Assessment of GNSS Galileo Contribution to the Modernization of CROPOS's Services.

CROPOS, as the Croatian GNSS network, was modernized and upgraded to support the Galileo system in 2019. Two of CROPOS's services-VPPS (Network RTK service) and GPPS (post-processing service)-were assessed for the contribution of the Galileo system to their performance. A station used for field testing was previously examined and surveyed to determine the local horizon and to carry out a detailed mission planning. The whole day of observation was divided into several sessions, each with a different visibility of Galileo satellites. A special observation sequence was designed: VPPS (GPS-GLO-GAL), VPPS (GAL-only), and GPPS (GPS-GLO-GAL-BDS). All observations were taken on the same station with the same GNSS receiver, Trimble R12. Each static observation session was post-processed in Trimble Business Center (TBC) in two different ways: considering all available systems (GGGB) and considering GAL-only observations. A daily static solution based on all systems (GGGB) was considered as the reference for the accuracy assessment of all obtained solutions. The results obtained with VPPS (GPS-GLO-GAL) and VPPS (GAL-only) were analyzed and assessed; the results obtained with GAL-only have shown a slightly higher scatter. It was concluded that the inclusion of the Galileo system in CROPOS has contributed to the availability and reliability of solutions but not to their accuracy. By complying with the observation rules and taking redundant measurements, the accuracy of GAL-only results can be improved.

Migration Testing of GPPS and HIPS Polymers: Swelling Effect Caused by Food Simulants Compared to Real Foods.

Migration kinetic data from general purpose polystyrene (GPPS) and high impact polystyrene (HIPS) were generated for a set of model substances as well as styrene monomer and oligomers at different temperatures (20 °C, 40 °C, 60 °C) using food simulants stipulated in the European Regulation (EU) 10/2011 and real foods like milk, cream and olive oil (20 °C, 40 °C). The extent of polymer swelling was characterized gravimetrically and visual changes of the test specimens after migration contact were recorded. Isooctane and 95% ethanol caused strong swelling and visual changes of HIPS, overestimating real migration into foods especially at high temperatures; GPPS was affected by isooctane only at 60 °C. With 50% ethanol, after 10 days contact at 60 °C or 40 °C both polymers were slightly swollen. Contrary, most of the real foods analyzed caused no detectable swelling or visual changes of the investigated polymers. This study demonstrates that the recommendations provided by EU regulations are not always in agreement with the physicochemical properties of styrenic polymers. The critical point remains the selection of adequate food simulants/testing conditions, since the high overestimation of aggressive media can lead to non-compliance of polystyrene materials even if the migration into real food would be of no concern.

Photosynthetic generation of heterologous terpenoids in cyanobacteria.

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene ß-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. "Fusion constructs as protein overexpression vectors" were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and ß-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Cloning and Expression Analysis of Key Enzyme Gene CoGPPS Involved in Iridoid Glycoside Synthesis in Cornus officinalis.

Cornus iridoid glycosides (CIGs), including loganin and morroniside, are the main active components of Cornus officinalis. As one of the key enzymes in the biosynthesis of CIGs, geranyl pyrophosphate synthase (GPPS) catalyzes the formation of geranyl pyrophosphate, which is the direct precursor of CIGs. In this study, the C. officinalis geranyl pyrophosphate synthase (CoGPPS) sequence was cloned from C. officinalis and analyzed. The cDNA sequence of the CoGPPS gene was 915 bp (GenBank No. OR725699). Phylogenetic analysis showed that CoGPPS was closely related to the GPPS sequence of Actinidia chinensis and Camellia sinensis, but relatively distantly related to Paeonia lactiflora and Tripterygium wilfordii. Results from the quantitative real-time PCR showed the spatiotemporal expression pattern of CoGPPS; that is, CoGPPS was specifically expressed in the fruits. Subcellular localization assay proved that CoGPPS was specifically found in chloroplasts. Loganin and morroniside contents in the tissues were detected by high-performance liquid chromatography, and both compounds were found to be at higher levels in the fruits than in leaves. Thus, this study laid the foundation for further studies on the synthetic pathway of CIGs.

Isoprenyl diphosphate synthases of terpenoid biosynthesis in rose-scented geranium (Pelargonium graveolens).

The essential oil of Pelargonium graveolens (rose-scented geranium), an important aromatic plant, comprising mainly mono- and sesqui-terpenes, has applications in food and cosmetic industries. This study reports the characterization of isoprenyl disphosphate synthases (IDSs) involved in P. graveolens terpene biosynthesis. The six identified PgIDSs belonged to different classes of IDSs, comprising homomeric geranyl diphosphate synthases (GPPSs; PgGPPS1 and PgGPPS2), the large subunit of heteromeric GPPS or geranylgeranyl diphosphate synthases (GGPPSs; PgGGPPS), the small subunit of heteromeric GPPS (PgGPPS.SSUI and PgGPPS.SSUII), and farnesyl diphosphate synthases (FPPS; PgFPPS).All IDSs exhibited maximal expression in glandular trichomes (GTs), the site of aroma formation, and their expression except PgGPPS.SSUII was induced upon treatment with MeJA. Functional characterization of recombinant proteins revealed that PgGPPS1, PgGGPPS and PgFPPS were active enzymes producing GPP, GGPP/GPP, and FPP respectively, whereas both PgGPPS.SSUs and PgGPPS2 were inactive. Co-expression of PgGGPPS (that exhibited bifunctional G(G)PPS activity) with PgGPPS.SSUs in bacterial expression system showed lack of interaction between the two proteins, however, PgGGPPS interacted with a phylogenetically distant Antirrhinum majus GPPS.SSU. Further, transient expression of AmGPPS.SSU in P. graveolens leaf led to a significant increase in monoterpene levels. These findings provide insight into the types of IDSs and their role in providing precursors for different terpenoid components of P. graveolens essential oil.

Establishing an Antimicrobial Stewardship Program in Sierra Leone: A Report of the Experience of a Low-Income Country in West Africa.

Antimicrobial Resistance (AMR) is a growing global health challenge that threatens to undo gains in human and animal health. Prevention and control of AMR requires functional antimicrobial stewardship (AMS) program, which is complex and often difficult to implement in low- and middle-income countries. We aimed to describe the processes of establishing and implementing an AMS program at Connaught Hospital in Sierra Leone. The project involved the setting up of an AMS program, capacity building and performing a global point prevalence survey (GPPS) at Sierra Leone's national referral hospital. Connaught Hospital established a multidisciplinary AMS subcommittee in 2021 to provide AMS services such as awareness campaigns, education and training and review of guidelines. We performed a GPPS on 175 patients, of whom more than half (98, 56.0%) were prescribed an antibiotic: 63 (69.2%) in the surgical wards and 53 (51.2%) in the medical wards. Ceftriaxone (60, 34.3%) and metronidazole (53, 30.3%) were the most common antibiotics prescribed to patients. In conclusion, it is feasible to establish and implement an AMS program in low-income countries, where most hospitalized patients were prescribed an antibiotic.

Feasibility properties of the EQ-5D-3L and 5L in the general population: evidence from the GP Patient Survey on the impact of age.

BACKGROUND: There is evidence to suggest that the proportion of missing values is slightly higher in the older population resulting in lower completion rates of the EQ-5D. However, existing studies rarely provide a within-sample comparison of feasibility properties across age groups to quantify this difference. Hence, this study examines feasibility properties of the EQ-5D-3L and 5L in the general population and explores the impact of age on the completion of EQ-5D instruments. METHODS: We pool five waves from the English GP Patient Survey, where respondents self-report their health in either EQ-5D-3L or 5L. Descriptive analysis was undertaken to analyse the distribution and proportion of missing values and completion rates stratified by age and EQ-5D version; logistic regression models were specified to quantify the impact of age, gender and potential long-term conditions on the completion of each of the EQ-5D instruments. RESULTS: The total sample comprises ~ 4.36 million observations, of which 2.88 million respondents report their health in 5L and 1.47 million in 3L, respectively. Respondents over 64 years have slightly more missing values in each dimension than younger respondents. The highest share was observed for the oldest age group in the dimension anxiety/depression (3L 9.1% vs. 5L 7.6%), but was otherwise below 5%. Consequently, completion rates (observed and predicted) decreased with older age and at a higher rate after the age of 64; this was more pronounced for the 3L. CONCLUSION: Evidence from our study suggests that both the EQ-5D-3L and 5L have good feasibility properties. In comparison to younger populations there appears to be a higher proportion of respondents with incomplete responses beyond the age of 64 years. Overall, the 5L version compares more favourably in terms of missing values, completion rates as well as with regard to the expected probability of an incomplete descriptive system.

Identification and Molecular Characterization of Geranyl Diphosphate Synthase (GPPS) Genes in Wintersweet Flower.

Geranyl diphosphate synthase (GPPS) is a plastid localized enzyme that catalyzes the biosynthesis of Geranyl diphosphate (GPP), which is a universal precursor of monoterpenes. Wintersweet (Chimonanthus praecox L.), a famous deciduous flowering shrub with a strong floral scent character, could have GPPS-like homologs that are involved in monoterpenes biosynthesis, but it remains unclear. In the present study, five full-length GPPS and geranylgeranyl diphosphate synthases (GGPPS) genes were identified in the wintersweet transcriptome database. The isolated cDNAs showed high protein sequence similarity with the other plants GPPS and GGPPS. The phylogenetic analysis further classified these cDNAs into four distinct clades, representing heterodimeric GPPS small subunits (SSU1 and SSU2), homodimeric GPPS, and GGPPS. Analysis of temporal expression revealed that all genes have the highest transcript level at the full-open flower stage. From tissue-specific expression analysis, CpGPPS.SSU1 and CpGGPPS1 were predominantly expressed in petal and flower, whereas CpGPPS.SSU2, GPPS, and GGPPS2 showed a constitutive expression. Additionally, the subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins, and the yeast two-hybrid assay showed that both CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS proteins. Taken together, these preliminary results suggest that the heterodimeric GPPS can regulate floral scent biosynthesis in wintersweet flower.

PkGPPS.SSU interacts with two PkGGPPS to form heteromeric GPPS in Picrorhiza kurrooa: Molecular insights into the picroside biosynthetic pathway.

Geranyl geranyl pyrophosphate synthase (GGPPS) is known to form an integral subunit of the heteromeric GPPS (geranyl pyrophosphate synthase) complex and catalyzes the biosynthesis of monoterpene in plants. Picrorhiza kurrooa Royle ex Benth., a medicinally important high altitude plant is known for picroside biomolecules, the monoterpenoids. However, the significance of heteromeric GPPS in P. kurrooa still remains obscure. Here, transient silencing of PkGGPPS was observed to reduce picroside-I (P-I) content by more than 60% as well as picroside-II (P-II) by more than 75%. Thus, PkGGPPS was found to be involved in the biosynthesis of P-I and P-II besides other terpenoids. To unravel the mechanism, small subunit of GPPS (PkGPPS.SSU) was identified from P. kurrooa. Protein-protein interaction studies in yeast as well as bimolecular fluorescence complementation (BiFC) in planta have indicated that large subunit of GPPS PkGPPS.LSUs (PkGGPPS1 and PkGGPPS2) and PkGPPS.SSU form a heteromeric GPPS. Presence of similar conserved domains such as light responsive motifs, low temperature responsive elements (LTRE), dehydration responsive elements (DREs), W Box and MeJA responsive elements in the promoters of PkGPPS.LSU and PkGPPS.SSU documented their involvement in picroside biosynthesis. Further, the tissue specific transcript expression analysis vis-à-vis epigenetic regulation (DNA methylation) of promoters as well as coding regions of PkGPPS.LSU and PkGPPS.SSU has strongly suggested their role in picroside biosynthesis. Taken together, the newly identified PkGPPS.SSU formed the heteromeric GPPS by interacting with PkGPPS.LSUs to synthesize P-I and P-II in P. kurrooa.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production.

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Resource or waste? A perspective of plastics degradation in soil with a focus on end-of-life options.

'Capable-of-being-shaped' synthetic compounds are prevailing today over horn, bone, leather, wood, stone, metal, glass, or ceramic in products that were previously left to natural materials. Plastic is, in fact, economical, simple, adaptable, and waterproof. Also, it is durable and resilient to natural degradation (although microbial species capable of degrading plastics do exist). In becoming a waste, plastic accumulation adversely affects ecosystems. The majority of plastic debris pollutes waters, accumulating in oceans. And, the behaviour and the quantity of plastic, which has become waste, are rather well documented in the water, in fact. This review collects existing information on plastics in the soil, paying particular attention to both their degradation and possible re-uses. The use of plastics in agriculture is also considered. The discussion is organised according to their resin type and the identification codes used in recycling programs. In addition, options for post-consumer plastics are considered. Acknowledged indicators do not exist, and future study they will have to identify viable and shared methods to measure the presence and the degradation of individual polymers in soils.

Genotoxicity of styrene oligomers extracted from polystyrene intended for use in contact with food.

Here, we conducted in vitro genotoxicity tests to evaluate the genotoxicity of styrene oligomers extracted from polystyrene intended for use in contact with food. Styrene oligomers were extracted with acetone and the extract was subjected to the Ames test (OECD test guideline No. 471) and the in vitro chromosomal aberration test (OECD test guideline No. 473) under good laboratory practice conditions. The concentrations of styrene dimers and trimers in the concentrated extract were 540 and 13,431 ppm, respectively. Extraction with acetone provided markedly higher concentrations of styrene oligomers compared with extraction with 50% ethanol aqueous solution, which is the food simulant currently recommended for use in safety assessments of polystyrene by both the United States Food and Drug Administration and the European Food Safety Authority. And these high concentrations of styrene dimers and trimers were utilized for the evaluation of genotoxicity in vitro. Ames tests using five bacterial tester strains were negative both in the presence or absence of metabolic activation. The in vitro chromosomal aberration test using Chinese hamster lung cells (CHL/IU) was also negative. Together, these results suggest that the risk of the genotoxicity of styrene oligomers that migrate from polystyrene food packaging into food is very low.

Derivation of safe health-based exposure limits for potential consumer exposure to styrene migrating into food from food containers.

Residual styrene present in polystyrene food packaging may migrate into food at low levels. To assure safe use, safe exposure levels are derived for consumers potentially exposed via food using No/Low Adverse Effect Levels from animal and human studies and assessment factors proposed by European organisations (EFSA, ECHA, ECETOC). Ototoxicity and developmental toxicity in rats and human ototoxicity and effects on colour discrimination have been identified as the most relevant toxicological properties for styrene health assessments. Safe exposure levels derived from animal studies with assessment factors of EFSA and ECHA were expectedly much lower than those using the ECETOC approach. Comparable safe exposure levels were obtained from human data with all sets of assessment factors while ototoxicity in rats led to major differences. The safe exposure levels finally selected based on criteria of science and health protection converged to the range of 90-120 mg/person/d. Assuming a consumption of 1 kg food/d for an adult, this translates to 90 mg styrene migration into 1 kg food as safe for consumers. This assessment supports a health based Specific Migration Limit of 90 ppm, a value somewhat higher than the current overall migration limit of 60 ppm in the European Union.