Development of a Universal Second-Tier Newborn Screening LC-MS/MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids.

First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.

Energy budget, carbon and water footprint in perennial agro and natural ecosystems inside a Natura 2000 site as provisioning and regulating ecosystem services.

Energy flows in perennial agro and natural ecosystems are essential and could be related to greenhouse gas emissions and increasing water demands. Energy indices and carbon and water footprint are useful tools in assessing the interaction between production systems and environmental impact inside Natura 2000 sites and enhancing ecosystem services. The main aims of this study were to determine and compare energy indices, carbon and water footprint of olive groves, orange and kiwi orchards, and grasslands within and between 2 years, located in a Natura 2000 site, using an adjusted life cycle assessment (LCA) method; and to illustrate the importance of farming systems to ecosystem services. Proportional stratified sampling was used to select 36 farm types (12 olive groves, 12 oranges, and 12 kiwi orchards) and 12 grasslands from the municipality of Amfilochia, western Greece, during 2018 and 2019. Descriptive data analysis, correlational analysis, ANOVA, and hierarchical cluster analysis (HCA) were used to summarize and analyze the data. The main energy contributors for the three farm types were fertilizers and fuel consumption. The only input for the grasslands was animal manure. Energy efficiency showed the highest performance in olive groves followed in descending order by kiwi orchards, grasslands, and orange orchards. Concerning carbon-footprint, the values were the highest for orange orchards, followed in descending order by kiwi orchards, olive groves, and grasslands. Total water footprint values were the highest for grasslands and the lowest for olive groves. For both years, regarding HCA, each cluster consisted of the same ecosystems. According to the abovementioned indices, olive groves and grasslands are the most environmental friendly ecosystems. They could contribute in the environmental protection of the Amvrakikos Gulf, a Natura 2000 site. Kiwi orchards, although they are intensive along with the orange ones, seem to be more friendly to the environment than them and their cultivation in this Natura 2000 area is recommended. Energy indices and carbon and water footprint could be used, in order to highlight the related ecosystem (dis)services.

Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris.

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

Could energy equilibrium and greenhouse gas emissions in agroecosystems play a key role in crop replacement? A case study in orange and kiwi orchards.

The development of agriculture is linked to energy resources. Consequently, energy analysis in agroecosystems could be a useful tool for monitoring some measures in the agricultural sector to mitigate greenhouse gas emissions. The objectives of this study were to (a) evaluate differences of energy indices in orange and kiwi orchards, and (b) point out whether inputs, outputs, efficiency, productivity, and carbon footprint can play a key role in crop replacement. Proportional stratified random sampling was used to select 26 orchards (10 oranges, 16 kiwis) from the Prefecture of Arta, western Greece, during 2015 and 2016. Univariate statistical methods were combined with multivariate ones. Nitrogen, Mg, Zn, herbicides, insecticides, fungicides, renewable energy inputs, fruit production, total outputs, and energy efficiency and productivity were statistically significantly high in the orange orchards. Phosphorus, Ca, irrigation, machinery, total inputs, intensity, non-renewable energy consumption, and carbon footprint were statistically significantly high in the kiwi orchards. The most important energy inputs for both fruit crops were fertilizers, fuels, irrigation, machinery, and herbicides. The orange orchards seem to be more friendly to the environment than the kiwi orchards by having low total energy inputs 32,210.3 MJ ha-1, intensity 1.4, consumption of non-renewable energy 0.7 MJ kg-1 and CO2 equivalent/fruit production 0.08 kg kg-1, and high energy outputs 105,120.0 MJ ha-1 and fruit production 53,648.0 kg ha-1. The findings of the present study show a relation between climate change and the production of farming systems, which can be a tool for decision makers. The correlation of the abovementioned parameters ensure higher profits and could help in achieving the best possible sustainable management of the agricultural ecosystems.

Current Status of Newborn Screening in Southeastern Europe.

Significant part of Southeastern Europe (with a population of 76 million) has newborn screening (NBS) programs non-harmonized with developed European countries. Initial survey was conducted in 2013/2014 among 11 countries from the region (Albania, Bulgaria, Bosnia and Herzegovina (BIH), Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia, and Slovenia) to assess the main characteristics of their NBS programs and their future plans. Their cumulative population at that time was ~52,5 million. At that time, none of the countries had an expanded NBS program, while phenylketonuria screening was not introduced in four and congenital hypothyroidism in three of 11 countries. We repeated the survey in 2020 inviting the same 11 countries, adding Cyprus, Greece, Hungary, and Malta (due to their geographical position in the wider region). The aims were to assess the current state, to evaluate the change in the period, and to identify the main obstacles impacting the implementation of expanded NBS and/or reaching a wider population. Responses were collected from 12 countries (BIH-Federation of BIH, BIH-Republic of Srpska, Bulgaria, Croatia, Greece, Hungary, Kosovo, North Macedonia, Malta, Montenegro, Romania, Serbia, Slovenia) with a population of 68.5 million. The results of the survey showed that the regional situation regarding NBS only modestly improved in this period. All of the surveyed countries except Kosovo screened for at least congenital hypothyroidism, while phenylketonuria was not screened in four of 12 countries. Croatia and Slovenia implemented an expanded NBS program using tandem mass spectrometry from the time of last survey. In conclusion, the current status of NBS programs in Southeastern Europe is very variable and is still underdeveloped (or even non-existent) in some of the countries. We suggest establishing an international task-force to assist with implementation and harmonization of basic NBS services where needed.

Structure-based design and application of a nucleotide coenzyme mimetic ligand: Application to the affinity purification of nucleotide dependent enzymes.

In the present study, a structure-based approach was exploited for the in silico design of a nucleotide coenzyme mimetic ligand. The enzyme formate dehydrogenase (FDH) was employed as a model in our study. The biomimetic ligand was designed and synthesized based on a tryptamine/3-aminopropylphosphonic acid bi-substituted 1,3,5-triazine (Trz) scaffold (Tra-Trz-3APP), which potentially mimics the interactions of NAD+-FDH complex. Molecular docking studies of the biomimetic ligand predicted that it can occupy the same binding site as the natural coenzyme. Molecular modeling and dynamics simulations revealed that the ligand binds in an energetically more stable pose in the FDH binding site, as it adopts a more twisty conformation, compared to the natural coenzyme. Study of the FDH/Tra-Trz-3APP-Sepharose interaction, through adsorption equilibrium studies and site-directed mutagenesis of selected FDH coenzyme binding residues, provided additional experimental evidences of the specificity of the interaction. The Tra-Trz-3APP-Sepharose biomimetic adsorbent was further evaluated towards a range of different dehydrogenases and was exploited for the development of a single-step purification protocol for FDH. The protocol afforded enzyme with high yield and purity, suitable for analytical and industrial purposes.