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Summary: Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors.For complete details on the use and execution of this protocol, please refer to Pham et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments—specifically, the GFP1-10 and GFP11 fragments—we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.

IntroductionThe continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action.MethodsUtilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin.ResultsA proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2.DiscussionOur study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

Abstract Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare disease, characterised by the aplasia of vagina and uterus in women with a 46,XX karyotype. Most cases are sporadic, but familial recurrence has also been described. Herein, we investigated an Italian cohort of 36 unrelated MRKH patients to explore the presence of pathogenic copy number variations (CNVs) by array-CGH and MLPA assays. On the whole, aberrations were found in 9/36 (25%) patients. Interestingly, one patient showed a novel heterozygous microduplication at Xp22.33, not yet described in MRKH patients, containing the PRKX gene. Moreover, a novel duplication of a specific SHOX enhancer was highlighted by MLPA. To predict the potential significance of CNVs in MRKH pathogenesis, we provided a network analysis for protein-coding genes found in the altered genomic regions. Although not all of these genes taken individually showed a clear clinical significance, their combination in a computational network highlighted that the most relevant biological connections are related to the anatomical structure development. In conclusion, the results described in the present study identified novel genetic alterations and interactions that may be likely involved in MRKH phenotype determination, so adding new insights into the complex puzzle of MRKH disease.

The increasing amounts of municipal solid waste and their management in landfills caused an increase in the production of leachate, a liquid formed by the percolation of rainwater through the waste. Leachate creates serious problems to municipal wastewater treatment plants; indeed, its high levels of ammonia are toxic for bacterial cells and drastically reduce the biological removal of nitrogen by activated sludge. In the present work, we studied, using a metagenomic approach based on next-generation sequencing (NGS), the microbial composition of sludge in the municipal wastewater treatment plant of Porto Sant’Elpidio (Italy). Through activated sludge enrichment experiments based on the Repetitive Re-Inoculum Assay, we were able to select and identify a minimal bacterial community capable of degrading high concentrations of ammonium (NH4+-N ≅ 350 mg/L) present in a leachate-based medium. The analysis of NGS data suggests that seven families of bacteria (Alcaligenaceae, Nitrosomonadaceae, Caulobacteraceae, Xanthomonadaceae, Rhodanobacteraceae, Comamonadaceae and Chitinophagaceae) are mainly responsible for ammonia oxidation. Furthermore, we isolated from the enriched sludge three genera (Klebsiella sp., Castellaniella sp. and Acinetobacter sp.) capable of heterotrophic nitrification coupled with aerobic denitrification. These bacteria released a trace amount of both nitrite and nitrate possibly transforming ammonia into gaseous nitrogen. Our findings represent the starting point to produce an optimized microorganisms’s mixture for the biological removal of ammonia contained in leachate.

Neurite atrophy with loss of neuronal polarity is a pathological hallmark of Alzheimer’s disease (AD) and other neurological disorders. While there is substantial agreement that disruption of intracellular vesicle trafficking is associated with axonal pathology in AD, comparatively less is known regarding its role in dendritic atrophy. This is a significant gap of knowledge because, unlike axons, dendrites are endowed with the complete endomembrane system comprising endoplasmic reticulum (ER), ER–Golgi intermediate compartment (ERGIC), Golgi apparatus, post-Golgi vesicles, and a recycling-degradative route. In this study, using live-imaging of pGOLT-expressing vesicles, indicative of Golgi outposts and satellites, we investigate how amyloid-β (Aβ) oligomers affect the trafficking of Golgi-like organelles in the different dendritic compartments of cultured rat hippocampal neurons. We found that short-term (4 h) treatment with Aβ led to a decrease in anterograde trafficking of Golgi vesicles in dendrites of both resting and stimulated (with 50 mM KCl) neurons. We also characterized the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi dynamics in dendrites. Mirtazapine treatment (10 μM) increased the number and both anterograde and retrograde motility, reducing the percentage of static Golgi vesicles. Finally, mirtazapine reverted the neurite atrophy induced by 24 h treatment with Aβ oligomers, suggesting that this drug is able to counteract the effects of Aβ by improving the dendritic trafficking of Golgi-related vesicles.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Two subclasses of ligand-gated ion channels (ASIC3 and P2X3) are both present at sensory neurons and might be therefore subject to receptor crosstalk. Here authors use electrophysiology, biochemistry and co-immunoprecipitation to show that the two ion channels interact and affect P2X3 currents.

ABSTRACT Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain. IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers’ microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic’s target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli, thereby eliciting antibiotic resistance in this human commensal bacterium.

This article investigates the use of translation notes to deal with translation problems. In Translation Studies, the presence of translation notes in a translation is considered particularly significant because they clearly indicate what features of the source text the translator considered important for the comprehension of the text and therefore necessary to retain or explain. In the field of comics in translation, the use of T/N is rather uncommon, and can be considered the main translation strategy that distinguishes scanlation from other types of translations. In the first part of this article, the structure of the English-language manga scanlation communities is examined; following this, the way culture-specific items are dealt with by manga scanlators is analysed; and finally, an explanatory hypothesis linking the broader structure of participation to individual translation strategies is presented. The argument put forward in this article is that translation notes are used in scanlation both to solve translation problems and as a way for scanlators to communicate directly with their readers, thereby foregrounding their mediating presence directly on the pages of scanlated manga.