ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

Orchestration of phosphate homeostasis by the ITPK1-type inositol phosphate kinase in the liverwort Marchantia polymorpha.

Land plants have evolved sophisticated sensing mechanisms and signalling pathways to adapt to phosphate-limited environments. While molecular players contributing to these adaptations in flowering plants have been described, how non-vascular bryophytes regulate phosphate (Pi) homeostasis remained largely unknown. In this study, we present findings that both male and female plants of the liverwort Marchantia polymorpha respond to altered phosphate availability through substantial developmental changes. We show that the second messenger inositol pyrophosphates (PP-InsPs) respond more quickly to changes in cellular Pi status than the lower inositol phosphates, highlighting a functional relationship between PP-InsP and Pi homeostasis in M. polymorpha. To further corroborate the possible involvement of PP-InsP in Pi homeostasis, we characterized M. polymorpha INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (MpITPK1) that phosphorylates InsP6 to generate InsP7 both in vitro and in vivo. Consistent with the role of PP-InsPs in Pi homeostasis, M. polymorpha lines with enhanced MpITPK1 expression leading to the accumulation of 5-InsP7 and an InsP8 isomer exhibit altered expression of phosphate starvation induced (PSI) genes and display attenuated responses to low phosphate. The characterization of MpPHO1-deficient plants with dramatically increased levels of 1,5-InsP8 further supports the role of PP-InsP in Pi homeostasis in this liverwort species. Notably, our study unveiled that MpITPK1 rescues the deregulated Pi homeostasis in Arabidopsis (Arabidopsis thaliana) ITPK1-deficient plants, suggesting that liverwort and eudicots share a functional ITPK1 homolog. In summary, our study provides insights into the regulation of Pi homeostasis by ITPK1-derived PP-InsPs in M. polymorpha.

Comprehensive Profiling of Rapamycin Interacting Proteins with Multiple Mass Spectrometry-Based Omics Techniques.

Profiling drug-protein interactions is critical for understanding a drug's mechanism of action and predicting the possible adverse side effects. However, to comprehensively profile drug-protein interactions remains a challenge. To address this issue, we proposed a strategy that integrates multiple mass spectrometry-based omics analysis to provided global drug-protein interactions, including physical interactions and functional interactions, with rapamycin (Rap) as a model. Chemoproteomics profiling reveals 47 Rap binding proteins including the known target protein FKBP12 with high confidence. Gen Ontology enrichment analysis suggested that the Rap binding proteins are implicated in several important cellular processes, such as DNA replication, immunity, autophagy, programmed cell death, aging, transcription modulation, vesicle-mediated transport, membrane organization, and carbohydrate and nucleobase metabolic processes. The phosphoproteomics profiling revealed 255 down-regulated and 150 up-regulated phosphoproteins responding to Rap stimulation; they mainly involve the PI3K-Akt-mTORC1 signaling axis. Untargeted metabolomic profiling revealed 22 down-regulated metabolites and 75 up-regulated metabolites responding to Rap stimulation; they are mainly associated with the synthesis processes of pyrimidine and purine. The integrative multiomics data analysis provides deep insight into the drug-protein interactions and reveals Rap's complicated mechanism of action.

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers.

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-ß-phosphate of inositol pyrophosphates and only minor activity against the ß-phosphates of 4-InsP7 and 6-InsP7. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-ß-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

Divergent improvement of two cultivated allotetraploid cotton species.

Interspecific genomic variation can provide a genetic basis for local adaptation and domestication. A series of studies have presented its role of interspecific haplotypes and introgressions in adaptive traits, but few studies have addressed their role in improving agronomic character. Two allotetraploid Gossypium species, Gossypium barbadense (Gb) and G. hirsutum (Gh) originating from the Americas, are cultivated independently. Here, through sequencing and the comparison of one GWAS panel in 229 Gb accessions and two GWAS panels in 491 Gh accessions, we found that most associated loci or functional haplotypes for agronomic traits were highly divergent, representing the strong divergent improvement between Gb and Gh. Using a comprehensive interspecific haplotype map, we revealed that six interspecific introgressions from Gh to Gb were significantly associated with the phenotypic performance of Gb, which could explain 5%-40% of phenotypic variation in yield and fibre qualities. In addition, three introgressions overlapped with six associated loci in Gb, indicating that these introgression regions were under further selection and stabilized during improvement. A single interspecific introgression often possessed yield-increasing potential but decreased fibre qualities, or the opposite, making it difficult to simultaneously improve yield and fibre qualities. Our study not only has proved the importance of interspecific functional haplotypes or introgressions in the divergent improvement of Gb and Gh, but also supports their potential value in further human-mediated hybridization or precision breeding.

Curcumin Reduces Neuroinflammation and Improves the Impairments of Anesthetics on Learning and Memory by Regulating the Expression of miR-181a-5p.

OBJECTIVE: The purpose of this study was to explore the role of curcumin (Cur) in isoflurane (ISO)-induced learning and memory dysfunction in Sprague-Dawley rats and further elucidate the mechanism of the protective effect produced by Cur. METHODS: Rat models of cognitive impairment were established by inhaling 3% ISO. The Morris water maze test was used to assess the cognitive function of rats. ELISA and qRT-PCR were used to analyze the protein levels of pro-inflammatory cytokines and expression levels of miR-181a-5p, respectively. RESULTS: Cur significantly improved the ISO-induced cognitive dysfunction in rats and alleviated the ISO-induced neuroinflammation. miR-181a-5p was overexpressed in ISO-induced rats, while Cur treatment significantly reduced the expression of miR-181a-5p. Overexpression of miR-181a-5p promoted the cognitive impairment and the release of inflammatory cytokines and reversed the neuroprotective effect of Cur. CONCLUSION: Cur has a protective effect on ISO-induced cognitive dysfunction, which may be achieved by regulating the expression of miR-181a-5p.

Probing Protein-Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns.

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis.

BACKGROUND: Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. RESULTS: In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.4 times. CONCLUSIONS: This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

Preparation of a monolithic column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction for capillary liquid chromatography.

A monolithic capillary column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in-situ copolymerization of a homemade monomer N,N-dimethyl-N-acryloxyundecyl-N-(3-sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed-mode stationary phase. It was demonstrated that the mixed-mode stationary phase displayed typic dual retention mechanisms of reversed-phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP3-4 kinase activity.

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.

Variations in Quinolinic Acid Levels in Tuberculosis Patients with Diabetes Comorbidity: A Pilot Prospective Cohort Study.

Objective: We aimed to investigate dysregulated metabolic pathways and identify diagnostic and therapeutic targets in patients with tuberculosis-diabetes (TB-DM). Methods: In our prospective cohort study, plasma samples were collected from healthy individuals, diabetic (DM) patients, untreated TB-only (TB-0)/TB-DM patients (TB-DM-0), and cured TB (TB-6)/TB-DM patients (TB-DM-6) to measure the levels of amino acids, fatty acids, and other metabolites in plasma using high-throughput targeted quantification methods. Results: Significantly different biological processes and biomarkers were identified in DM, TB-DM-0, and TB-DM-6 patients. Moreover, quinolinic acid (QA) showed excellent predictive accuracy for distinguishing between DM patients and TB-DM-0 patients, with an AUC of 1 (95% CI 1-1). When differentiating between TB-DM-0 patients and TB-DM-6 patients, the AUC was 0.9297 (95% CI 0.8460-1). Compared to those in DM patients, the QA levels were significantly elevated in TB-DM-0 patients and decreased significantly after antituberculosis treatment. We simultaneously compared healthy controls and untreated tuberculosis patients and detected an increase in the level of QA in the plasma of tuberculosis patients, which decreased following treatment. Conclusion: These findings improve the current understanding of tuberculosis treatment in patients with diabetes. QA may serve as an ideal diagnostic biomarker for TB-DM patients and contribute to the development of more effective treatments.

Phytate metabolism is mediated by microbial cross-feeding in the gut microbiota.

Dietary intake of phytate has various reported health benefits. Previous work showed that the gut microbiota can convert phytate to short-chain fatty acids (SCFAs), but the microbial species and metabolic pathway are unclear. Here we identified Mitsuokella jalaludinii as an efficient phytate degrader, which works synergistically with Anaerostipes rhamnosivorans to produce the SCFA propionate. Analysis of published human gut taxonomic profiles revealed that Mitsuokella spp., in particular M. jalaludinii, are prevalent in human gut microbiomes. NMR spectroscopy using 13C-isotope labelling, metabolomic and transcriptomic analyses identified a complete phytate degradation pathway in M. jalaludinii, including production of the intermediate Ins(2)P/myo-inositol. The major end product, 3-hydroxypropionate, was converted into propionate via a synergistic interaction with Anaerostipes rhamnosivorans both in vitro and in mice. Upon [13C6]phytate administration, various 13C-labelled components were detected in mouse caecum in contrast with the absence of [13C6] InsPs or [13C6]myo-inositol in plasma. Caco-2 cells incubated with co-culture supernatants exhibited improved intestinal barrier integrity. These results suggest that the microbiome plays a major role in the metabolism of this phytochemical and that its fermentation to propionate by M. jalaludinii and A. rhamnosivorans may contribute to phytate-driven health benefits.

Breathomics for diagnosing tuberculosis in diabetes mellitus patients.

Introduction: Individuals with diabetes mellitus (DM) are at an increased risk of Mycobacterium tuberculosis (Mtb) infection and progressing from latent tuberculosis (TB) infection to active tuberculosis disease. TB in the DM population is more likely to go undiagnosed due to smear-negative results. Methods: Exhaled breath samples were collected and analyzed using high-pressure photon ionization time-of-flight mass spectrometry. An eXtreme Gradient Boosting (XGBoost) model was utilized for breathomics analysis and TB detection. Results: XGBoost model achieved a sensitivity of 88.5%, specificity of 100%, accuracy of 90.2%, and an area under the curve (AUC) of 98.8%. The most significant feature across the entire set was m106, which demonstrated a sensitivity of 93%, specificity of 100%, and an AUC of 99.7%. Discussion: The breathomics-based TB detection method utilizing m106 exhibited high sensitivity and specificity potentially beneficial for clinical TB screening and diagnosis in individuals with diabetes.

Determination of 5-methyldeoxycytosine and oxidized derivatives by nano-liquid chromatography with zwitterionic monolithic capillary column.

DNA methylation is one of the epigenetic modifications at the 5-carbon of cytosine to form 5-methyl-2'-deoxycytidine (5mdC). In mammalian cells, 5mdC can be transferred to 5-hydroxymethyl-2'-deoxycytidine (5hmdC) by ten-eleven translocation (TET), and 5hmdC is further oxidized to 5-formyl-2'-deoxycytidine (5fodC) and 5-carboxyl-2'-deoxycytidine (5cadC). In the present work, we developed a highly sensitive nano liquid chromatographic method for the determination of 5mC and 5hmC with zwitterionic monolithic capillary column. The conditions for the preparation of zwitterionic monolithic capillary column were systematically optimized. The monolithic capillary column displayed high column efficiency for nucleoside dA (190,000 plates/m) and allowed the baseline separation of 10 standard nucleosides in HILIC mode. The detection sensitivity was improved significantly by using the large volume injection combined with sample stacking onto the head of the column when sample was dissolved in high content organic solvent (ACN: H2O = 97:3). The limit of detection for 5mdC and 5hmdC were determined as 4 nM and 3 nM, respectively, and the corresponding limit of quantification were determined as 12 nM and 10 nM, respectively. Further, the zwitterionic monolithic capillary column can be easily utilized in nano-LC and mass spectrometry coupling for qualitative analysis of 5mdC, 5hmdC, 5fodC and 5cadC in real mouse brain sample. The whole genomic DNA methylation levels in mouse brain sample can be easily determined with UV detection.

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81.

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues.

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones.

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP1, InsP2, and InsP3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these "lower" inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP2 and InsP3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP6 to Ins(1,2,3)P3, an understudied InsP3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP3 isomers, namely Ins(1,2,3)P3, Ins(1,2,6)P3 and/or Ins(2,3,4)P3.

Profiling protein interactions by purification with capillary monolithic affinity column in combination with label-free quantitative proteomics.

An approach for profiling protein-protein interactions by using affinity purification with capillary monolithic immobilized metal affinity chromatography column (cm-IMAC) in combination with label free quantitative proteomics was described in the present work. The cm-IMAC columns were prepared in a single step by copolymerization of the function monomer, namely (S)-2,2'-((1-carboxy-5-(pent­4-enamido)pentyl)azanediyl)diacetic acid which provide a nitrilotriacetate (NTA) moiety to form chelated complexation with Ni (II) ions, inside the fused silica capillaries. The His6-tagged bait protein can be easily immobilized on the cm-IMAC columns through the formation of chelating complexation with the NTA-Ni (II) functional groups of the matrix. The cm-IMAC columns were used to explore protein-protein interactions (PPIs) on a proteomic scale when combined with label-free proteomics. A known interaction pair of proteins, namely NDP52 (amino acid sequence 10-126) and NAP1 (33-75) as well as Bcl-2 family proteins were used for proof of concept. New interactors of Bcl-XL were identified and validated by co-immunoprecipitation.

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were utilized in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to investigate InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR-down to individual enantiomers-which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme contributing to cellular homeostasis. Detailed characterization of MINPP1 activity in vitro and in cells showcased the unique reactivity of this phosphatase. Our results demonstrate that metabolic labeling with stable isotopomers in conjunction with NMR spectroscopy and CE-MS constitutes a powerful tool to annotate InsP networks in a variety of biological contexts.

[Effect of elevated atmospheric CO2 concentration and temperature on volatile halogenated organic compound content in soils of Schima superba and Cunninghamia lanceolata seedlings.] / 大气CO2浓度和温度升高对木荷和杉木幼苗土壤卤代烃含量的影响.

Global changes caused by the increases of atmospheric CO2 concentration and temperature have important effects on soil biogeochemical processes. The synthesis and release of volatile halogenated organic compounds (VOXs) is an important pathway for soil to participate in the global material cycle and energy flow. In this study, Schima superba and Cunninghamia lanceolata seedlings in the southern subtropics were selected as the research objects. Four treatments, including control (CK), elevated CO2 concentration (EC), elevated temperature (ET) and elevated both factors (EC+ET) were set up. The effects of EC and ET on soil VOXs formation were studied by an open-top chamber system coupled with a purging and trapping gas chromatography/mass spectrometry. The results showed that VOXs content in the soil of S. superba seedlings was 0.065-0.252 ng·g-1, which was higher than that of C. lanceolata (0.038-0.136 ng·g-1). At the EC, ET and EC+ET treatments, VOXs contents were reduced in soils of both species. The effect of ET was the most significant, with the decrease rates of 74.2% and 72.1% in both soils, respectively. The change of VOXs content with increasing temperature mainly attributed to the changes of soil moisture and nitrogen content. The content of VOXs in the soils of S. superba seedlings decreased more than that of C. lanceolata under different treatments. In CK, EC, ET and EC+ET treatment, bromodichloromethane (BDCM) (27.5%, 36.7%, 32.9%, 32.6%) and tetrachloromethane (TCM) (9.0%, 16.8%, 22.7%, 15.8%) were the main VOXs in the soil of S. superba seedlings, respectively, while BDCM and dibromomethane (DBM) were the main VOXs in the soil of C. lanceolata seedlings. BDCM accounted for 31.9%, 38.2%, 40.9% and 37.2% of the VOXs content in each treatment, and DBM accounted for 17.9%, 16.5%, 19.2% and 16.0% of the VOXs content, respectively. Simulating elevated atmospheric CO2 concentration and temperature was conducive to more comprehensive reflection of the ecological effect of global climate change, and it could provide data support for improving the VOCs flux model.