New Clinical Markers of Oxidized Lipid-Associated Protein Damage in Children and Adolescents with Obesity.

Obesity in children and adolescents has been associated with oxidative stress (OS). The lipid hydroperoxides (LOOH) and the malondialdehyde (MDA) and thiobarbituric reactive substances (TBARS) that oxidatively modify proteins (Pr) (i.e., PrMDA and PrTBARS, respectively) represent markers of OS-associated lipid peroxidation. We aimed to assess OS in children and adolescents with obesity using-for the first time-markers involved in the early and late lipid oxidation process. LOOH, PrMDA, and PrTBARS were investigated in 41 children and adolescents with obesity and 31 controls. Obesity was defined as BMI > 95% for age and sex. The PrMDA/PrTBARS pair, which reflects a late peroxidation stage, was found to be significantly high (39%/180%) in children and adolescents with obesity compared to controls (p < 0.001). Similarly, the early LOOH peroxidation stage marker was increased by 30%. The studied OS parameters were not influenced by sex or age. Our study introduces LOOH, PrTBARS, and PrMDA as markers for evaluating OS in children and adolescents with obesity. LOOH, PrTBARS, and PrMDA may also hold promise as prognostic markers for potential obesity-associated long-term complications.

Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Rationale and Design of Heart Failure Prevalence and Evolution of Heart Failure in Diabetes Mellitus Type II Patients at High Risk (HF-LanDMark Study).

(1) Background: Patients with diabetes mellitus (DM) are at increased risk for heart failure (HF). Accurate data regarding the prevalence of HF stages among diabetics in Greece are scarce. (2) Aim: The present study will examine the prevalence and evolution of HF stages among patients with type II DM (T2DM) diagnosed in the past 10 years, with no previous history of HF and at high CV risk, in Greece, as well as will explore the potential determinants of the development of symptomatic HF in these patients. (3) Methods: Through a non-interventional, epidemiological, single-country, multi-center, prospective cohort study design, a sample of 300 consecutive patients will be enrolled in 11 cardiology departments that are HF centers of excellence. Patients will be either self-referred or referred by primary or secondary care physicians and will be followed for up to 24 months. Demographic, clinical, echocardiography, electrocardiography, cardiac biomarkers (troponin, NT-proBNP) and health-related quality of life questionnaire data will be recorded as well as clinical events, including mortality, HF hospitalizations and HF-related healthcare resource utilization. The primary outcomes are the proportion of patients diagnosed with symptomatic HF (ACC/AHA Stage C) at enrolment in the overall study population and the proportions of patients with HF stages A, B and C, as well as by NYHA functional classification in the overall study population. (4) Conclusions: The HF-LanDMark study is the first epidemiological study that will assess the prevalence of HF among T2DM patients in Greece that could potentially enhance prompt therapeutic interventions shown to delay the development of HF in the T2DM patient population (HF-LanDMark, Clinical Trials.gov number, NCT04482283).

Organic Catalytic Activity as a Method for Agnostic Life Detection.

An ideal life detection instrument would have high sensitivity but be insensitive to abiotic processes and would be capable of detecting life with alternate molecular structures. In this study, we propose that catalytic activity can be the basis of a nearly ideal life detection instrument. There are several advantages to catalysis as an agnostic life detection method. Demonstrating catalysis does not necessarily require culturing/growing the alien life and in fact may persist even in dead biomass for some time, and the amplification by catalysis is large even by minute amounts of catalysts and, hence, can be readily detected against abiotic background rates. In specific, we propose a hydrolytic catalysis detection instrument that could detect activity in samples of extraterrestrial organic material from unknown life. The instrument uses chromogenic assay-based detection of various hydrolytic catalytic activities, which are matched to corresponding artificial substrates having the same, chromogenic (preferably fluorescent) upon release, group; D- and L-enantiomers of these substrates can be used to also answer the question whether unknown life is chiral. Since catalysis is a time-proportional product-concentration amplification process, hydrolytic catalytic activity can be measured on a sample of even a minute size, and with instruments based on, for example, optofluidic chip technology.

Novel oxidized LDL-based clinical markers in peritoneal dialysis patients for atherosclerosis risk assessment.

Maintenance peritoneal dialysis (PD) is commonly associated with cardiovascular diseases (CVDs), whose risk is assessed via LDL-C. Nonetheless, oxidized LDL (oxLDL), as being a key component of atherosclerotic lesions, could be also associated with atherosclerosis and related CVDs. However, its predictive value for CVDs risk assessment is subject of research studies due to the lack of specific methods to measure oxLDL status from its individual lipid/protein components. In the present study, six novel oxLDL markers, representative of certain oxidative modifications on the LDL protein and lipid components, are measured in atherosclerosis-prone PD patients (39) versus those in chronic kidney disease patients (61) under hemodialysis (HD) and healthy controls (40). LDL from serum of PD, HD and control subjects were isolated and fractionated into cholesteryl esters, triglycerides, free cholesterol, phospholipids and apolipoprotein B100 (apoB100). Subsequently the oxLDL markers cholesteryl ester hydroperoxides (-OOH), triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100 malondialdehyde and apoB100 dityrosines were measured. LDL carotenoid levels and LDL particle serum concentration were also measured. The levels of all oxLDL lipid-OOH markers were significantly elevated in PD patients versus control, while the levels of cholesteryl ester-/triglyceride-/free cholesterol-OOH were significantly elevated in PD versus HD patients, regardless of patients' underlying medical conditions, sex, age, PD type, clinical biochemical markers and medication. It should be noted that all fractionated lipid-OOH levels were inversely correlated with LDL-P concentration, while LDL-P concentration was not correlated with LDL-C in PD patients. Moreover, LDL carotenoids were significantly lower in PD patients versus control. The increased levels of oxLDL status specific markers in both PD and HD patients (compared to control), support a potential prognostic value of oxLDL regarding CVD risk assessment in both patient groups. Lastly, the study introduces the oxLDL peroxidation markers free cholesterol-OOH and cholesteryl ester-OOH as complementary to LDL-P number, and as possible alternatives to LDL-C.

Time-Related Evidence of Intestinal Oxidative Stress in Obstructive Jaundice-Induced Rats.

INTRODUCTION: Obstructive jaundice is known to affect intestinal permeability and facilitate bacterial translocation through related mechanisms. This study was conducted to evaluate the alterations concerning blood biochemistry and levels of several markers of oxidative stress (OS) in blood and intestinal mucosa caused by obstructive jaundice and how these fluctuate over time, in order to further explore the possibility of intervening in the OS path in future experiments. METHODS: A total of 54 albino Wistar rats were randomly divided into three groups (control, sham operated, and bile duct ligation) and sacrificed at specific time intervals (12 h and 2, 7, and 14 days). The intestinal barrier function was evaluated by measuring endotoxin levels in portal, aortic, and peripheral blood. Also, basic biochemical parameters were simultaneously measured in peripheral blood. Tissue samples collected from the terminal ileum were homogenized for determining the OS markers, lipid peroxidation, and protein-free radical-induced oxidation. RESULTS: We designed this experiment to examine the alterations in enteric mucosa primarily in relation to OS in a period of 14 days. During this time period, we investigated in specific time intervals not only OS fluctuations but also other liver function parameters, as well as CRP and endotoxin levels. The alterations were monitored in relation to time after bile duct ligation. CONCLUSION: Bile duct ligation in rats causes OS versus post-ligation time progression of the common bile duct. OS was increased by ∼50% compared to control/sham and peaked at 7 days and at least up to 14 days post-ligation. This phenomenon was accompanied with a deranging of liver function after ligation, as anticipated, but not in all measured parameters; biochemical and endotoxin levels followed the same pattern.

Parental Lifestyle Changes and Correlations with Children's Dietary Changes during the First COVID-19 Lockdown in Greece: The COV-EAT Study.

The aim of this study was to investigate changes in the eating behaviour of parents during the first lockdown implemented in Greece due to COVID-19 and to explore possible associations with corresponding changes in the eating behaviour of their children. A quantitative cross-sectional study was performed using an online questionnaire. The study sample consisted of 397 parents with children aged 2−18 years, who were recruited from 63 municipalities in Greece. It was observed that the percentage of parents and children reporting consumption of breakfast during the lockdown period increased by 10.6% and 5%, respectively. Also, 75% of the parents increased their snack consumption and 61% their sweets consumption. Parents increased home-cooking during lockdown (6.4 times/week), compared to 5.6 times/week before (p < 0.001), which was associated with decreased consumption of fast foods for both parents and children (p < 0.001 for all comparisons) and also correlated with increased consumption of fruit and vegetables for children (p < 0.05). More than half parents tried to lose weight during lockdown (58.4%). In conclusion, both favourable (home-cooking) and unfavourable (increased snacking) lifestyle changes during the first COVID-19 lockdown in Greece were reported for parents.

Time progression and regional expression of brain oxidative stress induced by obstructive jaundice in rats.

BACKGROUND: Obstructive jaundice induces oxidative changes in the brain parenchyma and plays significant role in clinical manifestations of hepatic encephalopathy. We aim to study the progression of the brain oxidative status over time and the differences of its pattern over the hemispheres, the brainstem and the cerebellum. We use an experimental model in rats and measuring the oxidative stress (OS) specific biomarkers protein malondialdehyde (PrMDA) and protein carbonyls (PrC = O). RESULTS: Hyperbilirubinemia has been confirmed in all study groups as the result of common bile duct obstruction. We confirmed increase in both PrMDA and PrC = O biomarkers levels with different type of changes over time. We also confirmed that the oxidative process develops differently in each of the brain areas in study. CONCLUSIONS: The present study confirms the progressive increase in OS in all brain areas studied using markers indicative of cumulative protein modification.

Maternal Calorie Restriction Induces a Transcriptional Cytoprotective Response in Embryonic Liver Partially Dependent on Nrf2.

BACKGROUND: Calorie restriction is known to enhance Nrf2 signaling and longevity in adult mice, partially by reducing reactive oxygen species, but calorie restriction during pregnancy leads to intrauterine growth retardation. The latter is associated with fetal reprogramming leading to increased incidence of obesity, metabolic syndrome and diabetes in adult life. Transcription factor Nrf2 is a central regulator of the antioxidant response and its crosstalk with metabolic pathways is emerging. We hypothesized that the Nrf2 pathway is induced in embryos during calorie restriction in pregnant mothers. METHODS: From gestational day 10 up to day 16, 50% of the necessary mouse diet was provided to Nrf2 heterozygous pregnant females with fathers being of the same genotype. Embryos were harvested at the end of gestational day 16 and fetal liver was used for qRT-PCR and assessment of oxidative stress (OS). RESULTS: Intrauterine calorie restriction led to upregulation of mRNA expression of antioxidant genes (Nqo1, Gsta1, Gsta4) and of genes related to integrated stress response (Chac1, Ddit3) in WT embryos. The expression of a key gluconeogenic (G6pase) and two lipogenic genes (Acacb, Fasn) was repressed in calorie-restricted embryos. In Nrf2 knockout embryos, the induction of Nqo1 and Gsta1 genes was abrogated while that of Gsta4 was preserved, indicating an at least partially Nrf2-dependent induction of antioxidant genes after in utero calorie restriction. Measures of OS showed no difference (superoxide radical and malondialdehyde) or a small decrease (thiobarbituric reactive substances) in calorie-restricted WT embryos. CONCLUSIONS: Calorie restriction during pregnancy elicits the transcriptional induction of cytoprotective/antioxidant genes in the fetal liver, which is at least partially Nrf2-dependent, with a physiological significance that warrants further investigation.

Detection of superoxide radical in all biological systems by Thin Layer Chromatography.

The study presents a new method that detects O2•-, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε+) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε+ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε+) and ethidium (Ε+). Quantification of 2-OH-E+ is based on its ex/em maxima at 290/540 nm, and of di-E+ and E+ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E+ (as well as di-E+ and E+) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε+ (by neutralizing the strong binding between its quaternary N+ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O2•- levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε+ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O2•- generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).

Methods on LDL particle isolation, characterization, and component fractionation for the development of novel specific oxidized LDL status markers for atherosclerotic disease risk assessment.

The present study uses simple, innovative methods to isolate, characterize and fractionate LDL in its main components for the study of specific oxidations on them that characterize oxidized low-density lipoprotein (oxLDL) status, as it causatively relates to atherosclerosis-associated cardiovascular disease (CVD) risk assessment. These methods are: (a) A simple, relatively time-short, low cost protocol for LDL isolation, to avoid shortcomings of the currently employed ultracentrifugation and affinity chromatography methodologies. (b) LDL purity verification by apoB100 SDS-PAGE analysis and by LDL particle size determination; the latter and its serum concentration are determined in the present study by a simple method more clinically feasible as marker of CVD risk assessment than nuclear magnetic resonance. (c) A protocol for LDL fractionation, for the first time, into its main protein/lipid components (apoB100, phospholipids, triglycerides, free cholesterol, and cholesteryl esters), as well as into LDL carotenoid/tocopherol content. (d) Protocols for the measurement, for the first time, of indicative specific LDL component oxidative modifications (cholesteryl ester-OOH, triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100-MDA, and apoB100-DiTyr) out of the many (known/unknown/under development) that collectively define oxLDL status, which contrasts with the current non-specific oxLDL status evaluation methods. The indicative oxLDL status markers, selected in the present study on the basis of expressing early oxidative stress-induced oxidative effects on LDL, are studied for the first time on patients with end stage kidney disease on maintenance hemodialysis, selected as an indicative model for atherosclerosis associated diseases. Isolating LDL and fractionating its protein and main lipid components, as well as its antioxidant arsenal comprised of carotenoids and tocopherols, paves the way for future studies to investigate all possible oxidative modifications responsible for turning LDL to oxLDL in association to their possible escaping from LDL's internal antioxidant defense. This can lead to studies to identify those oxidative modifications of oxLDL (after their artificial generation on LDL), which are recognized by macrophages and convert them to foam cells, known to be responsible for the formation of atherosclerotic plaques that lead to the various CVDs.

Oxidized lipid-associated protein damage in children and adolescents with type 1 diabetes mellitus: New diagnostic/prognostic clinical markers.

BACKGROUND: Type 1 diabetes mellitus (DM1), a chronic metabolic disorder of autoimmune origin, has been associated with oxidative stress (OS), which plays a central role in the onset, progression, and long-term complications of DM1. The markers of OS lipid peroxidation products, lipid hydroperoxides (LOOH), and also malondialdehyde (MDA) and thiobarbituric reactive substances (TBARS) that oxidatively modify proteins (Pr) (i.e., PrMDA and PrTBARS, respectively), have been associated with DM2, DM1, diabetic neuropathy, and microalbuminuria. OBJECTIVE/SUBJECTS: Here, we investigated LOOH, PrMDA and PrTBARS in 50 children and adolescents with DM1 and 21 controls. RESULTS: The novel OS marker PrTBARS was assessed for the first time in children and adolescents with DM1. LOOH and the pair PrMDA/PrTBARS, representing early and late peroxidation stages, respectively, are found to be significantly higher (130%, 50/90%, respectively, at p < 0.001) in patients with DM1 compared to controls. The studied OS parameters did not differ with age, age at diagnosis, sex, duration of DM1, presence of recent ketosis/ketoacidosis, or mode of treatment. CONCLUSIONS: We propose that LOOH, PrMDA and the new marker PrTBARS could serve as potential diagnostic clinical markers for identifying OS in children and adolescents with DM1, and may, perhaps, hold promise as a prognostic tool for future complications associated with the disease.

Oxidative Stress and NADPH Oxidase: Connecting Electromagnetic Fields, Cation Channels and Biological Effects.

Electromagnetic fields (EMFs) disrupt the electrochemical balance of biological membranes, thereby causing abnormal cation movement and deterioration of the function of membrane voltage-gated ion channels. These can trigger an increase of oxidative stress (OS) and the impairment of all cellular functions, including DNA damage and subsequent carcinogenesis. In this review we focus on the main mechanisms of OS generation by EMF-sensitized NADPH oxidase (NOX), the involved OS biochemistry, and the associated key biological effects.

Lifestyle Changes and Determinants of Children's and Adolescents' Body Weight Increase during the First COVID-19 Lockdown in Greece: The COV-EAT Study.

Previous studies showed that the coronavirus disease 2019 (COVID-19) lockdown imposed changes in adults' lifestyle behaviors; however, there is limited information regarding the effects on youth. The COV-EAT study aimed to report changes in children's and adolescents' lifestyle habits during the first COVID-19 lockdown and explore potential associations between changes of participants' lifestyle behaviors and body weight. An online survey among 397 children/adolescents and their parents across 63 municipalities in Greece was conducted in April-May 2020. Parents self-reported changes of their children's lifestyle habits and body weight, as well as sociodemographic data of their family. The present study shows that during the lockdown, children's/adolescents' sleep duration and screen time increased, while their physical activity decreased. Their consumption of fruits and fresh fruit juices, vegetables, dairy products, pasta, sweets, total snacks, and breakfast increased, while fast-food consumption decreased. Body weight increased in 35% of children/adolescents. A multiple regression analysis showed that the body weight increase was associated with increased consumption of breakfast, salty snacks, and total snacks and with decreased physical activity. The COV-EAT study revealed changes in children's and adolescents' lifestyle behaviors during the first COVID-19 lockdown in Greece. Effective strategies are needed to prevent excessive body weight gain in future COVID-19 lockdowns.

Omega-3 fatty acids supplementation protects the retina from age-associated degeneration in aged C57BL/6J mice.

OBJECTIVE: To evaluate the therapeutic effects of omega-3 (ω3) fatty acids in the retina of aged mice when the blood arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is maintained between 1.0 and 1.5. METHODS AND ANALYSIS: Aged (24-month-old) wild-type C57BL/6J mice were allocated to two groups: ω3 treated and untreated. Treatment with ω3 was by daily gavage administration of EPA and docosahexaenoic acid for 60 days. Gas chromatography was used to identify and quantify fatty acids in the blood and retina. To count lipofuscin granules and measure the photoreceptor layer, eyecups were examined histologically using transmission electron microscopy and light microscopy. We also analysed eyecups using mass spectrometry-based proteomics. RESULTS: AA levels were lower, and EPA levels were higher, in the blood and retinas of the ω3-treated group than in the untreated group, resulting in a lower AA/EPA ratio. The ω3-treated group also showed significantly fewer lipofuscin granules and a thicker outer nuclear layer than the untreated group. Proteomic analysis revealed significantly greater expression of myelin basic protein, myelin regulatory factor-like protein, myelin proteolipid protein and glial fibrillar acidic protein in the ω3-treated group than in the untreated group. Three different pathways were significantly affected by ω3 treatment: fatty acid elongation, biosynthesis of unsaturated fatty acids and metabolic pathways. CONCLUSION: Two months of ω3 supplementation (when the blood AA/EPA~1.0-1.5) in aged mice reduced lipofuscin granule formation in the retina and protected the photoreceptor layer, suggesting that ω3 supplementation slows normal age-related retinal degeneration.

Abdominal pain caused by a giant liver hydatid cyst and the management of the residual cavity in a tertiary public hospital in Greece.

Diagnosis and treatment of liver hydatid cysts may be challenging. Many surgical techniques have been proposed for the treatment of liver hydatid cysts, but the problem of the residual cavity still remains controversial and challenging, especially in giant liver hydatid cysts which are rare entities that have not been widely described in the literature so far. Capitonnage, external tube drainage and omentoplasty are the most commonly used techniques. Herein, we report the case of a 70-year-old man with a mild upper quadrant pain that proved to have a giant liver hydatid cyst, 21*14 cm2, occupying the entire right lobe of the liver. We describe a successful surgical approach with cyst unroofing and careful evacuation of the multiple daughter cysts by aspiration, and the effective management of the residual cavity by the combination of all three aforementioned techniques.

The Oxygen Release Instrument: Space Mission Reactive Oxygen Species Measurements for Habitability Characterization, Biosignature Preservation Potential Assessment, and Evaluation of Human Health Hazards.

We describe the design of an instrument, the OxR (for Oxygen Release), for the enzymatically specific and non-enzymatic detection and quantification of the reactive oxidant species (ROS), superoxide radicals (O2•-), and peroxides (O22-, e.g., H2O2) on the surface of Mars and Moon. The OxR instrument is designed to characterize planetary habitability, evaluate human health hazards, and identify sites with high biosignature preservation potential. The instrument can also be used for missions to the icy satellites of Saturn's Titan and Enceladus, and Jupiter's Europa. The principle of the OxR instrument is based on the conversion of (i) O2•- to O2 via its enzymatic dismutation (which also releases H2O2), and of (ii) H2O2 (free or released by the hydrolysis of peroxides and by the dismutation of O2•-) to O2 via enzymatic decomposition. At stages i and ii, released O2 is quantitatively detected by an O2 sensor and stoichiometrically converted to moles of O2•- and H2O2. A non-enzymatic alternative approach is also designed. These methods serve as the design basis for the construction of a new small-footprint instrument for specific oxidant detection. The minimum detection limit of the OxR instrument for O2•- and O22- in Mars, Lunar, and Titan regolith, and in Europa and Enceladus ice is projected to be 10 ppb. The methodology of the OxR instrument can be rapidly advanced to flight readiness by leveraging the Phoenix Wet Chemical Laboratory, or microfluidic sample processing technologies.

Functional Properties of Amino Acid Side Chains as Biomarkers of Extraterrestrial Life.

The present study proposes to search our solar system (Mars, Enceladus, Europa) for patterns of organic molecules that are universally associated with biological functions and structures. The functions are primarily catalytic because life could only have originated within volume/space-constrained compartments containing chemical reactions catalyzed by certain polymers. The proposed molecular structures are specific groups in the side chains of amino acids with the highest catalytic propensities related to life on Earth, that is, those that most frequently participate as key catalytic groups in the active sites of enzymes such as imidazole, thiol, guanidinium, amide, and carboxyl. Alternatively, these or other catalytic groups can be searched for on non-amino-acid organic molecules, which can be tested for certain hydrolytic catalytic activities. The first scenario assumes that life may have originated in a similar manner as the terrestrial set of α-amino acids, while the second scenario does not set such a requirement. From the catalytic propensity perspective proposed in the first scenario, life must have invented amino acids with high catalytic propensity (His, Cys, Arg) in order to overcome, and be complemented by, the low catalytic propensity of the initially available abiogenic amino acids. The abiogenic and the metabolically invented amino acids with the lowest catalytic propensity can also serve as markers of extraterrestrial life when searching for patterns on the basis of the following functional propensities related to protein secondary/quaternary structure: (1) amino acids that are able to form α-helical intramembrane peptide domains, which can serve as primitive transporters in protocell membrane bilayers and catalysts of simple biochemical reactions; (2) amino acids that tend to accumulate in extremophile proteins of Earth and possibly extraterrestrial life. The catalytic/structural functional propensity approach offers a new perspective in the search for extraterrestrial life and could help unify previous amino acid-based approaches.

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower protein quantity (2.5 µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay.

Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay.

A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biological source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral (ntr) pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with ethyl acetate/hexane wash. The ntrDNPH assay is more reliable and sensitive than the standard (std) DNPH photometric assay because it eliminates its main limitations: (i) interfering unreacted DNPH (pKa 1.55) that is nonspecifically bound to the TCA (pKa 0.7)-protein pellet is not effectively removed after wash with EtOH: ethyl acetate because it is positively charged, (ii) acid (TCA-induced) hydrolysis of the protein carbonyl-DNPH hydrazone, (iii) sample protein concentration re-determination, (iv) loss of sample acid (TCA)-soluble proteins, (v) DNA interference, and (vi) requires high protein quantity samples (≥ 1 mg). Considering ntrDNPH assay's very low protein limit (1 µg), its cumulative and functional sensitivities are 2600- and 2000-fold higher than those of the stdDNPH assay, respectively. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aqueous, membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl determination on defined cell protein fractions, and to eliminate assay interference from protein samples containing (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate precipitation). Lastly, the ntrDNPH assay determines carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens.