The influence of in situ synthesis parameters on the formation of ZnO nanoparticles and the UPF value of cotton fabric

The aim of this research was to investigate different parameters of the in situ synthesis of ZnO nanoparticles on cotton in order to achieve a high ultraviolet protection factor (UPF). In the fi rst part of the research the influence of different reducing agents (Na2CO3, KOH, and NaOH) and their molar concentrations (0.1 M and 1 M) on the formation of ZnO nanoparticles and on the UPF values of cotton fabric were studied. The second part of the research was focused on the other parameters of in situ synthesis, such as the synthesis time ratio (time ratio between the treatment of the fabric in the precursor (ZnCl2) and treatment after the reducing agent was added) and drying period duration after the in situ synthesis. Using UV/Vis spectroscopy, high UPF values (UPF 50+) were measured for cotton fabrics where in situ synthesis was performed using NaOH and KOH, both at 1 M molar concentration. Inductively coupled plasma mass spectrometry (ICP-MS) revealed a higher content of zinc on the fabric when NaOH was used. Scanning electron microscopy (SEM) showed that use of this reducing agent resulted in cotton fabric completely covered with small, round shaped nanoparticles. From the second part of the research, it was found that longer treatment times after the reducing agent was added produced functionalised cotton fabric with higher UPF values. The drying period duration after in situ synthesis did not signifi cantly affect the UPF value of the fabric, but it did influence the morphology of the synthesised nanoparticles. With a longer drying time the nanoparticles were more rounded. The samples had poor wash fastness even after the fi rst wash, which was found through low UPF values

Electrochemical Stability and Degradation of Commercial Pd/C Catalyst in Acidic Media

Palladium has attracted significant attention as a catalyst or co-catalyst for many electrochemical reactions in energy conversion devices. We have studied electrochemical stability of a commercial Pd/C sample in an acidic electrolyte by exposing it to an accelerated stress test (AST) to mimic potential spikes in fuel cells and electrolyzers during start/stop events. AST consisted of extensive rapid potential cycling (5000 cycles, 1 V/s) in two potential regions, namely AST1 was performed between 0.4 and 1.4 VRHE, while AST2 was performed between 0.05 and 1.4 VRHE. Degradation of Pd/C was monitored by the changes in Pd electrochemical surface area, while the hydrogen evolution reaction (HER) was used as a test reaction to observe the corresponding impact of the degradation on the activity of Pd/C. Significant Pd/C degradation and HER activity loss were observed in both potential regions. Coupling of the electrochemical flow cell with an inductively coupled plasma mass spectrometry device showed substantial Pd dissolution during both ASTs. Identical location scanning electron microscopy revealed that Pd dissolution is followed by redeposition during both ASTs, resulting in particle size growth. Particle size growth was seen as especially dramatic in the case of AST2, when particularly large Pd nanostructures were obtained on top of the catalyst layer. According to the results presented in this work, (in)stability of Pd/C and other Pd-based nanocatalysts should be studied systematically as it may present a key factor limiting their application in energy conversion devices

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value

Reference-grade genome and large linear plasmid of Streptomyces rimosus: pushing the limits of Nanopore sequencing

[EN] Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtainedSIThis work was supported as part of the European project “Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients” (grant ID 720793, European Union’s Horizon 2020 Research and Innovation Program) and by the Slovenian Research Agency (P4-0116, P4-0077, and P1-0034). L.S. is supported by a Slovenian Research Agency young researcher grant (35220200570), and M.T. is supported by grant C3330-19-952047 funded by Republic of Slovenia Ministry of Education, Science, and Sport and the European Union European Regional Development Fun

Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus : Pushing the Limits of Nanopore Sequencing

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters