Monitoring the metabolite content of seasoned zucchinis during storage by NMR-based metabolomics.

This study applied 1H NMR metabolomics to monitor the metabolite content of baked and seasoned zucchinis packaged in both compostable and plastic trays. Polar extracts of samples stored at 4 °C up to 35 days were investigated to check for metabolite changes upon shelf life. The evolution of the integral values of only the main metabolites responsible for sample differentiation (lactate, acetate, malate, α and ß glucose and sucrose) were further analysed and compared. In particular, the evaluation of lactate and acetate amount, considered markers of fermentation progress, showed a comparable performance for the two types of packaging in preserving the freshness of seasoned zucchinis, confirming the maintenance of food product composition within the declared shelf life period in the recommended storage conditions. Albeit preliminary, the results support the NMR metabolomics as a tool for identifying candidate metabolites to monitor the shelf life of foods, thereby improving the understanding of molecular changes during storage.

HR MAS 1H NMR and chemometrics as useful tool to assess the geographical origin of cocoa beans - Comparison with HR 1H NMR.

Chocolate and cocoa-based products are among the goods with higher added value. A current trend of the cocoa market is to offer to the consumers high quality cocoa products, namely mono-origin cocoa. However, a reliable analytical method able to trace the geographical origin of cocoa is lacking. In this work we tested the capability of HR MAS 1H NMR combined with chemometrics to assess the geographical origins of 60 fermented and dried cocoa beans of 23 different cocoa producing countries from the three major crop-growing areas (Africa, Central/South America, Asia/Oceania). Metabolic profiling was determined by HR MAS 1H NMR directly on cocoa powder after the method optimization. The same samples were also subjected to extraction and analysis with HR 1H NMR. HR MAS 1H NMR, as 1H NMR analysis, allowed the simultaneous detection of amino acids, polyalcohols, organic acids, sugars, methylxanthines, catechins. Moreover, HR MAS allows the detection of lipids, not present in the aqueous extract utilized for 1H NMR. The data set obtained is therefore representative of all classes of cocoa compounds. Untargeted HR MAS 1H NMR and 1H NMR datasets were utilized as fingerprint of the samples and elaborated with multivariate statistical methods. A targeted quantitative approach of selected metabolites was possible only with HR 1H NMR data, because HR MAS 1H NMR does not give reliable quantitative results. All the approaches adopted showed a discrimination of the cocoa origins. HR MAS presents the advantages to obtain a very rapid picture of the samples, comprising both lipophilic and hydrophilic components, avoiding any sample manipulation.

Cannabinoid-free Cannabis sativa L. grown in the Po valley: evaluation of fatty acid profile, antioxidant capacity and metabolic content.

Within a project aimed to reintroduce non-drug hemp cultivars in the Italian Po valley, for fibre but also high added-value nutraceutical production, investigation on locally grown plants has been performed, in order to assess their oil and metabolic content. This study provides useful information regarding three different hemp cultivars, from two sites, in view of their potential industrial application. The oil was characterised by a high unsaturated/saturated fatty acid ratio and by an almost perfect balance of ω-3 and ω-6 fatty acids, as requested for healthy foods. The alcoholic extracts, for which a high content of amino acids and phenolic compounds has been highlighted, could provide dietary supplements to help in preventing oxidative stress. By investigating the Carmagnola cultivar, six known and four new lignanamides have been identified, confirming and assessing the general metabolic pattern in the seeds of these locally grown plants.

Nuclear magnetic resonance and chemometrics to assess geographical origin and quality of traditional food products.

In this globalization era, the opening of the markets has put at almost everybody's disposal a wide variety of foods, allowing everybody to taste food flavors and aromas from different nations. Notwithstanding this opportunity, countries try to preserve their markets by developing protection policies. A few countries have adopted different denominations to label their "typical food" products in order to give them additional value. Besides, the term "typical food" is widely thought of as something anchored to the local traditions, with geographical meaning and made with typical raw materials. Then a "typical food" starts to be considered "traditional" when it is made following specific and old recipes. As a matter of fact, these products acquire particular organoleptic characteristics that are not reproducible when produced in different places. In this review, NMR studies coupled to multivariate statistical analysis are presented with the aim of determining geographical origin and key quality characteristics.

Evaluation of the production year in Italian and Chinese tomato paste for geographical determination using O2PLS models.

Nuclear magnetic resonance (NMR) is nowadays largely used as valid tool in metabolomic applications. In this study, the metabolite content of Italian and Chinese tomato paste at different concentration rates of two production years (2007 and 2008) was investigated with the aim of building a robust geographical differentiation statistical model. A total of 119 tomato paste samples were analyzed by (1)H NMR and multivariate data analysis tools, in particular using bidirectional orthogonal projection to latent structures-discriminant analysis (O2PLS-DA). This technique is well-suited for noisy and correlated variables and was recently adopted to obtain robust classification models, having a clear interpretation of the systematic variation useful to characterize each class. In the present study, the analysis of latent space underlying the classification model allowed us to understand the role played by the production year on geographical discrimination. The O2PLS-DA model performed considering only tomato paste samples of 2007 was capable of predicting the geographical origin of all analyzed samples. The effect of the production year therefore resulted in not affecting the geographical origin discrimination.

Triple concentrated tomato paste: discrimination between Italian and Chinese products.

(1)H NMR spectroscopy was applied to discriminate triple concentrated tomato paste coming from two different countries. Notwithstanding different tomato cultivars and ripening stages employed to obtain the final product, significant discrimination between Italian and Chinese samples was obtained by combining NMR data and principal component analysis. Supervised orthogonal projection to latent structure discriminant analysis (OPLS-DA) technique was used to build robust classification models, while S-plot was employed to identify statistically significant variables responsible for class separation. Citrate content resulted in being the most relevant chemical compound for Chinese and Italian sample differentiation. In order to highlight other compounds able to contribute to sample differentiation, citrate content was excluded, and a new classification model was built. This latter model indicated aspartate, glutamine, and sugars as important variables in sample differentiation.

Ripening and geographical characterization of Parmigiano Reggiano cheese by 1H NMR spectroscopy.

Samples of Italian Parmigiano Reggiano cheese of different ripening stages (14, 24 and 30 months) were analyzed by (1)H NMR spectroscopy and the water-soluble metabolites content compared with samples of "Grana type" cheese from east Europe countries. Different multivariate statistical protocols were examined to build up a stable model for both ripening and geographical discrimination among the samples. The discriminant approach revealed a larger metabolite content increasing with ripening process; in particular younger samples were characterized by a higher content in leucine and isoleucine, while 30 months samples by a larger extent of threonine. The use of a classification approach resulted in a better discrimination of geographical samples. Foreign "Grana type" samples resulted well differentiated with respect to all Italian samples of Parmigiano Reggiano cheese. Foreign samples were characterized by a large amount of leucine and isoleucine, compounds that typified young Italian samples (14 months), and also by lactate, butanoate and acetate, thus suggesting short ripening for foreign samples. Parmigiano Reggiano samples were instead characterized by a large amount of all other compounds, in particular by threonine, which typified old samples (30 months), and other amino acids.

NMR and chemometric methods: a powerful combination for characterization of Balsamic and Traditional Balsamic Vinegar of Modena.

This work presents the capability of NMR spectroscopy combined with Chemometrics in predicting the ageing of Balsamic and Traditional Balsamic Vinegar of Modena. The need of an analytical method is an important requirement for both research oriented and commercial evaluation of these very valuable products. (1)H NMR spectroscopy, based on the advantage of rapid sample analysis without any manipulation or derivatization, is here proposed as a valid tool to describe Balsamic and Traditional Balsamic Vinegar of Modena. For this purpose, 72 reliable samples, were divided into three different groups according to their ageing process: young (<12 years), old (>12 and <25 years) and extra old (>25 years). Hierarchical Projection to Latent Structures Discriminant Analysis (PLS-DA) allowed us to characterize the ageing process. Variables showing the largest VIP (Variable Importance in the Projection) were extracted from PLS-DA model, thus shedding lights onto the role played by specific compounds in this complex ageing process. Two robust classification models, were built by PLS-DA and Naïve Bayes classifier and compared to prove the accuracy of the representation on both training and test sets. The predictions obtained for 41 "unknown" vinegar samples with these both methods gave more than 80% agreement among them.

NMR relaxation data for quality characterization of Balsamic vinegar of Modena.

In this paper, 22 samples consisting of Balsamic vinegar of Modena and two Traditional Balsamic vinegar of Modena samples have been analyzed by means of (1)H NMR spectroscopy. Some selected resonances have been used for quantification and for T1 (spin-lattice relaxation time) measurements. Statistical protocols applied to NMR data for quantification of selected resonances revealed the possibility to differentiate the samples according to their ageing process. Additionally, T1 measurements revealed a strong correlation with the ageing process and these new data were added for improving the statistical model, which was used to predict T1 relaxation time of selected compounds for Balsamic vinegar samples. Our results indicate that the use of NMR data and statistical methods is a valid approach that can be successfully used for ageing characterization of Balsamic vinegar of Modena samples.

Effect of all-trans-retinoic acid on the hypercoagulable state of patients with breast cancer.

To evaluate whether all-trans-retinoic acid (ATRA) is able to modulate the hemostatic system in patients with solid tumors, we studied patients with locally advanced breast cancer who were enrolled in a Phase Ib study of ATRA +/- Tamoxifen (Tam). In this study, two groups of 15 patients/each were treated for 21 days before operation with ATRA at three doses (15, 45, or 75 mg/m(2)/day on alternate days) given alone (group 1) or in combination with Tam (group 2). One additional group received Tam alone. Plasma samples were evaluated for hypercoagulation markers (FVIIa, F1+2, TAT, D-dimer), fibrinolysis proteins (t-PA, PAI-1), and coagulation inhibitors (protein C, AT). At baseline, cancer patients had FVIIa, F1+2, TAT, and PAI-1 significantly greater than control subjects. During treatment, in the patients given ATRA alone, hypercoagulation markers appeared unmodified. Instead, subjects given Tam alone had a significant elevation of FVIIa, F1+2, and TAT versus baseline. However, in the ATRA + Tam groups, hypercoagulation markers were decreased compared with Tam alone. These results suggest that in selected conditions, pre-operative ATRA may modulate the hypercoagulable state of breast cancer patients.

Capillary electrophoresis investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors.

We recently reported the use of a deconvolution strategy to identify the best chiral selectors for Nalpha-2,4-dinitrophenyl (Dnp) amino acid racemates from a combinatorial library composed of thousands of homodetic cyclohexapeptides. Selection was based on the capillary electrophoresis (CE) enantioresolution for a set of Dnp-amino acids. The groups involved in the chiral discrimination were assessed by nuclear magnetic resonance (NMR) spectroscopy, which revealed a strong involvement of one of the aromatic rings of the cyclopeptide in the binding with the analyte. In order to better understand the recognition mechanism, and thus extend the applicability of the analytical system, modifications on both analyte and selector structure were introduced. The effects on separation were evaluated in terms of resolution values and mobility variation.

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

Chiral capillary electrophoresis and nuclear magnetic resonance investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors.

In the present work, synthetic cyclohexa- and cycloheptapeptides previously singled out by a combinatorial chemistry approach have been evaluated as chiral selectors in capillary electrophoresis. By applying the countercurrent migration technique and employing a new adsorbed coating, a series of dinitrophenyl amino acids as well as some chiral compounds of pharmaceutical interest have been evaluated for enantiorecognition. The results thus obtained led to a deeper investigation of the chiral discrimination process, by carrying out nuclear magnetic resonance (NMR) studies on selected cyclopeptide-analyte complexes. These studies shed light on the chemical groups involved in the analyte-selector interaction and provided useful information for a wider application of these cyclopeptides in the separation of other drug enantiomers.

New adsorbed coatings for capillary electrophoresis.

New acrylic polymers bearing oxirane groups were synthesized to be used in the production of coated capillaries. A fully automated coating procedure was devised based on the use of diluted water solutions of these polymers. The whole procedure required less than 30 min. The new polymers rapidly adsorbed from water onto the capillary wall, thus suppressing electroosmotic flow (EOF) to a negligible value. The adsorbed coatings were stable for hundreds of hours at high pH, temperature, and in the presence of 8 M urea. Efficient separations of acidic and basic proteins were achieved in the new phases.

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies.

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.

A single-point mutation in the extreme heat- and pressure-resistant sso7d protein from sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core.

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation.

Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed.

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure.

CB2, a CNBr peptide of 36 residues from type I collagen alpha1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1J(NH)=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (delta delta/delta T of -3.6 ppb/degrees C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354-7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N(epsilon) hydrogen, leaving the four N(eta) hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric alpha1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17 degrees C.

Cancer procoagulant and tissue factor are differently modulated by all-trans-retinoic acid in acute promyelocytic leukemia cells.

All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4. 306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] alpha), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARbeta) and CD437 (selective for RARgamma), both lacking this capability. Cells were treated with either ATRA or the analogues (10(-6) to 10(-8) mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARalpha agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARbeta agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both alpha and beta nuclear retinoid receptors.

The effect of very-low-dose warfarin on markers of hypercoagulation in metastatic breast cancer: results from a randomized trial.

Malignancy is a risk factor for thromboembolism and anti-cancer chemotherapy can increase this risk. Prophylaxis of thrombosis with very-low-dose warfarin given concurrently with chemotherapy has a significantly reduced rate of thromboembolism in a randomized trial in women with stage IV breast cancer. In a group of 32 patients randomized in one center (16 subjects on warfarin and 16 on placebo), we have prospectively studied the plasma levels of: 1. Markers of 'in vivo' clotting activation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+2 [F1+2] and D-dimer), 2. Factor VII (FVII), and 3. Natural anticoagulants (protein C [PC] and antithrombin [AT]). The aims of this study were: 1. to examine whether laboratory tests predicted those patients who developed thrombosis, and 2. to evaluate the effect of very-low-dose warfarin on hemostatic variables. The patients' hemostatic parameters were evaluated before entry into the study and after starting chemotherapy +/- prophylaxis, before each course for nine courses. Before-treatment results were compared to those of a sex and age-matched non-cancer control group. There was a significant elevation of plasma levels of TAT (p <0.001), F1+2 (p <0.001), D-dimer (p <0.0001) and FVIIa (p <0.05), as well as an increase of FVII proteolysis (p <0.05), whereas plasma PC and AT concentrations were not different from controls. After starting chemotherapy, markers of clotting activation were progressively lower in the group receiving warfarin prophylaxis compared to the group on placebo. Differences between the groups became statistically significant (p <0.01) after the 4th course of chemotherapy. Deep vein thrombosis occurred in two patients in the placebo arm. The results of this study indicate that before therapy, an hypercoagulable state is present in stage IV breast cancer, and after starting chemotherapy, abnormalities of hypercoagulation markers persist, however they are reduced by very-low-dose-warfarin. None of the laboratory variables could predict thrombosis in the single patient.