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The authors describe a colorimetric method for determination of kanamycin by using gold nanoparticles (AuNPs) as the element of signal-conversion and by applying hybridization chain reaction-assisted signal amplification. The assay is carried out by monitoring the absorbance change and color change adding salt to the reaction solution containing kanamycin (analyte), hairpin DNA probe, and AuNPs. Three hairpin DNA probes with sticky ends were absorbed on the AuNPs via their sticky ends. Cating with DNA prevents them from salt-induced aggregation (which leads to a color change from red to blue) in the complete absence of kanamycin. In contrast, in the presence of kanamycin, the aptamer hairpin DNA probe binds kanamycin, and the newly exposed section of DNA triggers a cascade of hybridization chain reactions with formation of numerous dsDNAs. On addition of salt, the AuNPs form blue aggregates due to the repulsion between dsDNA and AuNPs. Under optimal conditions, the ration of absorbance at 520 and 630 nm drops with the kanamycin concentration in the range from 1 to 40 μM, and the limit of detection is 0.68 μM. The assay can selectively distinguish kanamycin from other antibiotics. The method was applied to kanamycin detection in (spiked) milk samples and gave excellent recoveries. Graphical abstract Schematic presentation of colorimetric method for kanamycin detection using gold nanoparticles modified with hairpin DNA probes and hybridization chain reaction-assisted amplification.

The WHO End TB Strategy requires drug susceptibility testing and treatment of all people with tuberculosis, but second-line diagnostic testing with line-probe assays needs to be done in experienced laboratories with advanced infrastructure. Fewer than half of people with drug-resistant tuberculosis receive appropriate treatment. We assessed the diagnostic accuracy of the rapid Xpert MTB/XDR automated molecular assay (Cepheid, Sunnyvale, CA, USA) to overcome these limitations. We did a prospective study involving individuals presenting with pulmonary tuberculosis symptoms and at least one risk factor for drug resistance in four sites in India (New Delhi and Mumbai), Moldova, and South Africa between July 31, 2019, and March 21, 2020. The Xpert MTB/XDR assay was used as a reflex test to detect resistance to isoniazid, fluoroquinolones, ethionamide, amikacin, kanamycin, and capreomycin in adults with positive results for Mycobacterium tuberculosis complex on Xpert MTB/RIF or Ultra (Cepheid). Diagnostic performance was assessed against a composite reference standard of phenotypic drug-susceptibility testing and whole-genome sequencing. This study is registered with ClinicalTrials.gov, number NCT03728725. Of 710 participants, 611 (86%) had results from both Xpert MTB/XDR and the reference standard for any drug and were included in analysis. Sensitivity for Xpert MTB/XDR detection of resistance was 94% (460 of 488, 95% CI 92–96) for isoniazid, 94% (222 of 235, 90–96%) for fluoroquinolones, 54% (178 of 328, 50–61) for ethionamide, 73% (60 of 82, 62–81) for amikacin, 86% (181 of 210, 81–91) for kanamycin, and 61% (53 of 87, 49–70) for capreomycin. Specificity was 98–100% for all drugs. Performance was equivalent to that of line-probe assays. The non-determinate rate of Xpert MTB/XDR (ie, invalid M tuberculosis complex detection) was 2·96%. The Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

Neomycin phosphotransferase II ( nptII ) is a selectable marker gene that is commonly used in plant molecular genetics and crop improvement, helping researchers to identify and select transgenically modified plants. The NPTII enzyme binds to and phosphorylates the aminoglycoside family of antibiotics, which are known translation inhibitors. Once the aminoglycoside is phosphorylated it is unable to bind to the ribosome and can no longer disrupt translation. Currently, the most widely used selection agent for screening NPTII expressing seedlings is kanamycin. Because the nptII transgene is frequently silenced epigenetically, kanamycin can be too toxic to seedlings that weakly express NPTII, leading to false negatives and making it harder to accurately identify transgenic plants. In this study we investigate the related aminoglycoside, paromomycin, as an alternative, non-lethal, selection agent to kanamycin across a series of transgenic Arabidopsis lines with varying NPTII expression. We investigated phenotypes of transgenic seedlings during and after antibiotic exposure. Seedling pigmentation and size are useful phenotypes for selecting seedlings with low nptII expression. Additionally, monitoring the photosynthetic efficiency and flowering time can help reduce the risk of false-positive results when treating seedlings with paromomycin.

Highly photoactive 3D nanoflower-like FeIn.sub.2S.sub.4/CdS heterostructures were synthesized by hydrothermal treatment and low-temperature cation exchange. The FeIn.sub.2S.sub.4/CdS displayed 14.5 times signal amplification in contrast to FeIn.sub.2S.sub.4 alone. It was applied as a photoactive substrate to construct a label-free photoelectrochemical (PEC) aptasensor for ultrasensitive determination of kanamycin (KAN). Under the optimal conditions, the constructed PEC aptasensor displayed a wide linear range (5.0 x 10.sup.-4 ~ 5.0 x 10.sup.1 ng mL.sup.-1) and a low detection limit (S/N = 3) of 40.01 fg mL.sup.-1. This study provides some constructive insights for preparation of advanced photoactive materials and exhibits great potential for quantitative determination of antibiotics in foods and environmental samples. Graphical

Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N 1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties. Here the authors use fast kinetics, X-ray crystallography, and cryo-EM to uncover the mechanism of ribosome inhibition by amikacin and kanamycin. They find that amikacin binds near the P-site tRNA, offering new strategies to fight antibiotic resistance.

The sites of mycobacterial infection in the lungs of tuberculosis (TB) patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations, resulting in compromised TB treatment response and resistance development. Quantifying lesion-specific drug uptake and pharmacokinetics (PKs) in TB patients is necessary to optimize treatment regimens at all infection sites, to identify patients at risk, to improve existing regimens, and to advance development of novel regimens. Using drug-level data in plasma and from 9 distinct pulmonary lesion types (vascular, avascular, and mixed) obtained from 15 hard-to-treat TB patients who failed TB treatments and therefore underwent lung resection surgery, we quantified the distribution and the penetration of 7 major TB drugs at these sites, and we provide novel tools for treatment optimization. A total of 329 plasma- and 1,362 tissue-specific drug concentrations from 9 distinct lung lesion types were obtained according to optimal PK sampling schema from 15 patients (10 men, 5 women, aged 23 to 58) undergoing lung resection surgery (clinical study NCT00816426 performed in South Korea between 9 June 2010 and 24 June 2014). Seven major TB drugs (rifampin [RIF], isoniazid [INH], linezolid [LZD], moxifloxacin [MFX], clofazimine [CFZ], pyrazinamide [PZA], and kanamycin [KAN]) were quantified. We developed and evaluated a site-of-action mechanistic PK model using nonlinear mixed effects methodology. We quantified population- and patient-specific lesion/plasma ratios (RPLs), dynamics, and variability of drug uptake into each lesion for each drug. CFZ and MFX had higher drug exposures in lesions compared to plasma (median RPL 2.37, range across lesions 1.26-22.03); RIF, PZA, and LZD showed moderate yet suboptimal lesion penetration (median RPL 0.61, range 0.21-2.4), while INH and KAN showed poor tissue penetration (median RPL 0.4, range 0.03-0.73). Stochastic PK/pharmacodynamic (PD) simulations were carried out to evaluate current regimen combinations and dosing guidelines in distinct patient strata. Patients receiving standard doses of RIF and INH, who are of the lower range of exposure distribution, spent substantial periods (>12 h/d) below effective concentrations in hard-to-treat lesions, such as caseous lesions and cavities. Standard doses of INH (300 mg) and KAN (1,000 mg) did not reach therapeutic thresholds in most lesions for a majority of the population. Drugs and doses that did reach target exposure in most subjects include 400 mg MFX and 100 mg CFZ. Patients with cavitary lesions, irrespective of drug choice, have an increased likelihood of subtherapeutic concentrations, leading to a higher risk of resistance acquisition while on treatment. A limitation of this study was the small sample size of 15 patients, performed in a unique study population of TB patients who failed treatment and underwent lung resection surgery. These results still need further exploration and validation in larger and more diverse cohorts. Our results suggest that the ability to reach and maintain therapeutic concentrations is both lesion and drug specific, indicating that stratifying patients based on disease extent, lesion types, and individual drug-susceptibility profiles may eventually be useful for guiding the selection of patient-tailored drug regimens and may lead to improved TB treatment outcomes. We provide a web-based tool to further explore this model and results at http://saviclab.org/tb-lesion/.

Efficient selection of transgenic plants is essential in plant biotechnology, and development of effective selectable marker systems plays a crucial role, especially as multiple rounds of transformation and gene stacking may require multiple selectable markers. The present study aims to develop and evaluate an enhanced spectinomycin resistance construct to provide robust selection for transformation events. The new construct was compared in multiple plant species to the standard spectinomycin selectable marker gene currently used for soybean transformation and to the neomycin phosphotransferase ( nptII ) gene that confers kanamycin resistance. In Arabidopsis thaliana , potato, and citrus, the transformation efficiency provided by the enhanced spectinomycin marker was approximately equal to that of kanamycin and considerably better than that observed for the standard spectinomycin marker. In soybean, the enhanced spectinomycin resistance marker preformed as well as the standard spectinomycin gene. These results demonstrate that the enhanced spectinomycin construct provides strong resistance, comparable to kanamycin in multiple species, offering an alternative tool for transgenic plant selection.

Eis (Enhanced intracellular survival) protein is an aminoglycoside acetyltransferase enzyme classified under the family – GNAT (GCN5-related family of N-acetyltransferases) secreted by Mycobacterium tuberculosis (Mtb). The enzymatic activity of Eis results in the acetylation of kanamycin, thereby impairing the drug’s action. In this study, we expressed and purified recombinant Eis (rEis) to determine the enzymatic activity of Eis and its potential inhibitor. Glide-enhanced precision docking was used to perform molecular docking with chosen ligands. Quercetin was found to interact Eis with a maximum binding affinity of -8.379 kcal/mol as compared to other ligands. Quercetin shows a specific interaction between the positively charged amino acid arginine in Eis and the aromatic ring of quercetin through π-cation interaction. Further, the effect of rEis was studied on the antibiotic activity of kanamycin A in the presence and absence of quercetin. It was observed that the activity of rEis aminoglycoside acetyltransferase decreased with increasing quercetin concentration. The results from the disk diffusion assay confirmed that increasing the concentration of quercetin inhibits the rEis protein activity. In conclusion, quercetin may act as a potential Eis inhibitor.

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.