Alterations in energy metabolism, total protein, uric and nucleic acids in African sharptooth catfish (Clarias gariepinus Burchell) exposed to crude oil and fractions.

Water contamination by crude oil is a growing challenge and little is known about the probabilistic and non-probabilistic ecosystem and species consequences. Therefore, research aimed at understanding species survival strategy in crude oil-contaminated environments with focus on cellular metabolic alterations and dynamics is vital. This study assessed the alterations in lactate dehydrogenase (LDH), glucose (GLU), glucose-6-phosphate dehydrogenase (G-6-PDH), total protein (TP), uric and nucleic acids (UA, RNA, and DNA) in the liver, heart, kidney, blood supernatants, and muscle homogenates of African sharptooth catfish ([ASC] Clarias gariepinus) exposed to varying bonny-light crude oil concentrations to understand the underlying cause of their delayed development as well as potential health and wellbeing. Three concentrations (20, 50, and 100 mg/L) of diluted whole bonny-light crude oil (DWC), water-soluble (WSF), and water-insoluble (WIF) fractions of bonny-light crude oil were used to grow ASC for 9 weeks at room temperature. Biochemical assessments revealed significant (at p < 0.05) elevations in heart LDH (48.57 ± 4.67 to 3011.34 ± 4.67 U/L) and blood G-6-PDH activities (54.86 ± 0.00 to 128 ± 18.29 mU/mL), GLU (0.22 ± 0.01 to 0.77 ± 0.01 mg/dL), TP (5.15 ± 0.14 to 22.33 ± 0.21 g/L), UA (0.29 ± 0.05 to 10.05 ± 0.27 mg/dL), as well as liver DNA (0.38 ± 0.02 to 2.33 ± 0.09 µg/mL) and RNA (12.52 ± 0.05 to 30.44 ± 0.02 µg/mL) levels for laboratory-grown ASC in DWC, WSF, WIF, and oil-impacted Ubeji river collected ASC relative to the control. Due to greater levels of cellular metabolic alterations in oil-impacted Ubeji River collected ASC, it is evident that bonny-light contamination levels in the river is greater than 100 mg/L. In conclusion, bonny-light crude oil is toxic to ASC and induces stress response. The ecological changes caused by bonny-light crude oil contamination may ultimately affect niche functioning and the development of organs in ASC.

Combining analyses of metabolite profiles and phosphorus fractions to explore high phosphorus utilization efficiency in maize.

Phosphorus (P) limitation is a significant factor restricting crop production in agricultural systems, and enhancing the internal P utilization efficiency (PUE) of crops plays an important role in ensuring sustainable P use in agriculture. To better understand how P is remobilized to affect crop growth, we first screened P-efficient (B73 and GEMS50) and P-inefficient (Liao5114) maize genotypes at the same shoot P content, and then analyzed P pools and performed non-targeted metabolomic analyses to explore changes in cellular P fractions and metabolites in maize genotypes with contrasting PUE. We show that lipid P and nucleic acid P concentrations were significantly lower in lower leaves of P-efficient genotypes, and these P pools were remobilized to a major extent in P-efficient genotypes. Broad metabolic alterations were evident in leaves of P-efficient maize genotypes, particularly affecting products of phospholipid turnover and phosphorylated compounds, and the shikimate biosynthesis pathway. Taken together, our results suggest that P-efficient genotypes have a high capacity to remobilize lipid P and nucleic acid P and promote the shikimate pathway towards efficient P utilization in maize.

Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4.

Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA-protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.

Metabolic reprograming confers tamoxifen resistance in breast cancer.

Breast cancer is the most common cancer among females and the leading cause of cancer-related deaths. Approximately 70 % of breast cancers are estrogen receptor (ER) positive. An ER antagonist such as tamoxifen is used as adjuvant therapy in ER-positive patients. The major problem with endocrine therapy is the emergence of acquired resistance in approximately 40 % of patients receiving tamoxifen. Metabolic alteration is one of the hallmarks of cancer cells. Rapidly proliferating cancer cells require increased nutritional support to fuel various functions such as proliferation, cell migration, and metastasis. Recent studies have established that the metabolic state of cancer cells influences their susceptibility to chemotherapeutic drugs and that cancer cells reprogram their metabolism to develop into resistant phenotypes. In this review, we discuss the major findings on metabolic pathway alterations in tamoxifen-resistant (TAMR) breast cancer and the molecular mechanisms known to regulate the expression and function of metabolic enzymes and the respective metabolite levels upon tamoxifen treatment. It is anticipated that this in-depth analysis of specific metabolic pathways in TAMR cancer might be exploited therapeutically.

Rumen and Serum Metabolomes in Response to Endophyte-Infected Tall Fescue Seed and Isoflavone Supplementation in Beef Steers.

Fescue toxicosis impacts beef cattle production via reductions in weight gain and muscle development. Isoflavone supplementation has displayed potential for mitigating these effects. The objective of the current study was to evaluate isoflavone supplementation with fescue seed consumption on rumen and serum metabolomes. Angus steers (n = 36) were allocated randomly in a 2 × 2 factorial arrangement of treatments including endophyte-infected (E+) or endophyte-free (E-) tall fescue seed, with (P+) or without (P-) isoflavones. Steers were provided a basal diet with fescue seed for 21 days, while isoflavones were orally administered daily. Following the trial, blood and rumen fluid were collected for metabolite analysis. Metabolites were extracted and then analyzed by UPLC-MS. The MAVEN program was implemented to identify metabolites for MetaboAnalyst 4.0 and SAS 9.4 statistical analysis. Seven differentially abundant metabolites were identified in serum by isoflavone treatment, and eleven metabolites in the rumen due to seed type (p < 0.05). Pathways affected by treatments were related to amino acid and nucleic acid metabolism in both rumen fluid and serum (p < 0.05). Therefore, metabolism was altered by fescue seed in the rumen; however, isoflavones altered metabolism systemically to potentially mitigate detrimental effects of seed and improve animal performance.

Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes.

RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins. Abbreviations: ACTB/ß-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.

Metatranscriptomic exploration of microbial functioning in clouds.

Clouds constitute the uppermost layer of the biosphere. They host diverse communities whose functioning remains obscure, although biological activity potentially participates to atmospheric chemical and physical processes. In order to gain information on the metabolic functioning of microbial communities in clouds, we conducted coordinated metagenomics/metatranscriptomics profiling of cloud water microbial communities. Samples were collected from a high altitude atmospheric station in France and examined for biological content after untargeted amplification of nucleic acids. Living microorganisms, essentially bacteria, maintained transcriptional and translational activities and expressed many known complementary physiological responses intended to fight oxidants, osmotic variations and cold. These included activities of oxidant detoxification and regulation, synthesis of osmoprotectants/cryoprotectants, modifications of membranes, iron uptake. Consistently these energy-demanding processes were fueled by central metabolic routes involved in oxidative stress response and redox homeostasis management, such as pentose phosphate and glyoxylate pathways. Elevated binding and transmembrane ion transports demonstrated important interactions between cells and their cloud droplet chemical environments. In addition, polysaccharides, potentially beneficial for survival like exopolysaccharides, biosurfactants and adhesins, were synthesized. Our results support a biological influence on cloud physical and chemical processes, acting notably on the oxidant capacity, iron speciation and availability, amino-acids distribution and carbon and nitrogen fates.

In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR.

In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.

3'-End labeling of nucleic acids by a polymerase ribozyme.

A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways are currently being targeted for clinical application in oncology.

Advances in Stimulus-Responsive Polymeric Materials for Systemic Delivery of Nucleic Acids.

Polymeric materials that respond to a variety of endogenous and external stimuli are actively developed to overcome the main barriers to successful systemic delivery of therapeutic nucleic acids. Here, an overview of viable stimuli that are proved to improve systemic delivery of nucleic acids is provided. The main focus is placed on nucleic acid delivery systems (NADS) based on polymers that respond to pathological or physiological changes in pH, redox state, enzyme levels, hypoxia, and reactive oxygen species levels. Additional discussion is focused on NADS suitable for applications that use external stimuli, such as light, ultrasound, and local hyperthermia.

Antifungal Activity of Ramulus cinnamomi Explored by ¹H-NMR Based Metabolomics Approach.

A ¹H nuclear magnetic resonance (NMR)-based approach to metabolomics combined bioassay was used to elucidate the antifungal activity of cinnamaldehyde (the main active compound of Ramulus cinnamomi) isolated from Ramulus cinnamomi (RC). Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) of NMR data was constructed to analyze all the P. italicum data acquired from the control and treatment groups at 4, 8, and 12 h. Metabolic profiles disclosed metabolic changes that were related to the antifungal effects of cinnamaldehyde against P. italicum including oxidative stress, disorder of energy metabolism, amino acids, and nucleic acids metabolism in treatment group. This integrated metabolomics approach provided an effective way to detect the antifungal effects of cinnamaldehyde against P. italicum dynamically.

Performance and consistency of a fluorescence-based high-throughput screening assay for use in Babesia drug screening in mice.

In this study, we evaluated the validity of a fluorescence-based assay using SYBR Green I (SG I) stain for screening antibabesial compounds against B. microti in mice. Two different hematocrits (HCTs; 2.5% and 5%) were used. Correlating relative fluorescence units (RFUs) with parasitemia showed significant linear relationships with R2 values of 0.97 and 0.99 at HCTs of 2.5% and 5%, respectively. Meanwhile, the Z' factors in a high-throughput screening (HTS) assay were within the permissible limit (≥0.5) at 2.5% HCT and lower than this value at 5% HCT. Taken together, the highest signal-to-noise (S/N) ratios were obtained at 2.5% HCT; therefore, we concluded that 2.5% was the best HCT for applying fluorescence assay in antibabesial drug screening in mice. Additionally, positive control mice and those treated with diminazene aceturate, pyronaridine tetraphosphate, and an allicin/diminazene aceturate combination showed peak parasitemia and fluorescence values on the same day post-inoculation. Moreover, using different concentrations of SG I revealed that the optimal concentration was 2x. In summary, considering that all experiments were applied under optimal laboratory conditions, fluorescence assay at 2.5% HCT using 2x SG I for B. microti parasite offers a novel approach for drug screening in mice.

Molecular aspects of cancer chemopreventive and therapeutic efficacies of tea and tea polyphenols.

The natural dietary product tea (Camellia sinensis) and its bioactive polyphenols such as epigallocatechin gallate (EGCG) and theaflavin (TF) demonstrated potential anticancer effects in different preclinical and clinical studies. The aim of the present review was to understand the molecular mechanisms of the tea and tea polyphenol-mediated cancer prevention and therapy. In the setting of in vivo cancer prevention studies, administration of the tea and tea polyphenols at preinitiation stages only showed partial prevention, whereas continuous administration showed potential effect in restriction of carcinogenesis in the body's multiple organs at early premalignant stages throughout the experiment. Similar to different in vitro cancer cell models, treatment after initiation stages showed potential therapeutic efficacy in vivo. But, the mechanisms of prevention and therapy were found to be similar regardless of tea and its polyphenols. They mainly serve as antioxidants and induce the detoxification system, thereby inhibiting carcinogen metabolism and cancer initiation. Additionally, they could inhibit self-renewal, proliferation, and survival of the tumor-initiating population in restriction of the carcinogenesis progression from cancer initiation and promotion. This might be a result of the modulation of membrane organization, interaction with DNA/RNA/proteins and epigenetic modifications, as well as regulation of cellular replicative potential by the tea polyphenols.

Recent developments in deuterium oxide tracer approaches to measure rates of substrate turnover: implications for protein, lipid, and nucleic acid research.

PURPOSE OF REVIEW: Methods that inform on dynamic metabolism that can be applied to clinical populations to understand disease progression and responses to therapeutic interventions are of great importance. This review perspective will highlight recent advances, development, and applications of the multivalent stable isotope tracer deuterium oxide (D2O) to the study of substrate metabolism with particular reference to protein, lipids, and nucleic acids, and how these methods can be readily applied within clinical and pharmaceutical research. RECENT FINDINGS: Advances in the application of D2O techniques now permit the simultaneous dynamic measurement of a range of substrates (i.e. protein, lipid, and nucleic acids, along with the potential for OMICs methodologies) with minimal invasiveness further creating opportunities for long-term 'free living' measures that can be used in clinical settings. These techniques have recently been applied to ageing populations and further in cancer patients revealing altered muscle protein metabolism. Additionally, the efficacy of numerous drugs in improving lipoprotein profiles and controlling cellular proliferation in leukaemia have been revealed. SUMMARY: D2O provides opportunities to create a more holistic picture of in-vivo metabolic phenotypes, providing a unique platform for development in clinical applications, and the emerging field of personalized medicine.

Mycobacterium phlei cell wall-nucleic acid complex in the treatment of nonmuscle invasive bladder cancer unresponsive to bacillus Calmette-Guerin.

INTRODUCTION: Current guidelines recommend cystectomy in patients with high risk NMIBC who fail to respond to BCG. However due to the significant morbidity and mortality of the procedure, many are not candidates or refuse it. No new treatments for this indication have been approved by the US FDA since 1998. AREAS COVERED: A cell wall-nucleic acid complex (MCNA) from M. phlei has been investigated for possible application in patients with BCG refractory NMIBC. The development of this biological from the original studies is reviewed, together with the clinical trials leading to a submission to the FDA. Its efficacy and safety are presented together with comparative analysis of alternative treatments, most of which are used off-label. In addition, new combinations of standard therapies are described as well as single agents exhibiting activity against these tumors. EXPERT OPINION: MCNA has shown activity against high risk BCG refractory bladder cancer and offers an alternative to current treatments. The clinical experience remains limited and the optimal therapeutic regimen (dose, frequency) have not been firmly established. Patients and clinicians would welcome the introduction of a compound that may delay or prevent the risks and negative impact in quality of life of cystectomy and urinary diversion.

Application of liquid chromatography/electrospray ionization ion trap tandem mass spectrometry for the evaluation of global nucleic acids: methylation in garden cress under exposure to CuO nanoparticles.

RATIONALE: A full understanding of the biological impact of nanomaterials demands analytical procedures suitable for the detection/quantification of epigenetic changes that occur in the exposed organisms. Here, the effect of CuO nanoparticles (NPs) on global methylation of nucleic acids in Lepidium sativum was evaluated by liquid chromatography/ion trap mass spectrometry. Enhanced selectivity toward cytosine-containing nucleosides was achieved by using their proton-bound dimers formed in positive electrospray ionization (ESI(+)) as precursor ions for multiple reaction monitoring (MRM) quantification based on one or two ion transitions. METHODS: Plants were exposed to CuO NPs (0-1000 mg L(-1)); nucleic acid extracts were washed with bathocuproine disulfate; nucleosides were separated on a Luna C18 column coupled via ESI(+) to an AmaZon SL mass spectrometer (Bruker Daltonics). Cytidine, 2´-deoxycytidine, 5-methylcytidine, 5-methyl-2´-deoxycytidine and 5-hydroxymethyl-2´-deoxycytidine were quantified by MRM based on MS(3) ([2M+H](+)/[M+H](+)/[M+H-132](+) or [M+H-116](+)) and MS(2) ([2M+H](+)/[M+H](+) ). RESULTS: Bathocuproine disulfate, added as Cu(I) complexing agent, allowed for elimination of [2M+Cu](+) adducts from the mass spectra. Poorer instrumental detection limits were obtained for MS(3) (20-120 fmol) as compared to MS(2) (9.0-41 fmol); however, two ion transitions helped to eliminate matrix effects in plant extracts. The procedure was tested by analyzing salmon sperm DNA (Sigma) and applied for the evaluation of DNA and RNA methylation in plants; in the absence of NPs, 13.03% and 0.92% methylated cytosines were found in DNA and RNA, respectively; for NPs concentration >50 mg L(-1), DNA hypomethylation was observed with respect to unexposed plants. RNA methylation did not present significant changes upon plant exposure; 5-hydroxymethyl-2´-deoxycytidine was not detected in any sample. CONCLUSIONS: The MRM quantification proposed here of cytosine-containing nucleosides using their proton-bound homo-dimers as precursor ions proved its utility for the assessment of global methylation of DNA and RNA in plants under stress imposed by CuO NPs. Detection of copper adducts with cytosine-containing ions, and their elimination by washing extracts with Cu(I) chelator, calls for further investigation.

Cross-species conservation of complementary amino acid-ribonucleobase interactions and their potential for ribosome-free encoding.

The role of amino acid-RNA nucleobase interactions in the evolution of RNA translation and protein-mRNA autoregulation remains an open area of research. We describe the inference of pairwise amino acid-RNA nucleobase interaction preferences using structural data from known RNA-protein complexes. We observed significant matching between an amino acid's nucleobase affinity and corresponding codon content in both the standard genetic code and mitochondrial variants. Furthermore, we showed that knowledge of nucleobase preferences allows statistically significant prediction of protein primary sequence from mRNA using purely physiochemical information. Interestingly, ribosomal primary sequences were more accurately predicted than non-ribosomal sequences, suggesting a potential role for direct amino acid-nucleobase interactions in the genesis of amino acid-based ribosomal components. Finally, we observed matching between amino acid-nucleobase affinities and corresponding mRNA sequences in 35 evolutionarily diverse proteomes. We believe these results have important implications for the study of the evolutionary origins of the genetic code and protein-mRNA cross-regulation.