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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.

Paenibacillus polymyxa , a Gram-positive bacterium commonly found in soil and plant roots, plays an important role in the environment due to its nitrogen-fixing ability and is renowned for producing antibiotics like polymyxin. In this study, we present a robust framework for investigating the evolutionary and taxonomic connections of strains belonging to P. polymyxa available at the National Center for Biotechnology Information, as well as five new additional strains isolated at the University of Camerino (Italy), through pangenome analysis. These strains can produce secondary metabolites active against Staphylococcus aureus and Klebsiella pneumoniae . Employing techniques such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) estimation, OrthoFinder, and ribosomal multilocus sequence typing, we consistently divided these P. polymyxa strains into four clusters, which differ significantly in terms of ANI and dDDH percentages, both considered as reference indices for separating bacterial species. Moreover, the strains of Cluster 2 were re-classified as belonging to the Paenibacillus ottowii species. By comparing the pangenomes, we identified the core genes of each cluster and analyzed them to recognize distinctive features in terms of biosynthetic/metabolic potential. The comparison of pangenomes also allowed us to pinpoint differences between clusters in terms of genetic variability and the percentage of the genome dedicated to core and accessory genes. In conclusion, the data obtained from our analyses of strains belonging to the P. polymyxa species converge toward a necessary reclassification, which will require a fundamental contribution from microbiologists in the near future., Importance: The development of sequencing technologies has led to an exponential increase in microbial sequencing data. Accurately identifying bacterial species remains a challenge because of extensive intra-species variability, the need for multiple identification methods, and the rapid rate of taxonomic changes. A substantial contribution to elucidating the relationships among related bacterial strains comes from comparing their genomic sequences. This comparison also allows for the identification of the "pangenome," which is the set of genes shared by all individuals of a species, as well as the set of genes that are unique to subpopulations. Here, we applied this approach to Paenibacillus polymyxa , a species studied for its potential as a biofertilizer and biocontrol agent and known as an antibiotic producer. Our work highlights the need for a more efficient classification of this bacterial species and provides a better delineation of strains with different properties., Competing Interests: The authors declare no conflict of interest.

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 GFP1-10 fast . We detail the expression and purification of the GFP1-10 fast 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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

The antimicrobial peptide LR GG (LLRLLRRGGRRLLRLL-NH2) was designed and chemically synthesized in a study conducted by Jia et al. Gram-negative bacteria were found to be sensitive to LR GG and exhibited a high therapeutic index. Genetic engineering methods were used to create the prokaryotic fusion expression vector pQE-GFP-LR GG , and the resulting corresponding fusion protein GFP-LR GG was subsequently expressed and purified. The precursor GFP was then removed by TEV proteolysis, and pure LR GG was obtained after another round of purification and endotoxin removal. The prokaryotic-expressed antimicrobial peptide LR GG displays a broad-spectrum antibacterial effect on Gram-negative bacteria, and its minimum inhibitory activity (MIC) against Escherichia coli can reach 2 μg/mL. Compared to the chemically synthesized LR GG, the prokaryotic-expressed LR GG exhibits similar temperature, pH, salt ion, serum stability, and cell selectivity. Furthermore, prokaryotic-expressed LR GG showed excellent therapeutic effects in both the infection model of cell selectivity and no embryotoxicity in a Galleria mellonella infection model. The mechanism by which LR GG causes bacterial death was found to be the disruption of the Gram-negative cell membrane.