Small molecule agonist of mitochondrial fusion repairs mitochondrial dysfunction.

Membrane dynamics are important to the integrity and function of mitochondria. Defective mitochondrial fusion underlies the pathogenesis of multiple diseases. The ability to target fusion highlights the potential to fight life-threatening conditions. Here we report a small molecule agonist, S89, that specifically promotes mitochondrial fusion by targeting endogenous MFN1. S89 interacts directly with a loop region in the helix bundle 2 domain of MFN1 to stimulate GTP hydrolysis and vesicle fusion. GTP loading or competition by S89 dislodges the loop from the GTPase domain and unlocks the molecule. S89 restores mitochondrial and cellular defects caused by mitochondrial DNA mutations, oxidative stress inducer paraquat, ferroptosis inducer RSL3 or CMT2A-causing mutations by boosting endogenous MFN1. Strikingly, S89 effectively eliminates ischemia/reperfusion (I/R)-induced mitochondrial damage and protects mouse heart from I/R injury. These results reveal the priming mechanism for MFNs and provide a therapeutic strategy for mitochondrial diseases when additional mitochondrial fusion is beneficial.

Green tea powder inclusion promoted hatchability through increased yolk antioxidant activity.

Dietary supplementation of green tea powder (GTP) changes egg quality of hens, however, whether these changes affect incubation is still unknown. This study was to compare the proteomic difference of incubated eggs from hens with GTP supplemented or not. Huainan partridge chickens (1,080) at 35 wk of age were allocated into 2 groups, one group fed basal diet (CG) and one group fed basal diet plus 1% GTP (EG). After 4 wk feeding, artificially fertilized eggs were collected for yolk cholesterol determination and incubation. During incubation, 6 embryos from each group were randomly selected in each day for yolk protein extraction and quantification. Yolk cholesterol content was significantly lower, while the hatchability was significantly higher in EG than that of the CG group (P < 0.05). Yolk protein concentration at embryonic days (ED) of 0, 2, 6, and 13 showed significant changes and were selected for proteomic analysis by 2-dimensional gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Fifty-one differentially expressed (DE) protein spots were identified among different incubation stages between CG and EG group which were mainly classified into vitellogenin, immunoglobulin, and ovoinhibitor, and occupied 45.1, 23.5, and 15.7%, respectively, to the total DE proteins. Ovotransferrin, participated in extracellular sequestering of iron ion process, was significantly lower in EG group than that of the CG group (P < 0.05). Ig light chain precursor (Immunoglobulin) exhibited higher expression at ED6 in EG group as compared with that of the CG group, and was participated in immune response related processes. Ovoinhibitor, mainly involved in protease binding activity, showed lower abundance at ED13 in EG group as compared with that of the CG group. Vitellogenin-3, showed lower expression in EG group as compared with that of the CG group, was mainly participated in lipid transportation and localization according to GO enrichment. Chickens fed diet with GTP provided eggs more antioxidant ability that increased hatchability, indicated that GTP could be considered as additive in breeding layer.

Revisiting imidazolium receptors for the recognition of anions: highlighted research during 2010-2019.

Imidazolium based receptors selectively recognize anions, and have received more and more attention. In 2006 and 2010, we reviewed the mechanism and progress of imidazolium salt recognition of anions, respectively. In the past ten years, new developments have emerged in this area, including some new imidazolium motifs and the identification of a wider variety of biological anions. In this review, we discuss the progress of imidazolium receptors for the recognition of anions in the period of 2010-2019 and highlight the trends in this area. We first classify receptors based on motifs, including some newly emerging receptors, as well as new advances in existing receptor types at this stage. Then we discuss separately according to the types of anions, including ATP, GTP, DNA and RNA.

Chemosensing of Guanosine Triphosphate Based on a Fluorescent Dinuclear Zn(II)-Dipicolylamine Complex in Water.

Guanosine triphosphate (GTP) is a key biomarker of multiple cellular processes and human diseases. The new fluorescent dinuclear complex [Zn2(L)(S)][OTf]4, 1 (asymmetric ligand, L = 5,8-Bis{[bis(2-pyridylmethyl)amino] methyl}quinoline, S = solvent, and OTf = triflate anion) was synthesized and studied in-depth as a chemosensor for nucleoside polyphosphates and inorganic anions in pure water. Additions at neutral pH of nucleoside triphosphates, guanosine diphosphate, guanosine monophosphate, and pyrophosphate (PPi) to 1 quench its blue emission (λem = 410 nm) with a pronounced selectivity toward GTP over other anions, including adenosine triphosphate (ATP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The efficient quenching response by the addition of GTP was observed in the presence of coexisting species in blood plasma and urine with a detection limit of 9.2 µmol L-1. GTP also shows much tighter binding to the receptor 1 on a submicromolar level. On the basis of multiple spectroscopic tools (1H, 31P NMR, UV-vis, and fluorescence) and DFT calculations, the binding mode is proposed through three-point recognition involving the simultaneous coordination of the N7 atom of the guanosine motif and two phosphate groups to the two Zn(II) atoms. Spectroscopic studies, MS-ESI, and DFT suggested that GTP bound to 1 in 1:1 and 2:2 models with high overall binding constants of log ß1 (1:1) = 6.05 ± 0.01 and log ß2 = 10.91 ± 0.03, respectively. The optical change and selectivity are attributed to the efficient binding of GTP to 1 by the combination of a strong electrostatic contribution and synergic effects of coordination bonds. Such GTP selectivity of an asymmetric metal-based receptor in water is still rare.

Plasma-treated carbon-fiber microelectrodes for improved purine detection with fast-scan cyclic voltammetry.

Here, we developed N2 and O2 plasma-treated carbon-fiber microelectrodes (CFME) for improved purine detection with fast-scan cyclic voltammetry (FSCV). Plasma treatment affects the topology and functionality of carbon which impacts the electrode-analyte interaction. CFME's are less sensitive to purines compared to catecholamines. Knowledge of how the electrode surface drives purine-electrode interaction would provide insight into methods to improve purine detection. Here, plasma-treated CFME's with N2 and O2 plasma was used to investigate the extent to which the surface functionality and topology affects purine detection and to improve purine sensing with FSCV. On average, O2 plasma increased the oxidative current for adenosine and ATP by 6.0 ± 1.2-fold and 6.4 ± 1.6-fold, and guanosine and GTP by 2.8 ± 0.47-fold and 5.8 ± 1.4-fold, respectively (n = 9). The O2 plasma increased the surface roughness and oxygen functionality. N2 plasma increased the oxidative current for adenosine and ATP by 1.5 ± 0.15-fold and 1.9 ± 0.23-fold, and guanosine and GTP by 1.4 ± 0.20-fold and 1.5 ± 0.20-fold, respectively (n = 11). N2 plasma increased the nitrogen functionality with minimal increases in roughness. Both plasma treatments impacted purines more than dopamine. Langmuir isotherms revealed that both plasma gases impact the theoretical surface coverage and adsorption strength of purines at the electrode. Overall, we show that purine detection is improved at surfaces with increased surface roughness, and oxygen and amine functionality. Plasma-treated CFMEs could be used in the future to study the analyte-electrode interaction of other neurochemicals.

Determination of the Global Pattern of Gene Expression in Yeast Cells by Intracellular Levels of Guanine Nucleotides.

Correlations between gene transcription and the abundance of high-energy purine nucleotides in Saccharomyces cerevisiae have often been noted. However, there has been no systematic investigation of this phenomenon in the absence of confounding factors such as nutrient status and growth rate, and there is little hard evidence for a causal relationship. Whether transcription is fundamentally responsive to prevailing cellular energetic conditions via sensing of intracellular purine nucleotides, independently of specific nutrition, remains an important question. The controlled nutritional environment of chemostat culture revealed a strong correlation between ATP and GTP abundance and the transcription of genes required for growth. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into S. cerevisiae, permitting analysis of the transcriptional effect of an increased demand for these nucleotides. During steady-state growth using the fermentable carbon source glucose, the futile consumption of ATP led to a decrease in intracellular ATP concentration but an increase in GTP and the guanylate energy charge (GEC). Expression of transcripts encoding proteins involved in ribosome biogenesis, and those controlled by promoters subject to SWI/SNF-dependent chromatin remodelling, was correlated with these nucleotide pool changes. Similar nucleotide abundance changes were observed using a nonfermentable carbon source, but an effect on the growth-associated transcriptional programme was absent. Induction of the GTP-cycling pathway had only marginal effects on nucleotide abundance and gene transcription. The transcriptional response of respiring cells to glucose was dampened in chemostats induced for ATP cycling, but not GTP cycling, and this was primarily associated with altered adenine nucleotide levels.IMPORTANCE This paper investigates whether, independently of the supply of any specific nutrient, gene transcription responds to the energy status of the cell by monitoring ATP and GTP levels. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into the yeast Saccharomyces cerevisiae, and the effect of an increased demand for these purine nucleotides on gene transcription was analyzed. The resulting changes in transcription were most consistently associated with changes in GTP and GEC levels, although the reprogramming in gene expression during glucose repression is sensitive to adenine nucleotide levels. The results show that GTP levels play a central role in determining how genes act to respond to changes in energy supply and that any comprehensive understanding of the control of eukaryotic gene expression requires the elucidation of how changes in guanine nucleotide abundance are sensed and transduced to alter the global pattern of transcription.

New detection method for nucleoside triphosphates based on carbon dots: The distance-dependent singlet oxygen trapping.

The distance-dependent based sensing mechanism, such as fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) absorption of gold nanoparticles, has been used widely in visual detection. In this work, we report another distance-dependent detection method for nucleoside triphosphates (NTPs) based on carbon dots (CDs) (1O2 donor) and 9, 10-diphenylanthracene-2-boronic acid (DABA, 1O2 acceptor). The CDs can generate singlet oxygen (1O2) which allows diffusion within 200 nm. Thus, the distance between CDs and DABA decreased through binding of NTPs (<200 nm), leading to absorption changes of DABA under light irradiation due to 1O2 trapping. This sensing system (CDs@DABA) has high selectivity for the detection of NTPs due to the double molecular recognition and a linear response in the 0-80 µM concentration range was accomplished with the detection limit as low as 4.35 µM.

Frailty Status in Older Adults Is Related to Alterations in Indoleamine 2,3-Dioxygenase 1 and Guanosine Triphosphate Cyclohydrolase I Enzymatic Pathways.

BACKGROUND: Frailty is a multidimensional syndrome correlated to the loss of homeostasis and increased vulnerability to stressors, which is associated with increase in the risk of disability, comorbidity, hospitalization, and death in the elderly. It is based on the interplay of physiological, psychological, social, and environmental factors. OBJECTIVES: Because aging involves a detrimental immune response, this work aimed to assess the possible role of chronic low-grade immune stimulation on frailty status in the elderly. METHODS: Biomarkers involved in indoleamine 2,3-dioxygenase 1 and guanosine triphosphate cyclohydrolase I enzymatic pathways (namely neopterin, tryptophan, kynurenine, phenylalanine, tyrosine, and nitrite) were analyzed in a population of Spanish older adults aged 65 years and above, and their relationships with frailty status were evaluated. RESULTS: Significant increases in neopterin levels, kynurenine/tryptophan ratio, and phenylalanine/tyrosine ratio, and significant decreases in tryptophan, nitrite and tyrosine concentrations in frail individuals compared with nonfrail persons were obtained. Significant correlations were also observed between immune biomarkers, indicating they change in parallel, thus, pointing to interrelated causes. Besides, reference ranges for a number of immune biomarkers in the population of robust older adults were established for the first time. CONCLUSIONS: Results obtained in the present study are consistent with the idea that frailty status in the elderly is associated with an additional degree of immune stimulation, manifested in a more intense disturbance of indoleamine 2,3-dioxygenase 1 and guanosine triphosphate cyclohydrolase I pathways than in nonfrail or prefrail older adults.

Association between housing type and γ-GTP increase after the Great East Japan Earthquake.

BACKGROUND: It has been reported that alcohol consumption increases after natural disasters, with an impact on health. However, the impact of relocation upon drinking behavior has been unclear. The aim of this study was to clarify the association between housing type and the impact of alcohol consumption on health after the Great East Japan Earthquake (GEJE) of 2011. METHODS: We analyzed 569 residents living in devastated areas of Ishinomaki city, who had undergone assessment of their γ-GTP levels at health check-ups in both 2010 and 2013, and had given details of the type of housing they occupied in 2013. The housing types were categorized into five groups: "same housing as that before the GEJE", "prefabricated temporary housing", "privately rented temporary housing/rental housing", "homes of relatives", and "reconstructed housing". We used fixed-effect regression analysis to examine the association between housing type after the GEJE and changes in γ-GTP after adjustment for age, BMI, housing damage, number of people in household, smoking status, presence of illness, psychological distress, and social network. RESULTS: The mean age of the participants was 71.5 years and 46.2% of them were men. The proportion of individuals who drank heavily, and suffered from psychological distress and insomnia, was highest among those living in privately rented temporary housing/rental housing. Compared with individuals who continued to occupy the same housing as those before the GEJE, the effect of change in γ-GTP was significantly higher in individuals who had moved to privately rented temporary housing/rental housing (b = 9.5, SE = 4.4, p < 0.05). CONCLUSION: Our present findings reveal that disaster victims who have moved to privately rented temporary housing/rental housing are at highest risk of negative health effects due to alcohol drinking.

Luminescent vesicular nanointerface: a highly selective and sensitive "turn-on" sensor for guanosine triphosphate.

A novel amphiphilic Tb(3+) complex (TbL(3+)(I)) consisting of a +3 charged head and a hydrophobic alkyl chain has been developed. It spontaneously self-assembles in water and forms stable vesicles at neutral pH. TbL(3+)(I) has no aromatic groups (functioning as an antenna), and its intrinsic luminescence is thus minimized. These features lead to the self-assembling TbL(3+)(I) receptor molecules demonstrating an increased luminescence intensity upon binding of nucleotides. Upon addition of guanosine triphosphate (GTP), the luminescence from Tb(3+) was notably promoted (127-fold), as the light energy absorbed by the guanine group of GTP was efficiently transferred to the Tb(3+) center. In the case of guanosine diphosphate (GDP) and guanosine monophosphate (GMP), respectively, 78-fold and 43-fold increases in luminescence intensity were observed. This enhancement was less significant than that observed for GTP, due to fewer negative charges on GDP and GMP. No other nucleotides or the tested nonphosphorylated nucleosides affected the luminescence intensity to any notable extent. In marked contrast, all tested nucleotides, including guanine nucleotides, barely promoted the luminescence of molecularly dispersed receptors, TbL(3+)(II), indicating that the confinement and organization of molecules in a nanointerface play vital roles in improving the performance of a sensing system. This Tb(3+) complex nanointerface is successfully used for monitoring the GTP-to-GDP conversion.

In situ simultaneous monitoring of ATP and GTP using a graphene oxide nanosheet-based sensing platform in living cells.

Here we present a detailed protocol for in situ multiple fluorescence monitoring of adenosine-5'-triphosphate (ATP) and guanosine-5'-triphosphate (GTP) in MCF-7 breast cancer cells by using graphene oxide nanosheet (GO-nS) and DNA/RNA aptamers. FAM-labeled ATP aptamer and Cy5-modified GTP aptamer are used to construct the multiple aptamer/GO-nS sensing platform through 'π-π stacking' between aptamers and GO-nS. Binding of aptamers to GO-nS guarantees the fluorescence resonance energy transfer between fluorophores and GO-nS, resulting in 'fluorescence off'. When the aptamer/GO-nS are transported inside the cells via endocytosis, the conformation of the aptamers will change on interaction with cellular ATP and GTP. On the basis of the fluorescence 'off/on' switching, simultaneous sensing and imaging of ATP and GTP in vitro and in situ have been realized through fluorescence and confocal microscopy techniques. In this protocol, we describe the synthesis of GO and GO-nS, preparation of aptamer/GO-nS platform, in vitro detection of ATP and GTP, and how to use this platform to realize intracellular ATP and GTP imaging in cultured MCF-7 cells. The preparation of GO-nS is anticipated to take 7-14 d, and assays involving microscopy imaging and MCF-7 cells culturing can be performed in 2-3 d.

Self-phosphorylating deoxyribozyme initiated cascade enzymatic amplification for guanosine-5'-triphosphate detection.

The self-phosphorylating deoxyribozymes identified by in vitro selection can catalyze their own phosphorylation by utilizing phosphate donor guanosine-5'-triphosphate (GTP) which plays a critical role in a majority of cellular processes. On the basis of the unique properties of self-phosphorylating deoxyribozymes, we report a novel GTP sensor coupled with λ exonuclease cleavage reaction and nicking enzyme assisted fluorescence signal amplification process. The deoxyribozymes with special catalytic and structural characteristics display good stability compared to protein and RNA enzymes. We combined these properties with enzymatic recycling cleavage strategy to build a sensor which produced enhanced fluorescence signal. Sensitive and selective detection of GTP was successfully realized with the well-designed deoxyribozyme-based sensing platform by taking advantage of the self-phosphorylating ability of the kinase deoxyribozyme, efficient digestion capacity of λ exonuclease, and enzymatic recycling amplification of nicking enzyme. The method not only provides a platform for detecting GTP but also shows great potential in analyzing a variety of targets by combining deoxyribozymes with signal amplification strategy.

A sensitive colorimetric and fluorescent sensor based on imidazolium-functionalized squaraines for the detection of GTP and alkaline phosphatase in aqueous solution.

Imidazolium-functionalized squaraine ImSQ8 is synthesized as a sensitive colorimetric and fluorescent chemosensor for GTP in aqueous solution. The detection limit of GTP reaches 5.4 ppb. Its applications in the live-cell imaging and enzyme activity assay have also been demonstrated.

New Anticancer Target Identified: Cancer cells need MTH1 enzyme to thrive; inhibiting it kills them

No disponible

ATP and GTP hydrolysis assays (TLC).

Many biochemical reactions that occur within the cell are thermodynamically unfavorable. However, when these reactions are coupled to NTP (nucleoside triphosphate) hydrolysis, the energy derived from the hydrolysis of the phosphodiester bond helps drive the reaction in the favorable direction. Examples of such proteins can be found in almost all facets of cellular metabolism: glycolysis and the TCA cycle, protein biosynthesis, DNA and RNA metabolism, cellular trafficking, cell signaling, cell division etc. Thus, characterization of the NTPase activity of these proteins in vitro can help in understanding the role of the protein in complex cellular processes.

Simultaneous quantification of energetically important metabolites in various cell types by CZE.

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

Selective detection of guanosine-5'-triphosphate and iodide by fluorescent benzimidazolium-based cyclophanes.

New fluorescent benzimidazolium-based receptors selectively display the effective fluorescence quenching effect for biologically important anions, GTP and I(-), in aqueous solution of physiological pH 7.4. These affinities can be attributed to the strong ionic H-bonding along with additional interactions of fluorophore moieties with the nucleic base of GTP and I(-).

Turning a kinase deoxyribozyme into a sensor.

The vast majority of deoxyribozyme-based sensors are designed using modified RNA-cleaving deoxyribozymes and detect analytes that act as allosteric regulators of their catalytic activity. These sensors are susceptible to background signals due to catalytic activity in the absence of target or contaminant molecules that cleave the RNA substrate, mimicking the deoxyribozyme reaction. In this manuscript, we introduce a novel system that avoids these problems by using the analyte as the substrate for a deoxyribozyme catalyzed self-phosphorylation reaction. This reaction creates a modified deoxyribozyme product that can be circularized and subjected to massive signal amplification by rolling circle amplification, leading to a sensor system with high sensitivity and low background, which can be coupled to numerous reporter systems. As an example of the potential of this system, we used the self-phosphorylating deoxyribozyme Dk2 to detect as little as 25 nM GTP even in the presence of 1 mM ATP, a potential contaminant. To demonstrate the adaptive properties of this system, we appended another DNA sequence to Dk2, which, once amplified by RCA, codes for a fluorescence generating deoxyribozyme. This two-deoxyribozyme system was able to report the presence of GTP from 4 µM to 1 mM, with specificity over other NTP molecules. Using this model system, we were able to show that small molecule modifying deoxyribozymes can be converted to analyte sensors by coupling their catalytic activity to signal amplification and reporting.

Simultaneous detection of ATP and GTP by covalently linked fluorescent ribonucleopeptide sensors.

A noncovalent RNA complex embedding an aptamer function and a fluorophore-labeled peptide affords a fluorescent ribonucleopeptide (RNP) framework for constructing fluorescent sensors. By taking an advantage of the noncovalent properties of the RNP complex, the ligand-binding and fluorescence characteristics of the fluorescent RNP can be independently tuned by taking advantage of the nature of the RNA and peptide subunits, respectively. Fluorescent sensors tailored for given measurement conditions, such as a detection wavelength and a detection concentration range for a ligand of interest can be easily identified by screening of fluorescent RNP libraries. The noncovalent configuration of a RNP becomes a disadvantage when the sensor is to be utilized at very low concentrations or when multiple sensors are applied to the same solution. Here, we report a strategy to convert a fluorescent RNP sensor in the noncovalent configuration into a covalently linked stable fluorescent RNP sensor. This covalently linked fluorescent RNP sensor enabled ligand detection at a low sensor concentration, even in cell extracts. Furthermore, application of both ATP and GTP sensors enabled simultaneous detection of ATP and GTP by monitoring each wavelength corresponding to the respective sensor. Importantly, when a fluorescein-modified ATP sensor and a pyrene-modified GTP sensor were co-incubated in the same solution, the ATP sensor responded at 535 nm only to changes in the concentration of ATP, whereas the GTP sensor detected GTP at 390 nm without any effect on the ATP sensor. Finally, simultaneous monitoring by these sensors enabled real-time measurement of adenosine deaminase enzyme reactions.

A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).