Cation-amino acid interactions: Implications for protein destabilization.

The mechanism for protein stabilization or destabilization has long been an open quest. In the present study, we have studied the interactions between amino acids and guanidinium (Gdm+)/ammonium (NH4+) ions by using low field nuclear magnetic resonance (LF-NMR), where Gdm+ and NH4+ are denaturant and stabilizer for proteins, respectively. It shows that Gdm+ favors to bind to the thiol group or the hydroxyl group on the side chain but weakly interacts with the α-carboxyl group. In contrast, NH4+ prefers to bind to the α-carboxyl group but slightly interacts with the thiol group or the hydroxyl group on the side chain of amino acids. 1HNMR reveals the hydrogen bonding between NH4+ and the α-carboxyl group, which is not involved in the interactions between Gdm+ and cysteine. Our study demonstrates that the strong interactions between the denaturant and the sulfur atom or the disulfide bond promote the direct binding of the denaturant toward proteins, leading to the destabilization.

Design, synthesis, and biological evaluation of benzo[d]imidazole-2-carboxamides as new anti-TB agents.

Tuberculosis is the leading cause of death globally among infectious diseases. Due to the development of resistance of Mycobacterium tuberculosis to currently used anti-TB medicines and the TB-HIV synergism the urgent need to develop novel anti-mycobacterial agents has been realized. The drug-to-target path has been the successful strategy for new anti-TB drug development. All the six drug candidates that have shown promise during the clinical trials and some of these being approved for treatment against MDR TB are the results of phenotype screening of small molecule compound libraries. In search of compounds belonging to novel pharmacophoric class that could be subjected to whole cell assay to generate new anti-TB leads the benzo[d]imidazole-2-carboxamide moiety has been designed as a novel anti-TB scaffold. The design was based on the identification of the benzimidazole ring as a prominent substructure of the FDA approved drugs, the structural analysis of reported anti-TB benzimidazoles, and the presence of the C-2 carboxamido functionality in novel bioisoteric anti-TB benzothiazoles. Twenty seven final compounds have been prepared via NH4Cl-catalyzed amidation of ethyl benzo[d]imidazole-2-carboxylates, as the required intermediates, obtained through a green "all water" one-pot synthetic route following a tandem N-arylation-reduction-cyclocondensation procedure. All of the synthesised target compounds were assessed for anti-TB potential using H37Rv ATCC27294 strain. Thirteen compounds were found with better MIC (0.78-6.25 µg/mL) than the standard drugs and being non-cytotoxic nature (<50% inhibition against RAW 264.7 cell lines at 50 µg/mL). The compound 8e exhibited best anti-TB activity (MIC: 2.15 µM and selectivity index: > 60) and a few others e.g., 8a, 8f, 8k and 8o are the next best anti-TB hits (MIC: 1.56 µg/mL). The determination and analysis of various physiochemical parameters revealed favorable druglike properties of the active compounds. The compounds 8a-l and 8o, with MIC values of ≤ 6.25 µg/mL, have high LipE values (10.66-11.77) that are higher than that of the suggested value of > 6 derived from empirical evidence for quality drug candidates and highlight their therapeutic potential. The highest LipE value of 11.77 of the best active compound 8e with the MIC of 0.78 µg/mL indicates its better absorption and clearance as a probable clinical candidate for anti-TB drug discovery. These findings highlight the discovery of benzimidazole-2-carboxamides for further development as new anti-TB agents.

Intranasal immunization with O-2'-Hydroxypropyl trimethyl ammonium chloride chitosan nanoparticles loaded with Newcastle disease virus DNA vaccine enhances mucosal immune response in chickens.

BACKGROUND: There has been a great interest in developing strategies for enhancing antigen delivery to the mucosal immune system as well as identifying mucosal active immunostimulating agents. To elevate the potential of O-2'-Hydroxypropyl trimethyl ammonium chloride chitosan (O-2'-HACC) as an adjuvant and mucosal immune delivery carrier for DNA vaccine, we prepared the O-2'-HACC loaded with Newcastle disease virus (NDV) F gene plasmid DNA and C3d6 molecular adjuvant (O-2'-HACC/pFDNA microparticles). RESULTS: The O-2'-HACC/pFDNA exhibited a regular spherical morphology with a particle size of 202.3 ± 0.52 nm, a zeta potential of 50.8 ± 8.21 mV, encapsulation efficiency of 90.74 ± 1.10%, and a loading capacity of 49.84 ± 1.20%. The plasmid DNA could be sustainably released from the O-2'-HACC/pFDNA after an initial burst release. Intranasal vaccination of chickens immunized with O-2'-HACC/pFDNA not only induced higher anti-NDV IgG and sIgA antibody titers but also significantly promoted lymphocyte proliferation and produced higher levels of IL-2, IL-4, IFN-γ, CD4+, and CD8 + T lymphocytes compared with the NDV commercial live attenuated vaccine. Intranasal delivery of the O-2'-HACC/pFDNA enhanced humoral, cellular, and mucosal immune responses and protected chickens from the infection of highly virulent NDV compared with the intramuscular delivery. CONCLUSIONS: Collectively, our findings indicated that the O-2'-HACC could be used as a vaccine adjuvant and delivery system for mucosal immunity and have an immense application promise.

Combination of solvent extraction with deep eutectic solvent based dispersive liquid-liquid microextraction for the analysis of aflatoxin M1 in cheese samples using response surface methodology optimization.

A new extraction procedure based on combination of a solvent extraction and deep eutectic solvent based dispersive liquid-liquid microextraction has been introduced for the extraction of aflatoxin M1 from cheese samples. In this method, acetonitrile, deionized water, and n-hexane are added onto the sample and vortexed. Owning to different affinities of the substances in cheese toward the mentioned solvents, an efficient and selective extraction of the analyte is done in the acetonitrile phase. After centrifugation, the acetonitrile phase is removed and mixed with a new hydrophobic deep eutectic solvent prepared from N,N-diethanol ammonium chloride and carvacrol as an extraction solvent. The mixture is injected into deionized water, and a cloudy solution is obtained. Eventually, an aliquot of the organic phase is injected into high-performance liquid chromatography-fluorescence detection. After optimizing the effective parameters with the response surface methodology and a quadratic model, limits of detection and quantification were 0.74 and 2.56 ng/kg, respectively. The obtained extraction recovery and enrichment factor were 94% and 94, respectively. Also, intra- (n = 6) and interday (n = 4) precisions were less than or equal to 8.6% at a concentration of 5 ng/kg. The suggested method was applied to determine aflatoxin M1 in different cheese samples.

Fabrication of gelatin microspheres containing ammonium hydrogen carbonate for the tunable release of herbicide.

The major challenge in utilizing pesticides lies in identifying the precise application that would improve the efficiency of these pesticides and decline their environmental and health hazards at the same time. Such application requires the development of specific formulations that enable controlled, stimuli-responsive release of the pesticides. Gelatin is a relatively cheap material characterized by temperature-sensitivity and abundant amino acid groups, which makes it suitable for the storage and controlled release of pesticides. In this study, gelatin microspheres were prepared by emulsion and cross-linking, then they were loaded with 2,4-dichlorophenoxyacetic acid sodium (2,4-D Na) as a model herbicide. To achieve temperature-tunable release of 2,4-D Na from the microspheres, NH4HCO3 was added to the formulations at different concentrations. The prepared formulations were characterized by SEM, FTIR, and size distribution analyzes, and their drug loading capacities were determined. Based on bioassay experiments, the 2,4-D Na-NH4HCO3-loaded gelatin microspheres can effectively control the spread of dicotyledonous weeds. Therefore, the strategy proposed herein can be used to develop novel, effective herbicide formulations.

Novel Approach for the Synthesis of Chlorophosphazene Cycles with a Defined Size via Controlled Cyclization of Linear Oligodichlorophosphazenes [Cl(PCl2=N)n-PCl3]+[PCl6].

Despite a significant number of investigations in the field of phosphazene chemistry, the formation mechanism of this class of cyclic compounds is still poorly studied. At the same time, a thorough understanding of this process is necessary, both for the direct production of phosphazene rings of a given size and for the controlled cyclization reaction when it is secondary and undesirable. We synthesized a series of short linear phosphazene oligomers with the general formula Cl[PCl2=N]n-PCl3+PCl6- and studied their tendency to form cyclic structures under the influence of elevated temperatures or in the presence of nitrogen-containing agents, such as hexamethyldisilazane (HMDS) or ammonium chloride. It was established that linear oligophosphazenes are inert when heated in the absence of the mentioned cyclization agents, and the formation of cyclic products occurs only when these agents are involved in the process. The ability to obtain the desired size phosphazene cycle from corresponding linear chains is shown for the first time. Known obstacles, such as side interaction with the PCl6- counterion and a tendency of longer chains to undergo crosslinking elongation instead of cyclization are still relevant, and ways to overcome them are being discussed.

Cation Specific Effects on the Domain-Domain Interaction of Heterogeneous Dimeric Protein Revealed by FRET Analysis.

Specific monovalent cation effects on the domain-domain interaction of heterogeneous dimeric protein were investigated using green fluorescent protein (GFP)-glutathione-s-transferase (GST) fusion protein as a model protein. Conjugating N-terminal of GST domain with a fluorescence probe Cyanine3, complementary increase and decrease of fluorescence intensities of Cyanine3 and GFP were recognized on the exclusive excitation of GFP and further the fluorescence decay of GFP was remarkably accelerated to show that an excellent Förster type of resonance excitation energy transfer (FRET) pair was constructed between GFP- and GST-domain. The spectral overlap integral and critical distance of the FRET pair were estimated to be 5.96×1013 M-1cm3 and 62.5 Å, respectively. The FRET rate and efficiency evaluated by fluorescence lifetime of the energy donor, GFP, were influenced by the monovalent cations included in the buffer solution to suggest that the domain-domain interactions of GFP-GST fusion protein would be susceptible to cation species and their concentrations. The order affecting the domain-domain interaction was estimated to be Li+>NH4+ >Na+>K+>Cs+, almost corresponding to the reverse Hofmeister series.

Water Soluble Coumarin Quaternary Ammonium Chlorides: Synthesis and Biological Evaluation.

In the present study, coumarin-bearing three pyridinium and three tetra-alkyl ammonium salts were synthesized. The compounds were fully characterized by 1 H- and 13 C-NMR, LC/MS and IR spectroscopic methods and elemental analyses. The cytotoxic properties of all compounds were tested against human liver cancer (HepG2), human colorectal cancer (Caco-2) and non-cancer mouse fibroblast (L-929) cell lines. Some compounds performed comparable cytotoxicity with standard drug cisplatin. Antibacterial properties of the compounds were tested against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria, but the compounds did not have any antibacterial effect against both bacteria. Enzyme inhibitory properties of all compounds were tested on the activities of human carbonic anhydrase I and II, and xanthine oxidase. All compounds inhibited both enzymes more effectively than standard drugs, acetazolamide and allopurinol, respectively. The biological evaluation results showed that ionic and water soluble coumarin derivatives are promising structures for further investigations especially on enzyme inhibition field.

Ammonium chloride-catalyzed green multicomponent synthesis of dihydropyrazine and tetrahydrodiazepine derivatives "on water".

This research describes a simple and efficient one-pot synthetic approach for the preparation of tetrahydrodiazepine and dihydropyrazine (or dihydroquinoxaline) derivatives in high yields in the presence of a substoichiometric amount of ammonium chloride as a green accelerator on water at 50 °C within 1-3 h.

Correlations of Ion Composition and Power Efficiency in a Reverse Electrodialysis Heat Engine.

The main objective of this study is to explore the influence of ion composition on the trans-membrane potential across the ion exchange membrane (IEM), and thus offers a reference for the deep insight of "reverse electrodialysis heat engine" running in the composite systems. In comparison to the natural system (river water | seawater), the performance of the reverse electrodialysis (RED) stack was examined using NaHCO3, Na2CO3, and NH4Cl as the supporting electrolyte in the corresponding compartment. The effect of flow rates and the concentration ratio in the high salt concentration compartment (HCC)/low salt concentration compartment (LCC) on energy generation was investigated in terms of the open-circuit voltage (OCV) and power density per membrane area. It was found that the new system (0.49 M NaCl + 0.01 M NaHCO3|0.01 M NaHCO3) output a relatively stable power density (0.174 W·m-2), with the open-circuit voltage 2.95 V under the low flow rate of 0.22 cm/s. Meanwhile, the simulated natural system (0.5 M NaCl|0.01 M NaCl) output the power density 0.168 W·m-2, with the open-circuit voltage 2.86 V under the low flow rate of 0.22 cm/s. The findings in this work further confirm the excellent potential of RED for the recovery of salinity gradient energy (SGP) that is reserved in artificially-induced systems (wastewaters).

Short Total Synthesis of [15N5]-Cylindrospermopsins from 15NH4Cl Enables Precise Quantification of Freshwater Cyanobacterial Contamination.

Fresh water cyanobacterial algal blooms represent a major health risk because these organisms produce cylindrospermopsin, a toxic, structurally complex, zwitterionic uracil-guanidine alkaloid recognized by the EPA as a dangerous drinking water contaminant. At present, the ability to detect and quantify the presence of cylindrospermospin in water samples is severely hampered by the lack of an isotopically labeled standard for analytical mass spectrometry. Herein, we present a concise, scaled total synthesis of 15N cylindrospermosin from 15N ammonium chloride, which leverages a unique stereoselective intramolecular double conjugate addition step to assemble the tricyclic guanidine core. In addition to providing the first pure isotopically labeled probe for precise quantification of this potent biotoxin in fresh water sources, our results demonstrate how unique constraints associated with isotope incorporation compel novel solutions to synthesis design.

Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry.

Matrix-assisted laser desorption/ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) is an important technique for visualizing the spatial distribution of compounds directly on the surface of organisms such as microorganisms, insects, plants, animals, and human tissues. However, MALDI-imaging-HRMS and the stable isotope labeling approach have never been combined for the detection and simultaneous visualization of labeled and unlabeled compounds, their analogues and derivatives, as well as their precursors. Herein, we present a methodology that labels microbial secondary metabolites directly on agar with stable isotopes and allows concurrent spatial distribution analyses by MALDI-imaging-HRMS. Using a thin film of labeled agar supplemented with [1-13C]-l-proline, [methyl-D3]-l-methionine, 15NH4Cl, or [15N]-l-serine overlaid on unlabeled agar, we demonstrate the incorporation of labeled precursors into prodiginines and serratamolides produced by an endophytic bacterium, Serratia marcescens, by MALDI-imaging-HRMS and HPLC-HRMS. Further, we show the incorporation of CD3 into prodigiosin as well as its characteristic fragments directly by MALDI-imaging-HRMS2. Our methodology has several advantages over currently existing techniques. First, both labeled and unlabeled compounds can be visualized simultaneously in high spatial resolution along with their labeled and unlabeled precursors. Second, by using a thin film of labeled agar, we utilize minimum amounts of expensive labeled compounds (1-3 mg) ensuring a cost-effective method for investigating biosynthetic pathways. Finally, our method allows in situ visualization and identification of target and nontarget compounds without the need of isolating the compounds. This is important for compounds that are produced by microorganisms in low, physiologically, or ecologically relevant concentrations.

Expanding the Coverage of the Metabolome with Nitrogen-Based NMR.

Isotopically labeling a metabolite and tracing its metabolic fate has provided invaluable insights about the role of metabolism in human diseases in addition to a variety of other issues. 13C-labeled metabolite tracers or unlabeled 1H-based NMR experiments are currently the most common application of NMR to metabolomics studies. Unfortunately, the coverage of the metabolome has been consequently limited to the most abundant carbon-containing metabolites. To expand the coverage of the metabolome and enhance the impact of metabolomics studies, we present a protocol for 15N-labeled metabolite tracer experiments that may also be combined with routine 13C tracer experiments to simultaneously detect both 15N- and 13C-labeled metabolites in metabolic samples. A database consisting of 2D 1H-15N HSQC natural-abundance spectra of 50 nitrogen-containing metabolites are also presented to facilitate the assignment of 15N-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes with 15N1-ammonium chloride, 15N4-arginine, and 13C2-acetate. Efficient 15N and 13C metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.

Mitotic defects in fission yeast lipid metabolism 'cut' mutants are suppressed by ammonium chloride.

Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.

pH-dependent production of himeic acid A and its non-enzymatic conversions to himeic acids B and C.

The fungus Aspergillus japonicus MF275 produces himeic acid A (1), containing a 4-pyrone ring, along with its congeners, himeic acids B (2) and C (3). During culture, 1 was gradually converted to 3, the corresponding 4-pyridone derivative. A study of the relationship between the culture pH and the fungal metabolites showed that a decrease from pH 6.5 to pH 2 is essential for production of 1, while a subsequent increase to pH 5 is necessary for production of 3. In addition, we revealed that 1 was non-enzymatically converted to 3 by the incorporation of an ammonium nitrogen atom in a pH 5 buffer, and that 1 was converted to 2 at a conversion ratio of 50% during incubation in MeOH for five days.

Aquaporin 4 as a NH3 Channel.

Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels.

Quantitative detection of isotopically enriched E. coli cells by SERS.

It is clear that investigating how bacterial cells work by analysing their functional roles in microbial communities is very important in environmental, clinical and industrial microbiology. The benefits of linking genes to their respective functions include the reliable identification of the causative agents of various diseases, which would permit appropriate and timely treatment in healthcare systems. In industrial and municipal wastewater treatment and management, such knowledge may allow for the manipulation of microbial communities, such as through bioaugmentation, in order to improve the efficiency and effectiveness of bioremediation processes. Stable isotope probing coupled with identification techniques has emerged to be a potentially reliable tool for the discrimination, identification and characterization of bacteria at community and single cell levels, knowledge which can be utilized to link microbially mediated bioprocesses to phylogeny. Development of the surface-enhanced Raman scattering (SERS) technique offers an exciting alternative to the Raman and Fourier-transform infrared spectroscopic techniques in understanding the metabolic processes of microorganisms in situ. SERS employing Ag and Au nanoparticles can significantly enhance the Raman signal, making it an exciting candidate for the analysis of the cellular components of microorganisms. In this study, Escherichia coli cells were cultivated in minimal medium containing different ratios of 12C/13C glucose and/or 14N/15N ammonium chloride as the only carbon and nitrogen sources respectively, with the overall final concentrations of these substrates being constant. After growth, the E. coli cells were analyzed with SERS employing an in situ synthesis of Ag nanoparticles. This novel investigation of the SERS spectral data with multivariate chemometrics demonstrated clear clusters which could be correlated to the SERS spectral shifts of biomolecules from cells grown and hence labelled with 13C and 15N atoms. These shifts reflect the isotopic content of the bacteria and quantification of the isotope levels could be established using chemometrics based on partial least squares regression.

A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

BACKGROUND: The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. RESULTS: The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15NH4Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. CONCLUSIONS: Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15N-labelled amino acids could be obtained.

Osmium-Mediated Transformation of 4-Thiouridine to Cytidine as Key To Study RNA Dynamics by Sequencing.

To understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4-thiouridine (4sU), followed by thio-selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC-seq) that eliminates affinity purification and allows for direct assessment of 4sU-labeled RNA. It employs an OsO4 -mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse-labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC-seq as a straight-forward and highly versatile method for studies of cellular RNA dynamics.

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides.

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.