidade , Camundongos , Animais , Cobaias , Camundongos Endogâmicos CBA , Neomicina , Alopecia , Mamíferos
13.
Pharmacol Rep ; 75(5): 1276-1290, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37704832

RESUMO

BACKGROUND: Human serum albumin (HSA) is a valuable component of non-enzymatic and endogenous antioxidant mechanisms. The antioxidant activity of HSA can be modulated by ligands, including drugs. Although this is a central topic in the field of oxidation, there is still a lack of information about the protection against the effects of elevated free radical levels. METHODS: The aim of this study was to investigate the antioxidant activity of kanamycin (KAN) and neomycin (NEO) and their effect on the antioxidant potential of HSA using spectroscopic and microcalorimetric techniques. RESULTS: Despite the fact that kanamycin and neomycin interact with HSA, no changes in the secondary structure of the protein have been observed. The analysis of the aminoglycoside antibiotics showed their low antioxidant activity and a synergistic effect of the interaction, probably due to the influence of ligands (KAN, NEO) on the availability of HSA amino acid residues functional groups, such as the free thiol group (Cys-34). CONCLUSIONS: Based on the spectroscopic and microcalorimetric data, both KAN and NEO can be considered modulators of the HSA antioxidant activity.


Assuntos
Antioxidantes , Albumina Sérica Humana , Humanos , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Canamicina/farmacologia , Neomicina/farmacologia , Ligação Proteica , Albumina Sérica Humana/metabolismo
14.
BMC Genomics ; 24(1): 577, 2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37759187

RESUMO

BACKGROUND: The mechanism underlying cognitive impairment after hearing loss (HL) remains unclear. N6-methyladenosine (m6A) is involved in many neurodegenerative diseases; however, its role in cognitive impairment after HL has not yet been investigated. Therefore, we aimed to analyze the m6A modification profile of the mouse hippocampus after HL exposure. A mouse model of neomycin-induced HL was established. An auditory brainstem-response test was utilized for detecting hearing threshold. The passive avoidance test was served as the mean for evaluating cognitive function. The m6A-regulated enzyme expression levels were analyzed by using reverse transcription quantitative real-time polymerase chain reaction and western blot analyses. RNA sequencing (RNA-Seq) and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were performed with the aim of investigating gene expression differences and m6A modification in the mouse hippocampus. RESULTS: Neomycin administration induced severe HL in mice. At four months of age, the mice in the HL group showed poorer cognitive performance than the mice in the control group. METTL14, WTAP, and YTHDF2 mRNA levels were downregulated in the hippocampi of HL mice, whereas ALKBH5 and FTO mRNA levels were significantly upregulated. At the protein level, METTL3 and FTO were significantly upregulated. Methylated RNA immunoprecipitation sequencing analysis revealed 387 and 361 m6A hypermethylation and hypomethylation peaks, respectively. Moreover, combined analysis of mRNA expression levels and m6A peaks revealed eight mRNAs with significantly changed expression levels and methylation. CONCLUSIONS: Our findings revealed the m6A transcriptome-wide profile in the hippocampus of HL mice, which may provide a basis for understanding the association between HL and cognitive impairment from the perspective of epigenetic modifications.


Assuntos
Perda Auditiva , Animais , Camundongos , Metilação , Adenosina , Hipocampo , Neomicina , RNA Mensageiro
15.
J Immunol ; 211(10): 1550-1560, 2023 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-37772812

RESUMO

Extrahepatic viral infections are often accompanied by acute hepatitis, as evidenced by elevated serum liver enzymes and intrasinusoidal infiltration of CD8+ T cells, without direct infection of the liver. An example is infectious mononucleosis caused by primary infection with EBV. Previously, we demonstrated that airway infection of mice with murine γ-herpesvirus 68 (MHV68), a murine model of EBV, caused liver inflammation with elevated serum liver enzymes and intrahepatic infiltration of IFN-γ-producing CD8+ T cells and NK cells. Mechanistically, the expression of the CXCR3-ligand chemokines, which are commonly induced by IFN-γ and attract IFN-γ-producing Th1-type cells via CXCR3, was upregulated in the liver. Importantly, the liver inflammation was suppressed by oral neomycin, an intestine-impermeable aminoglycoside, suggesting an involvement of some products from the intestinal microbiota. In this study, we showed that the liver inflammation and the expression of the CXCR3-ligand chemokines in the liver were effectively ameliorated by i.p. administration of anti-TLR4 mAb or C34, a TLR4 blocker, as well as in TLR4-deficient mice. Conversely, intrarectal inoculation of Escherichia coli as an extraintestinal source of LPS aggravated liver inflammation in MHV68-infected mice with increased expression of the CXCR3-ligand chemokines in the liver. In contrast, the lung inflammation in MHV68-infected mice was not affected by oral neomycin, i.p. administration of C34, or TLR4 deficiency. Collectively, the LPS-TLR4 pathway plays a pivotal role in the liver inflammation of MHV68-infected mice at least in part by upregulating the CXCR3-ligand chemokines in the liver.


Assuntos
Hepatite , Hepatopatias , Animais , Camundongos , Quimiocinas/metabolismo , Inflamação , Ligantes , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neomicina , Receptores CXCR3/metabolismo , Receptor 4 Toll-Like
16.
Biochemistry (Mosc) ; 88(6): 723-730, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37748869

RESUMO

Investigation of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, we have identified and biochemically characterized three aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B in the strain Streptomyces rimosus ATCC 10970 (producing oxytetracycline), which is resistant to most natural aminoglycoside antibiotics. In the presented work, it was shown that the resistance of this strain to other AGs is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2') subfamily. Induction of the expression of the gene, designated by us as aac(2')-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and increases minimum inhibitory concentrations of these antibiotics.


Assuntos
Streptomyces rimosus , Paromomicina , Antibacterianos/farmacologia , Aminoglicosídeos/farmacologia , Neomicina , Escherichia coli
17.
Int J Colorectal Dis ; 38(1): 210, 2023 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-37555867

RESUMO

PURPOSE: Surgical site infections (SSIs) are common in colorectal surgery. Mechanical bowel preparation (MBP) in conjunction with oral antibiotics (OABs) have been shown to reduce SSI rates. It however is still unclear which OABs to use, and how this can be implemented in practice. METHODS: This is a prospective observational study carried out in Swansea Bay University Health Board during 2019-2021, evaluating the introduction of OABs in a stepwise manner on the incidence of SSI in major colorectal surgery. A control group having MBP only was compared to two OAB groups: one group had MBP plus metronidazole only and the second MBP plus metronidazole and neomycin. A 30-day follow-up after surgery was ascertained via chart review and telephone contact. Logistic regression was performed to estimate the relation between OAB use and SSI, with adjustment for confounding. In a subset of patients, faecal samples were analysed through 16S rRNA amplicon sequencing before and after OAB treatment, depicting the impact of the gut microbiome. RESULTS: In total 160 patients were analysed: 46 patients had MBP only, whilst 76 patients had MBP plus metronidazole only and 38 patients had MBP with metronidazole/neomycin. The SSI rate in the entire cohort was 33.8%, whilst the adjusted ORs for the single- and dual-OAB groups were 0.76 (95% CI: 0.17-1.81) and 0.50 (95% CI: 0.17-1.52). The microbial analysis demonstrated that the relative abundance for many bacterial genera was changed before and after OAB treatment, but no link with SSI development could be shown. CONCLUSIONS: Introduction of OABs in conjunction with MBP in colorectal surgery is feasible, and may potentially lead to lower rates of SSI, as well as altering the community structure of the faecal microbiome. More research is needed, especially considering different OABs and mechanistic studies of the gut microbiome in the context of colorectal surgery.


Assuntos
Antibacterianos , Cirurgia Colorretal , Humanos , Antibacterianos/uso terapêutico , Infecção da Ferida Cirúrgica/etiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Infecção da Ferida Cirúrgica/tratamento farmacológico , Metronidazol/uso terapêutico , Antibioticoprofilaxia , RNA Ribossômico 16S , Neomicina/uso terapêutico , Cuidados Pré-Operatórios/efeitos adversos , Procedimentos Cirúrgicos Eletivos/efeitos adversos , Administração Oral , Catárticos/uso terapêutico
18.
J Am Chem Soc ; 145(28): 15284-15294, 2023 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-37420313

RESUMO

Understanding how ligands bind to ribonucleic acids (RNA) is important for understanding RNA recognition in biological processes and drug development. Here, we have studied neomycin B binding to neomycin-sensing riboswitch aptamer constructs by native top-down mass spectrometry (MS) using electrospray ionization (ESI) and collisionally activated dissociation (CAD). Our MS data for a 27 nt aptamer construct reveal the binding site and ligand interactions, in excellent agreement with the structure derived from nuclear magnetic resonance (NMR) studies. Strikingly, for an extended 40 nt aptamer construct, which represents the sequence with the highest regulatory factor for riboswitch function, we identified two binding motifs for neomycin B binding, one corresponding to the bulge-loop motif of the 27 nt construct and the other one in the minor groove of the lower stem, which according to the MS data are equally populated. By replacing a noncanonical with a canonical base pair in the lower stem of the 40 nt aptamer, we can reduce binding to the minor groove motif from ∼50 to ∼30%. Conversely, the introduction of a CUG/CUG motif in the lower stem shifts the binding equilibrium in favor of minor groove binding. The MS data reveal site-specific and stoichiometry-resolved information on aminoglycoside binding to RNA that is not directly accessible by other methods and underscore the role of noncanonical base pairs in RNA recognition by aminoglycosides.


Assuntos
Neomicina , Riboswitch , Framicetina , Antibacterianos/metabolismo , Aminoglicosídeos , RNA , Espectrometria de Massas , Sítios de Ligação , Conformação de Ácido Nucleico , Ligantes
19.
Neurobiol Dis ; 183: 106176, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37263384

RESUMO

Aminoglycoside antibiotics (AGAs) are widely used in life-threatening infections, but they accumulate in cochlear hair cells (HCs) and result in hearing loss. Increases in adenosine triphosphate (ATP) concentrations and P2X7 receptor expression were observed after neomycin treatment. Here, we demonstrated that P2X7 receptor, which is a non-selective cation channel that is activated by high ATP concentrations, may participate in the process through which AGAs enter hair cells. Using transgenic knockout mice, we found that P2X7 receptor deficiency protects HCs against neomycin-induced injury in vitro and in vivo. Subsequently, we used fluorescent gentamicin-Fluor 594 to study the uptake of AGAs and found fluorescence labeling in wild-type mice but not in P2rx7-/- mice in vitro. In addition, knocking-out P2rx7 did not significantly alter the HC count and auditory signal transduction, but it did inhibit mitochondria-dependent oxidative stress and apoptosis in the cochlea after neomycin exposure. We thus conclude that the P2X7 receptor may be linked to the entry of AGAs into HCs and is likely to be a therapeutic target for auditory HC protection.


Assuntos
Aminoglicosídeos , Ototoxicidade , Animais , Camundongos , Aminoglicosídeos/toxicidade , Aminoglicosídeos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Ototoxicidade/metabolismo , Antibacterianos/toxicidade , Neomicina/toxicidade , Neomicina/metabolismo , Células Ciliadas Auditivas/metabolismo , Cóclea , Trifosfato de Adenosina/metabolismo
20.
Biochemistry ; 62(11): 1755-1766, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37172221

RESUMO

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, G4C4, in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G4C4, with thermal enhancement decreasing with increasing pH and/or Na+ concentration. Although G4C4 bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to G4C4 is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na+ concentration results in a decrease in G4C4 affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH3+ groups participating in electrostatic interactions with G4C4. These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.


Assuntos
Aminoglicosídeos , Neomicina , Neomicina/química , Neomicina/metabolismo , Aminoglicosídeos/metabolismo , Antibacterianos/química , DNA/química , Termodinâmica , Conformação de Ácido Nucleico , Sítios de Ligação
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