Phenotypic and transcriptomic acclimation of the green microalga Raphidocelis subcapitata to high environmental levels of the herbicide diflufenican.

Herbicide pollution poses a worldwide threat to plants and freshwater ecosystems. However, the understanding of how organisms develop tolerance to these chemicals and the associated trade-off expenses are largely unknown. This study aims to investigate the physiological and transcriptional mechanisms underlying the acclimation of the green microalgal model species Raphidocelis subcapitata (Selenastraceae) towards the herbicide diflufenican, and the fitness costs associated with tolerance development. Algae were exposed for 12 weeks (corresponding to 100 generations) to diflufenican at the two environmental concentrations 10 and 310 ng/L. The monitoring of growth, pigment composition, and photosynthetic performance throughout the experiment revealed an initial dose-dependent stress phase (week 1) with an EC50 of 397 ng/L, followed by a time-dependent recovery phase during weeks 2 to 4. After week 4, R. subcapitata was acclimated to diflufenican exposure with a similar growth rate, content of carotenoids, and photosynthetic performance as the unexposed control algae. This acclimation state of the algae was explored in terms of tolerance acquisition, changes in the fatty acids composition, diflufenican removal rate, cell size, and changes in mRNA gene expression profile, revealing potential fitness costs associated with acclimation, such as up-regulation of genes related to cell division, structure, morphology, and reduction of cell size. Overall, this study demonstrates that R. subcapitata can quickly acclimate to environmental but toxic levels of diflufenican; however, the acclimation is associated with trade-off expenses that result in smaller cell size.

Healthcare organization policy recommendations for the governance of surgical innovation: review of NHS policies.

BACKGROUND: The governance for introducing innovative surgical procedures/devices differs from the research requirements needed for new drugs. New invasive procedures/devices may be offered to patients outside of research protocols with local organization oversight alone. Such institutional arrangements exist in many countries and written policies provide guidance for their use, but little is known about their scope or standards. METHODS: One hundred and fifty acute NHS trusts in England and seven health boards in Wales were systematically approached for information about their policies. A modified framework approach was used to analyse when policies considered new procedures/devices to be within local organization remit and/or requiring research ethics committee (REC) approval. RESULTS: Of 113 policies obtained, 109 and 34 described when local organization and REC approval was required, respectively. Procedures/devices being used for the first time in the organization (n = 69) or by a clinician (n = 67) were commonly within local remit, and only 36 stated that evidence was required. Others stated limited evidence as a rationale for needing REC approval (n = 13). External guidance categorizing procedures as 'research only' was the most common reason for gaining REC approval (n = 15). Procedures/devices with uncertain outcomes (n = 28), requiring additional training (n = 26), and not previously used (n = 6) were within the remit of policies, while others recommended REC application in these situations (n = 5, 2 and 7, respectively). CONCLUSION: This study on NHS policies for surgical innovation shows variability in the introduction of procedures/devices in terms of local oversight and/or need for REC approval. Current NHS standards allow untested innovations to occur without the safety of research oversight and thus a standard approach is urgently needed.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Phenomics reveals a novel putative chloroplast fatty acid transporter in the marine diatom Skeletonema marinoi involved in temperature acclimation.

Diatoms are the dominant phytoplankton in temperate oceans and coastal regions and yet little is known about the genetic basis underpinning their global success. Here, we address this challenge by developing the first phenomic approach for a diatom, screening a collection of randomly mutagenized but identifiably tagged transformants. Based upon their tolerance to temperature extremes, several compromised mutants were identified revealing genes either stress related or encoding hypothetical proteins of unknown function. We reveal one of these hypothetical proteins is a novel putative chloroplast fatty acid transporter whose loss affects several fatty acids including the two omega-3, long-chain polyunsaturated fatty acids - eicosapentaenoic and docosahexaenoic acid, both of which have medical importance as dietary supplements and industrial significance in aquaculture and biofuels. This mutant phenotype not only provides new insights into the fatty acid biosynthetic pathways in diatoms but also highlights the future value of phenomics for revealing specific gene functions in these ecologically important phytoplankton.

Whole Genome Sequence of Marinobacter salarius Strain SMR5, Shown to Promote Growth in its Diatom Host.

Attempts to obtain axenic cultures of the marine diatom Skeletonema marinoi often result in poor growth, indicating the importance of the microbiome to the growth of its host. In order to identify the precise roles played by these associated bacteria, individual strains were isolated, cultured and sequenced. We report the genome of one such strain - SMR5, isolated from a culture of S. marinoi strain R05AC sampled from top layer sediments of the Swedish west coast. Its genome of 4,630,160 bp consists of a circular chromosome and one circular plasmid, and 4,263 CDSs were inferred in the annotation. Comparison of 16S rRNA sequences and other markers, along with phylotaxonomic analysis, leads us to place strain SMR5 in the taxon Marinobacter salarius. Pathway analysis and previous experimental work suggest that this strain may produce a growth factor, as well as improve iron availability for its host via siderophores.

Friends With Benefits: Exploring the Phycosphere of the Marine Diatom Skeletonema marinoi.

Marine diatoms are the dominant phytoplankton in the temperate oceans and coastal regions, contributing to global photosynthesis, biogeochemical cycling of key nutrients and minerals and aquatic food chains. Integral to the success of marine diatoms is a diverse array of bacterial species that closely interact within the diffusive boundary layer, or phycosphere, surrounding the diatom partner. Recently, we isolated seven distinct bacterial species from cultures of Skeletonema marinoi, a chain-forming, centric diatom that dominates the coastal regions of the temperate oceans. Genomes of all seven bacteria were sequenced revealing many unusual characteristics such as the existence of numerous plasmids of widely varying sizes. Here we have investigated the characteristics of the bacterial interactions with S. marinoi, demonstrating that several strains (Arenibacter algicola strain SMS7, Marinobacter salarius strain SMR5, Sphingorhabdus flavimaris strain SMR4y, Sulfitobacter pseudonitzschiae strain SMR1, Yoonia vestfoldensis strain SMR4r and Roseovarius mucosus strain SMR3) stimulate growth of the diatom partner. Testing of many different environmental factors including low iron concentration, high and low temperatures, and chemical signals showed variable effects on this growth enhancement by each bacterial species, with the most significant being light quality in which green and blue but not red light enhanced the stimulatory effect on S. marinoi growth by all bacteria. Several of the bacteria also inhibited growth of one or more of the other bacterial strains to different extents when mixed together. This study highlights the complex interactions between diatoms and their associated bacteria within the phycosphere, and that further studies are needed to resolve the underlying mechanisms for these relationships and how they might influence the global success of marine diatoms.

Complete Genome Sequence of the Diatom-Associated Bacterium Sphingorhabdus sp. Strain SMR4y.

The bacterial strain SMR4y belongs to the diverse microbiome of the marine diatom Skeletonema marinoi strain R05AC. After assembly of its genome, presented here, and subsequent analyses, we placed it in the genus Sphingorhabdus This strain has a 3,479,724-bp circular chromosome (with 3,340 coding sequences) and no known plasmids.

Genome Sequence of Kordia sp. Strain SMS9 Identified in a Non-Axenic Culture of the Diatom Skeletonema marinoi.

Initial efforts to sequence the genome of the marine diatom Skeletonema marinoi were hampered by the presence of genetic material from bacteria, and there was sufficient material from some of these bacteria to enable the assembly of full chromosomes. Here, we report the genome of strain SMS9, one such bacterial species identified in a non-axenic culture of S. marinoi strain ST54. Its 5,482,391 bp circular chromosome contains 4,641 CDSs, and has a G+C content of 35.6%. Based on 16S rRNA comparison, phylotaxonomic analysis, and the genome similarity metrics dDDH and OrthoANI, we place this strain in the genus Kordia, and to the best of our knowledge, this is the first Kordia species to be initially described from European waters. As attempts to culture this strain have failed, however, the specifics of its relationship with S. marinoi are still uncertain.

Skeletonema marinoi as a new genetic model for marine chain-forming diatoms.

Diatoms are ubiquitous primary producers in marine ecosystems and freshwater habitats. Due to their complex evolutionary history, much remains unknown about the specific gene functions in diatoms that underlie their broad ecological success. In this study, we have genetically transformed the centric diatom Skeletonema marinoi, a dominant phytoplankton species in temperate coastal regions. Transformation of S. marinoi is the first for a true chain-forming diatom, with the random genomic integration via nonhomologous recombination of a linear DNA construct expressing the resistance gene to the antibiotic zeocin. A set of molecular tools were developed for reliably identifying the genomic insertion site within each transformant, many of which disrupt recognizable genes and constitute null or knock-down mutations. We now propose S. marinoi as a new genetic model for marine diatoms, representing true chain-forming species that play a central role in global photosynthetic carbon sequestration and the biogeochemical cycling of silicates and various nutrients, as well as having potential biotechnological applications.

Genome Sequence of Arenibacter algicola Strain SMS7, Found in Association with the Marine Diatom Skeletonema marinoi.

Arenibacter algicola strain SMS7 was isolated from a culture of the marine diatom Skeletonema marinoi strain ST54, sampled from top-layer sediments in Kosterfjord, Sweden. Here, we present its 5,857,781-bp genome, consisting of a circular chromosome and one circular plasmid, in all containing 4,932 coding sequences.

Complete Genome Sequence of Novel Sulfitobacter pseudonitzschiae Strain SMR1, Isolated from a Culture of the Marine Diatom Skeletonema marinoi.

When studying diatoms, an important consideration is the role of associated bacteria in the diatom-microbiome holobiont. To that end, bacteria isolated from a culture of Skeletonema marinoi strain R05AC were sequenced, one of which being bacterial strain SMR1, presented here. The genome consists of a circular chromosome and seven circular plasmids, totalling 5,121,602 bp. After phylotaxonomic analysis and 16S rRNA sequence comparison, we place this strain in the taxon Sulfitobacter pseudonitzschiae on account of similarity to the type strain. The annotated genome suggests similar interactions between strain SMR1 and its host diatom as have been shown previously in diatom-associated Sulfitobacter, for example bacterial production of growth hormone for its host, and breakdown of diatom-derived DMSP by Sulfitobacter for use as a sulfur source.

Is hydrophobic interaction chromatography the most suitable technique to characterize site-specific antibody-drug conjugates?

Antibody drug conjugates (ADCs) belong to the fastest growing class of therapeutic agents for cancer therapy. In preclinical and clinical studies, there is a significant number of site-specific ADCs (also known as third generation ADCs), which are more homogeneous than their previous generations. These new ADC formats, in which the inter-chain disulphide bridges (hinge cysteines) are not reduced, also need to be deeply characterized. In particular, various quality attributes (QAs) have to be determined, such as free antibody level, average drug to antibody ratio (DAR) and drug distribution. In this contribution, a non-commercial site-specific conjugated ADC has been analyzed by RPLC. Our results demonstrated that RPLC has a huge potential to determine QAs and can replace the historically used HIC methods as RPLC provides better separation quality for such type of ADCs. Site-specific ADCs can be analyzed in RPLC at intact level without the need for sample preparation. A further advantage of RPLC is that it enables the direct coupling to MS and thus allows the fine identification of all eluting species.

Whole-Genome Sequence of the Novel Antarctobacter heliothermus Strain SMS3, Found in Association with the Marine Diatom Skeletonema marinoi.

As part of an ongoing investigation into the microbiome of the marine diatom Skeletonema marinoi, the bacterial strain SMS3 was isolated from a culture of S. marinoi strain ST54, which had been propagated from a sample of top layer marine sediments taken from the Swedish west coast. We present here the sequenced genome of this bacterium, which we place in the taxon Antarctobacter heliothermus, based on a phylotaxonomic analysis and its high 16S rRNA sequence similarity to the A. heliothermus type strain DSM 11445T. Its 5,331,190 bp genome consists of a circular chromosome and three circular plasmids, and contains 5,019 CDSs. Strain SMS3 contains a phosphatidylcholine synthase gene, as well as genes involved in DMSP degradation, both of which imply a potential symbiotic relationship with its host.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

Complete Genome Sequence of Loktanella vestfoldensis Strain SMR4r, a Novel Strain Isolated from a Culture of the Chain-Forming Diatom Skeletonema marinoi.

We report here the genome sequence of Loktanella vestfoldensis strain SMR4r, isolated from the marine diatom Skeletonema marinoi strain RO5AC. Its 3,987,360-bp genome consists of a circular chromosome and two circular plasmids, one of which appears to be shared with an S. marinoi-associated Roseovarius species.

Extending the limits of size exclusion chromatography: Simultaneous separation of free payloads and related species from antibody drug conjugates and their aggregates.

Size exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partially excluded molecules from the pores of the stationary phase, based on their difference in hydrodynamic volume. In this work, a baseline resolution was obtained between the monomeric antibody drug conjugate (ADC) and high molecular weight species (HMWS). Besides HMWS, small free payloads, linkers and linker-payloads of ADCs, which would not be discriminated solely based on their size (MW < 1.5 kDa), were also separated on the same SEC column by applying sequentially an acetonitrile gradient after the elution of the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amount under native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For this purpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 µm particles and 250 Špore size, was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was also developed to further extend the possibility offered by this experimental setup. The SECxRPLC multiple heart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 µL sampling loops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to be suitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SEC method was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC method facilitated separating some additional impurities. The streamlined methodology will further support the development and characterization of ADC products.

Genome Sequence of Roseovarius mucosus Strain SMR3, Isolated from a Culture of the Diatom Skeletonema marinoi.

We present the genome of Roseovarius mucosus strain SMR3, a marine bacterium isolated from the diatom Skeletonema marinoi strain RO5AC sampled from top layer sediments at 14 m depth. Its 4,381,426 bp genome consists of a circular chromosome and two circular plasmids and contains 4,178 coding sequences (CDSs).

Quantitative determination of salbutamol sulfate impurities using achiral supercritical fluid chromatography.

In the last years, supercritical fluid chromatography has largely been acknowledged as a singular and performing technique in the field of separation sciences. Recent studies highlighted the interest of SFC for the quality control of pharmaceuticals, especially in the case of the determination of the active pharmaceutical ingredient (API). Nevertheless, quality control requires also the determination of impurities. The objectives of the present work were to (i) demonstrate the interest of SFC as a reference technique for the determination of impurities in salbutamol sulfate API and (ii) to propose an alternative to a reference HPLC method from the European Pharmacopeia (EP) involving ion-pairing reagent. Firstly, a screening was carried out to select the most adequate and selective stationary phase. Secondly, in the context of robust optimization strategy, the method was developed using design space methodology. The separation of salbutamol sulfate and related impurities was achieved in 7min, which is seven times faster than the LC-UV method proposed by European Pharmacopeia (total run time of 50min). Finally, full validation using accuracy profile approach was successfully achieved for the determination of impurities B, D, F and G in salbutamol sulfate raw material. The validated dosing range covered 50 to 150% of the targeted concentration (corresponding to 0.3% concentration level), LODs close to 0.5µg/mL were estimated. The SFC method proposed in this study could be presented as a suitable fast alternative to EP LC method for the quantitative determination of salbutamol impurities.

Deletion of FtsH11 protease has impact on chloroplast structure and function in Arabidopsis thaliana when grown under continuous light.

The membrane-integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual-targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast-located proteins of unknown function (Tic22-like protein and YGGT-A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6-week-old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non-photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.