Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Distinctive metabolic profiles between Cystic Fibrosis mutational subclasses and lung function.

INTRODUCTION: Cystic fibrosis (CF) is a lethal multisystemic disease of a monogenic origin with numerous mutations. Functional defects in the cystic fibrosis transmembrane conductance receptor (CFTR) protein based on these mutations are categorised into distinct classes having different clinical presentations and disease severity. OBJECTIVES: The present study aimed to create a comprehensive metabolomic profile of altered metabolites in patients with CF, among different classes and in relation to lung function. METHODS: A chemical isotope labeling liquid chromatography-mass spectrometry metabolomics was used to study the serum metabolic profiles of young and adult CF (n = 39) patients and healthy controls (n = 30). Comparisons were made at three levels, CF vs. controls, among mutational classes of CF, between CF class III and IV, and correlated the lung function findings. RESULTS: A distinctive metabolic profile was observed in the three analyses. 78, 20, and 13 significantly differentially dysregulated metabolites were identified in the patients with CF, among the different classes and between class III and IV, respectively. The significantly identified metabolites included amino acids, di-, and tri-peptides, glutathione, glutamine, glutamate, and arginine metabolism. The top significant metabolites include 1-Aminopropan-2-ol, ophthalmate, serotonin, cystathionine, and gamma-glutamylglutamic acid. Lung function represented by an above-average FEV1% level was associated with decreased glutamic acid and increased guanosine levels. CONCLUSION: Metabolomic profiling identified alterations in different amino acids and dipeptides, involved in regulating glutathione metabolism. Two metabolites, 3,4-dihydroxymandelate-3-O-sulfate and 5-Aminopentanoic acid, were identified in common between the three anlayses and may represent as highly sensitive biomarkers for CF.

Rethinking experiments that explore multiple global change factors.

Our current capacity to predict the responses of ecosystem functions under global change factors is limited. We propose new and more efficient approaches to experimental design and modeling that utilize interactions between ecosystem functions to improve our understanding of their sensitivity to global change factors.

Effects of Water and Fertilizer Management Practices on Methane Emissions from Paddy Soils: Synthesis and Perspective.

Water and fertilizer management practices are considered to have great influence on soil methane (CH4) emissions from paddy fields. However, few studies have conducted a quantitative analysis of the effects of these management practices. Here, we selected 156 observations of water management from 34 articles and 288 observations of fertilizer management from 37 articles and conducted a global meta-analysis of the effects of water and fertilizer management practices on soil CH4 emissions in paddy fields. In general, compared with traditional irrigation (long-term flooding irrigation), water-saving irrigation significantly decreased soil CH4 emissions but increased rice yield. Among the different practices, intermittent irrigation had the fewest reductions in CH4 emissions but the greatest increase in rice yield. In addition, fertilization management practices such as manure, mixed fertilizer (mixture), and straw significantly enhanced CH4 emissions. Rice yields were increased under fertilization with a mixture, traditional fertilizer, and controlled release fertilizer. Our results highlight that suitable agricultural water and fertilizer management practices are needed to effectively reduce CH4 emissions while maintaining rice yields. We also put forward some prospects for mitigating soil CH4 emissions from paddy fields in the context of global warming in the future.

Reducing plant-derived ethylene concentrations increases the resistance of temperate grassland to drought.

Model predictions indicate that extreme drought events will occur more frequently by the end of this century, with major implications for terrestrial ecosystem functions such as plant productivity and soil respiration. Previous studies have shown that drought-induced ethylene produced by plants is a key factor affecting plant growth and development, but the impact of drought-induced ethylene on ecosystem functions in natural settings has not yet been tested. Here, we reduced the amount of plant-derived ethylene concentrations by adding the ethylene inhibitor aminoethoxyvinylglycine (AVG), and investigated in situ plant productivity, soil respiration and ethylene concentrations for two years in a semi-arid temperate grassland in Inner Mongolia, China. Drought significantly reduced plant productivity and soil respiration, but the application of AVG reduced ethylene concentrations and significantly increased aboveground plant productivity and soil respiration, effectively enhancing resistance to drought. The reason for this could be that AVG application increased the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and abundance of the acdS gene (the key gene for ACC deaminase), facilitating reduced ACC concentrations in the plant tissue and reduced in planta ethylene synthesis. In addition, there was a significant correlation between soil ACC deaminase activity and plant productivity. Given the global distribution of arid and semi-arid areas, and the expected increases in the frequency and intensity of drought stress, this is a significant concern. These results provide novel evidence of the impact of drought-induced plant ethylene production on ecosystem functions in semi-arid temperate grassland ecosystems.

Manipulation of soil methane oxidation under drought stress.

Drought events are predicted to occur more frequently, but comprehensive knowledge of their effects on methane (CH4) oxidation by soil methanotrophs in upland ecosystems remains elusive. Here, we put forward a new conceptual model in which drought influences soil CH4 oxidation through a direct pathway (i.e., positive effects of soil CH4 oxidation via increasing soil aeration) and through an indirect pathway (i.e., negative effects of in planta ethylene (C2H4) production on soil CH4 oxidation). Through measuring soil CH4 efflux along a gradient of drought stress, we found that drought increases soil CH4 oxidation, as the former outweighs the latter on soil CH4 oxidation, based on a mesocosm experiment employing distinct levels of watering and a long-term drought field trial created by rainfall exclusion in a subtropical evergreen forest. Moreover, we used aminoethoxyvinylglycine (AVG), a C2H4 biosynthesis inhibitor, to reduce in planta C2H4 production under drought, and found that reducing in planta C2H4 production increased soil CH4 oxidation under drought. To confirm these findings, we found that inoculation of plant growth-promoting rhizobacteria containing the 1-aminocyclopropane-1-carboxylate deaminase alleviated the negative effects of drought-induced in planta C2H4, thus increasing soil CH4 oxidation rates. All these results provide strong evidence for the hypothesis that in planta C2H4 production inhibits soil CH4 oxidation under drought. To our knowledge, this is the first study to manipulate the negative feedback between C2H4 production and CH4 oxidation under drought stress. Given the current widespread extent of arid and semiarid regions in the world, combined with the projected increased frequency of drought stress in future climate scenarios, we provide a reliable means for increasing soil CH4 oxidation in the context of global warming.

Soil aeration rather than methanotrophic community drives methane uptake under drought in a subtropical forest.

Little information is available about the effects of drought on soil methane (CH4) uptake and the underlying feedback of the soil microbial community in forest biomes. More importantly, a meta-analysis of the current literature on this topic revealed that there are virtually no data available in subtropical forests. To fill the abovementioned knowledge gap, we carried out a 3-year investigation of in situ CH4 efflux under drought in a subtropical forest, and found that drought significantly increased soil CH4 uptake (P < 0.001). However, drought did not change oxidation potentials and abundances of methanotrophs, and similar methanotrophic communities were observed between the drought and ambient control sites based on metagenomic sequencing analysis. Active methanotrophic communities were dominated by the genus Methylosinus based on DNA stable-isotope probing analysis. Structural equation model analysis indicated that direct drought-derived pathway, i.e., increasing soil aerations, outweighs the indirect pathway, i.e., altering methanotrophic communities and activities, and plays a predominant role in driving soil CH4 uptake in forest ecosystems. To our knowledge, our work is the first study to investigate the effects of drought on in situ CH4 efflux and the underlying microbial mechanisms in subtropical forests.

Metabolomics Profiling of Cystic Renal Disease towards Biomarker Discovery.

Cystic renal disease (CRD) comprises a heterogeneous group of genetic and acquired disorders. The cystic lesions are detected through imaging, either incidentally or after symptoms develop, due to an underlying disease process. In this study, we aim to study the metabolomic profiles of CRD patients for potential disease-specific biomarkers using unlabeled and labeled metabolomics using low and high-resolution mass spectrometry (MS), respectively. Dried-blood spot (DBS) and serum samples, collected from CRD patients and healthy controls, were analyzed using the unlabeled and labeled method. The metabolomics profiles for both sets of samples and groups were collected, and their data were processed using the lab's standard protocol. The univariate analysis showed (FDR p < 0.05 and fold change 2) was significant to show a group of potential biomarkers for CRD discovery, including uridine diphosphate, cystine-5-diphosphate, and morpholine. Several pathways were involved in CRD patients based on the metabolic profile, including aminoacyl-tRNA biosynthesis, purine and pyrimidine, glutathione, TCA cycle, and some amino acid metabolism (alanine, aspartate and glutamate, arginine and tryptophan), which have the most impact. In conclusion, early CRD detection and treatment is possible using a metabolomics approach that targets alanine, aspartate, and glutamate pathway metabolites.

Distinctive Metabolomics Patterns Associated With Insulin Resistance and Type 2 Diabetes Mellitus.

Obesity is associated with an increased risk of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) which is a multi-factorial disease associated with a dysregulated metabolism and can be prevented in pre-diabetic individuals with impaired glucose tolerance. A metabolomic approach emphasizing metabolic pathways is critical to our understanding of this heterogeneous disease. This study aimed to characterize the serum metabolomic fingerprint and multi-metabolite signatures associated with IR and T2DM. Here, we have used untargeted high-performance chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) to identify candidate biomarkers of IR and T2DM in sera from 30 adults of normal weight, 26 obese adults, and 16 adults newly diagnosed with T2DM. Among the 3633 peak pairs detected, 62% were either identified or matched. A group of 78 metabolites were up-regulated and 111 metabolites were down-regulated comparing obese to lean group while 459 metabolites were up-regulated and 166 metabolites were down-regulated comparing T2DM to obese groups. Several metabolites were identified as IR potential biomarkers, including amino acids (Asn, Gln, and His), methionine (Met) sulfoxide, 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate, serotonin, L-2-amino-3-oxobutanoic acid, and 4,6-dihydroxyquinoline. T2DM was associated with dysregulation of 42 metabolites, including amino acids, amino acids metabolites, and dipeptides. In conclusion, these pilot data have identified IR and T2DM metabolomics panels as potential novel biomarkers of IR and identified metabolites associated with T2DM, with possible diagnostic and therapeutic applications. Further studies to confirm these associations in prospective cohorts are warranted.

Rare rather than abundant microbial communities drive the effects of long-term greenhouse cultivation on ecosystem functions in subtropical agricultural soils.

Long-term greenhouse cultivation has an adverse effect on ecosystem functions such as soil carbon (C) and nitrogen (N) pools and greenhouse gas (GHG) emissions, but the underlying microbial mechanisms still remain unclear. Here, different sites under long-term greenhouse cultivation in a subtropical agricultural ecosystem were selected to measure soil C and N contents, extractable organic C (EOC) and N (EON) contents, and potential GHG emissions. Metagenomic analysis and 16S rRNA high-throughput sequencing were used to measure microbial communities. The results showed that long-term greenhouse cultivation increased soil salinity, and significantly increased soil total C and N contents, EOC and EON contents, and N2O emission potentials, although it significantly decreased CO2 emission and CH4 oxidation potential compared with the ambient control. Changes in soil CH4 oxidation and N2O emission potential exhibited similar patterns in the corresponding key functional genes based on according to our metagenomic analysis. In addition, long-term greenhouse cultivation did not change microbial diversity, although it clearly affected soil microbial community composition. Soil microbial communities were further classified into rare and abundant microbial taxa. Rare rather than abundant microbial taxa could adequately explain the changes in ecosystem functions, except for CH4 oxidation potential across the treatments. To our knowledge, this is the first study to quantify the importance of microbial subcommunities to ecosystem functions on the basis of microbial co-occurrence network analysis under greenhouse cultivation in agricultural ecosystems. Overall, our results indicated that rare rather than abundant microbial taxa could act as indicators of variations in ecosystem functions under long-term greenhouse cultivation in subtropical agricultural soils, which might be useful for better management practices and improving crop yields in agricultural ecosystems.

Obesity Connected Metabolic Changes in Type 2 Diabetic Patients Treated With Metformin.

Metformin is widely used in the treatment of Type 2 Diabetes Mellitus (T2DM). However, it is known to have beneficial effects in many other conditions, including obesity and cancer. In this study, we aimed to investigate the metabolic effect of metformin in T2DM and its impact on obesity. A mass spectrometry (MS)-based metabolomics approach was used to analyze samples from two cohorts, including healthy lean and obese control, and lean as well as obese T2DM patients on metformin regimen in the last 6 months. The results show a clear group separation and sample clustering between the study groups due to both T2DM and metformin administration. Seventy-one metabolites were dysregulated in diabetic obese patients (30 up-regulated and 41 down-regulated), and their levels were unchanged with metformin administration. However, 30 metabolites were dysregulated (21 were up-regulated and 9 were down-regulated) and then restored to obese control levels by metformin administration in obese diabetic patients. Furthermore, in obese diabetic patients, the level of 10 metabolites was dysregulated only after metformin administration. Most of these dysregulated metabolites were dipeptides, aliphatic amino acids, nucleic acid derivatives, and urea cycle components. The metabolic pattern of 62 metabolites was persistent, and their levels were affected by neither T2DM nor metformin in obesity. Interestingly, 9 metabolites were significantly dysregulated between lean and obese cohorts due to T2DM and metformin regardless of the obesity status. These include arginine, citrulline, guanidoacetic acid, proline, alanine, taurine, 5-hydroxyindoleacetic acid, and 5-hydroxymethyluracil. Understanding the metabolic alterations taking place upon metformin treatment would shed light on possible molecular targets of metformin, especially in conditions like T2DM and obesity.

Metabolomics Distinguishes DOCK8 Deficiency from Atopic Dermatitis: Towards a Biomarker Discovery.

Bi-allelic mutations in the dedicator of cytokinesis 8 (DOCK8) are responsible for a rare autosomal recessive primary combined immunodeficiency syndrome, characterized by atopic dermatitis, elevated serum Immunoglobulin E (IgE) levels, recurrent severe cutaneous viral infections, autoimmunity, and predisposition to malignancy. The molecular link between DOCK8 deficiency and atopic skin inflammation remains unknown. Severe atopic dermatitis (AD) and DOCK8 deficiency share some clinical symptoms, including eczema, eosinophilia, and increased serum IgE levels. Increased serum IgE levels are characteristic of, but not specific to allergic diseases. Herein, we aimed to study the metabolomic profiles of DOCK8-deficient and AD patients for potential disease-specific biomarkers using chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS). Serum samples were collected from DOCK8-deficient (n = 10) and AD (n = 9) patients. Metabolomics profiling using CIL LC-MS was performed on patient samples and compared to unrelated healthy controls (n = 33). Seven metabolites were positively identified, distinguishing DOCK8-deficient from AD patients. Aspartic acid and 3-hydroxyanthranillic acid (3HAA, a tryptophan degradation pathway intermediate) were up-regulated in DOCK8 deficiency, whereas hypotaurine, leucyl-phenylalanine, glycyl-phenylalanine, and guanosine were down-regulated. Hypotaurine, 3-hydroxyanthranillic acid, and glycyl-phenyalanine were identified as potential biomarkers specific to DOCK8 deficiency. Aspartate availability has been recently implicated as a limiting metabolite for tumour growth and 3HAA; furthermore, other tryptophan metabolism pathway-related molecules have been considered as potential novel targets for cancer therapy. Taken together, perturbations in tryptophan degradation and increased availability of aspartate suggest a link of DOCK8 deficiency to oncogenesis. Additionally, perturbations in taurine and dipeptides metabolism suggest altered antixidation and cell signaling states in DOCK8 deficiency. Further studies examining the mechanisms underlying these observations are necessary.

Development of a simple and efficient method of harvesting and lysing adherent mammalian cells for chemical isotope labeling LC-MS-based cellular metabolomics.

Chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) has a potential to become a very powerful platform for comprehensive and quantitative analysis of metabolites in cellular metabolomics. While CIL LC-MS with rationally designed reagents for labeling can provide much enhanced detection and quantification of metabolites, pre-analytical process such as cell harvest and cell lysis is also a critical step in the overall workflow for metabolomic analysis. In this study, we examined various combinations of cell harvest and cell lysis methods in order to develop a simple and efficient method for harvesting and lysing adherent mammalian cells tailored to CIL LC-MS-based cellular metabolomics. We evaluated and compared two cell harvest methods (physical scraping and trypsinization) and two cell lysis methods (glass-bead-assisted lysis and freeze-thaw-cycle lysis). We used two types of commonly used mammalian cells, HeLa and MCF-7 cells, to cross-validate the findings. LC-UV quantification of the labeled metabolites after dansylation of cell extracts was used to compare the overall cell harvest and cell lysis efficiencies among different combination methods. It was found that scraping combined with freeze-thaw-cycle lysis gave the highest total metabolite concentration. We also performed multivariate and univariate analyses of the amine/phenol submetabolome data generated from different preparations to investigate the impact of cell harvest and lysis methods on cellular metabolome profiles. Comparing to scraping, trypsinization caused more significant metabolome changes likely due to metabolite leakage and metabolite level changes. The cellular metabolomes obtained from the two lysis methods were found to be similar; however, freeze-thaw-cycle lysis gave a higher lysis efficacy, compared to the glass bead method. We concluded that the combination of scraping and freeze-thaw-cycle was optimal for harvesting and lysing adherent mammalian cells for CIL LC-MS metabolomics.

The hydrophilicity vs. ion interaction selectivity plot revisited: The effect of mobile phase pH and buffer concentration on hydrophilic interaction liquid chromatography selectivity behavior.

This work systematically investigates the selectivity changes on many HILIC phases from w(w)pH 3.7-6.8, at 5 and 25mM buffer concentrations. Hydrophilicity (kcytosine/kuracil) vs. ion interaction (kBTMA/kuracil) selectivity plots developed by Ibrahim et al. (J. Chromatogr. A 1260 (2012) 126-131) are used to investigate the effect of mobile phase changes on the selectivity of 18 HILIC columns from various classes. "Selectivity change plots" focus on the change in hydrophilicity and ion interaction that the columns exhibit upon changing mobile phase conditions. In general, the selectivity behavior of most HILIC columns is dominated by silanol activity. Minimal changes in selectivity are observed upon changing pH between w(w)pH 5 and 6.8. However, a reduction in ionic interaction is observed when the buffer concentration is increased at w(w)pH≥5.0 due to ionic shielding. Reduction of the w(w)pH to<5.0 results in decreasing cation exchange activity due to silanol protonation. Under all eluent conditions, the majority of phases show little change in their hydrophilicity.