13C NMR Research Articles

3-Benzoyl-1′,3′,6-trimethyl-2′H,3H,4H-spiro[furo[3,2-c]pyran-2,5′-pyrimidine]-2′,4,4′,6′(1′H,3′H)-tetraone

We describe an approach towards the synthesis of previously unknown 3-benzoyl-1′,3′,6-trimethyl-2′H,3H,4H-spiro[furo[3,2-c]pyran-2,5′-pyrimidine]-2′,4,4′,6′(1′H,3′H)-tetraone. The presented method is based on the cyclization of 5-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-2-oxo-2-phenylethyl)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione. It was shown that the presented reaction proceeds through the stage of bromination of the starting compound followed by O-nucleophilic attack. The structures of the obtained compound were established by 1H, 13C NMR and IR spectroscopy, and high-resolution mass spectrometry.

Copillar[5]arene Appended Pyrene Schiff Base: Photophysics, Aggregation Induced Emission and Picric Acid Recognition

Herein, we report the synthesis of copillar[5]arene‐based pyrene Schiff base 1 and its characterization by using 1H, 13C NMR, FT‐IR and mass spectrometry. UV‐vis absorption, steady‐state fluorescence and time‐resolved fluorescence are done to elucidate the photophysical behaviors of 1. To understand the electronic structure of 1, density functional theory (DFT) calculations are performed. Owing to the presence of pyrene via a Schiff base linkage, compound 1 exhibits aggregation‐induced emission (AIE) characteristics. It shows aggregation in aqueous THF and DMF. The aggregation behavior is successfully demonstrated by steady‐state fluorescence, dynamic light scattering (DLS) and time‐correlated single‐photon counting (TCSPC) experiments. Experimental findings reveal that hydrophobic effect is the driving force in the formation of aggregates. As application, the aggregated state of 1 in aqueous THF fluorimetrically recognizes picric acid (PA) selectively over a series of nitro‐ and nonnitroaromatics with a detection limit of 1.62 x 10‐7 M. The emission of the aggregated state is fully quenched upon interaction with PA.

Extraction and Characterization of Inulin-Like Fructans from Hydroponically Grown Stevia rebaudiana Roots for Food Applications.

Stevia rebaudiana is a plant native to South America known for producing steviol glycosides and fructans used in low-calorie and functional foods. This study aimed to cultivate and isolate inulin from hydroponically grown S. rebaudiana roots. Seedlings were grown hydroponically for 28 days; then, roots were dried and extracted at 121°C for 20min. Extracts were centrifuged and lyophilized, yielding supernatant (SSR-H) and precipitate (PSR-H) fractions. Fructan yields were 21.34% (SSR-H) and 2.91% (PSR-H). HPLC, FTIR, and NMR analyses confirmed inulin-like fructans with polymerization degrees of 16 and 31, respectively. The characteristic peaks of a fructan-like-inulin were observed in the FTIR, ¹H, and ¹³C NMR spectra. These results demonstrate effective inulin extraction from hydroponically grown S. rebaudiana roots, highlighting its potential applications in the food industry.

Beyond the Hawthorne Reaction: Li+ Induced Thermal Dehydrocoupling of closo-10-vertex Carborane Anions.

In the 1970s Hawthorne reported an electrochemical dehydrocoupling reaction of the closo-carborane anion [HCB9H91-] 1 to form the biscarborane [C2B18H182-] 2. In this Communication we show that the said "Hawthorne Reaction" can be achieved thermally and that it tolerates C-butylation. The new compound 2butyl was fully characterized by 11B, 1H, and 13C NMR spectroscopies, high-resolution mass spectrometry, and single-crystal X-ray diffraction. One interesting caveat is that 2 or 2butyl only form thermally when they are salts of Li+ and not NEt4+, Na+, K+, or Cs+. This observation means that Li+ in some way facilitates this process, introducing a new kind of Li+ effect.

Synthesis, Antibacterial, and Antibiofilm Activities of Imidazo[2,1-b]thiazole-Chalcone Derivatives: In Vitro and In Silico Studies.

In recent years, Imidazothiazole-Chalcone conjugates have emerged as notable pharmacophores with potential applications in discovering biologically active compounds. This study focuses on synthesizing novel imidazo[2,1-b]thiazole chalcone derivatives through a facile and conventional process adhering to several principles of green chemistry, facilitating scalable production. The synthesized compounds underwent comprehensive spectroscopic analysis, including 1H NMR, 13C NMR, LC-MS, and FT-IR techniques. Theoretical FT-IR and NMR analysis, Frontier Molecular Orbitals FMOs, and global reactivity descriptors were calculated and interpreted. Furthermore, MEP Surface, Mulliken atomic charge, ELF, LOL and QTAIM were analyzed. The newly synthesized compounds were screened in vitro for their antibacterial and antibiofilm activities. In addition, computational docking studies were performed to gain further insights into molecular interactions and found to support the results.

Molecular structure characteristic of coals of different rank.

Understanding the structural characteristics of coal at the molecular level is fundamental for its effective utilization. To explore the molecular structure characteristic, the long-flame coal from Daliuta (DLT), coking coal from Yaoqiao (YQ), and anthracite from Taixi (TX) were investigated using various techniques such as elemental analysis, Fourier transform infrared spectroscopy, solid-state 13C nuclear magnetic resonance spectroscopy, and X-ray photoelectron spectroscopy. Based on the structural parameters, the coal molecular model was constructed and optimized. The molecular formula of DLT was C193H178N2O47, that of YQ was C201H179N3O30S, and that of TX was C198H118N2O10. With an increase in the degree of metamorphism, the substitution of the benzene ring gradually shifted towards lower levels of substitution. The content of long chain in the aliphatic chain decreased while the content of branched chains kept increasing. The percentage of aromatic ether increased gradually, while the phenolic hydroxyl group initially decreased but then increased. The carboxyl group C = O decreased and eventually disappeared in anthracite coal. The proportion of pyrrole nitrogen gradually increased while that of pyridine nitrogen and protonated pyridine gradually decreased. The 2D planar structure of coal was constructed using ChemDraw, ACD/CNMR Predictor, and gNMR programs. The geometry optimization was performed using the COMPASS II force field within the Forcite module in Materials Studio 2020. The annealing process employed NVT ensemble at a simulation temperature of 298K. The Amorphous Cell module in Materials Studio was used to construct large-scale 3D molecular models, with the set parameters in this paper.

Glycerol-Derived Water-Lean Amines for Post-Combustion CO2 Capture: The Improvement in Capacity and Viscosity.

Water-lean absorbents are regarded as a new generation of post-combustion CO2 capture technology that could significantly relieve those drawbacks posed by traditional aqueous alkanolamines. However, the exponential increase in viscosity during CO2 absorption remains an urgent issue that needs to be resolved before their practical deployment. In this work, novel water-lean amines based on biomass glycerol have been devised as single-component CO2 absorbents with low viscosity (79~110 cP at 25 oC, 29~39 cP at 40 oC) under high capacity (12~18 wt% at 25 oC, 10~17 wt% at 40 oC). The captured CO2 could be smoothly released by thermal desorption. Results from preliminary stability test and 10 absorption-desorption cycles showed that such non-aqueous absorbents had significant structural toughness as well as reusability. Spectroscopic measurements including 13C NMR and in situ FTIR were performed to gain mechanistic insights by monitoring the entire CO2 absorption and desorption process, while DSC, VLE and DFT calculations provided rational interpretation for reaction kinetics and thermodynamics. The synergistic promotion of glycerol ether group on both CO2 chemical and physical absorption was also verified under high pressure conditions.

New Brusatol Derivatives as Anti-Settlement Agents Against Barnacles, Targeting HSP90: Design, Synthesis, Biological Evaluation, and Molecular Docking Investigations

The increasing challenge of marine biofouling, mainly due to barnacle settlement, necessitates the development of effective antifoulants with minimal environmental toxicity. In this study, fifteen derivatives of brusatol were synthesized and characterized using 13C-NMR, 1H-NMR, and mass spectrometry. All the semi-synthesized compounds obtained using the Multi-Target-Directed Ligand (MTDL) strategy, when evaluated as anti-settlement agents against barnacles, showed promising activity. Compound 3 exhibited the highest anti-settlement capacity, with an EC50 value of 0.1475 μg/mL, an LC50/EC50 ratio of 42.2922 (>15 indicating low toxicity), and a resuscitation rate of 71.11%, while it showed no significant phenotypic differences in the zebrafish embryos after treatment for 48 h. The toxicity screening of zebrafish also demonstrated the low ecotoxicity of the selected compounds. Furthermore, homology modeling of the HSP90 structure was performed based on related protein sequences in barnacles. Subsequently, molecular docking studies were conducted on HSP90 using these newly synthesized derivatives. Molecular docking analyses showed that most activated derivatives displayed low binding energies with HSP90, aligning well with the biological results. They were found to interact with key residues in the binding site, specifically ARG243, TYR101, and LEU73. These computational findings are anticipated to aid in predicting the enzyme targets of the tested inhibitors and their potential interactions, thus facilitating the design of novel antifoulants in future research endeavors.

Anti-Migratory Activity of Brazilin Chemodiversification on Breast Cancer Cells

Breast cancer is the most common and the leading cause of cancer death in women worldwide; treating invasive breast carcinomas is challenging due to the side effects of chemotherapeutics. Compounds isolated from natural sources have been proposed as potential molecules for cancer therapy; for instance, the homoisoflavonoid brazilin has shown pharmacological properties, including anti-tumoral and anti-inflammatory activities. In this study, we isolated brazilin from the heartwood of Haematoxylum brasiletto; then, we performed a semi-synthesis by adding three methyl or acetyl groups to the core structure of brazilin. We confirmed the identity of brazilin and its derivatives by spectroscopic data (1H NMR and 13C NMR) and measured their purity by optical rotation. Then, we analyzed the effects of brazilin and its derivatives in three mammary gland-derived cell lines: the TNBC MDA-MB-231, the ERα(+) MCF7, and the non-tumorigenic MCF10A. We evaluated the cell viability by MTT assays, cell migration by wound-healing assays, and focal adhesion kinase (FAK) activation by Western blot. Regarding biological assays, the MTT assay showed that these compounds showed cytotoxic effects on the MCF7 and MDA-MB-231 breast cancer cells at 20 µM but was not toxic in non-tumorigenic MCF10A mammary epithelial cells. Specifically, the greatest effects found from treatment with the compounds were in the MDA-MB-231 cell line, where the IC50 of brazilin was 49.92 μM, and for MCF7, the brazilin-(OAc)3 was 49.97 μM. These effects were dose- and time-dependent, as well as being associated with a decrease in the levels of cell migration and FAK activation.

Green Synthesis, Formulation and Test Field of Lymantria monacha L. (Lepidoptera: Lymantriidae) Sex Pheromone in East European Region

Green Synthesis, Formulation and Test Field of Lymantria monacha L. (Lepidoptera: Lymantriidae) Sex Pheromone in East European Region

In-cell NMR reveals metabolic adaptations in central carbon pathways driving antibiotic tolerance in Salmonella Typhimurium.

Antibiotic tolerance presents a significant challenge in eradicating bacterial infections, as tolerant strains can survive antibiotic treatment, contributing to the recurrence of infections and the development of resistance. However, unlike antibiotic resistance, tolerance is not detectable by standard susceptibility assays such as minimal inhibitory concentration (MIC) tests. Consequently, antibiotic tolerance often goes unnoticed in clinical settings. Bacterial metabolism is closely linked to antibiotic efficacy, and thus presents as a potential target for novel diagnostic methods. Recent advancements in nuclear magnetic resonance (NMR) spectroscopy, including dynamic nuclear polarization (DNP-NMR), enable a non-invasive real-time approach to analyzing bacterial metabolism. In this study, we applied both 1H and in-cell 13C NMR spectroscopy to investigate metabolic adaptations in a tolerance-evolved Salmonella Typhimurium strain, C10, developed through ten cycles of ampicillin treatment. Our results demonstrated that despite similar MICs and growth rates, the C10 strain exhibited a 25-fold increase in tolerance compared to the wild-type, while exhibiting lower metabolic activity. Under ampicillin stress, however, the C10 strain maintained higher metabolic activity and demonstrated greater resilience in glucose consumption and metabolite production relative to the wild-type. Using DNP-NMR, rapid metabolic shifts in the C10 strain were identified within 10 minutes of exposure to high concentrations of ampicillin, characterized by accumulation of key metabolites such as pyruvate and acetate. Overall, our findings underscore the potential of real-time NMR-based analyses to provide deeper insights into antibiotic tolerance and distinguish between susceptible and tolerant bacterial strains.

Synthesis and Biological Evaluation of Cyclohexane-1,2,3,5-Tetrol Analogs as Potential Lead Candidates for Cancer Therapy

Introduction: A preliminary biological evaluation of ten synthesized analogs, 5a-5j {[5-(E)-benzylidene amino]-1,3,4-thiadiazol-2-yl} cyclohexane-1,2,3,5-tetrol, revealed promising results, positioning these compounds as potential lead candidates for further optimization and preclinical exploration in cancer therapy. Incorporating the 1,3,4-thiadiazol moiety into the structure of the produced analogs considerably improved their anticancer activities, revealing the compound's high potential for cancer treatment. Spectroscopic approaches and structural analysis, such as elemental analysis, IR, ¹H NMR, ¹³C NMR, and MS, were crucial for properly establishing the molecular structures of these analogs and evaluating their anticancer activity. These methodologies laid a solid platform for rationally developing new anticancer medications with increased efficacy and fewer adverse effects. Furthermore, computational tools like molecular docking, ADMET prediction, and drug-likeness evaluation have accelerated the drug development by finding the most promising lead candidates for preclinical and clinical trials. This technique not only saves time and costs, but also raises the chances of producing successful anticancer drugs. Method: The main aim of the current study is to develop, synthesize, in-silico, in-vitro potentials of {5-[(E)-benzylidene amino]-1,3,4-thiadiazol-2-yl} cyclohexane-1,2,3,5-tetrol for a possible anticancer drug to improve their efficiency and selectivity against cancer cells, computational approaches aided in the rational design of these chemicals. Spectroscopic methods verified the chemical structures of the target compounds. The structures show the presence of 1,3,4, thiadiazol also responsible for anticancer activity. The 10 analogs were synthesized and showed encouraging anticancer efficacy in preliminary biological evaluation, suggesting they might be suitable lead candidates for more optimization and preclinical exploration Results: {5-[(E)-benzylidene amino]-1,3,4-thiadiazol-2-yl} cyclohexane-1,2,3,5-tetrol derivatives 5a-5j showed optimum IC50 values in invitro activity by SRB assay using MCF-7 as a strain, the few selected analogs. Conclusion: The study synthesized cyclohexane-1,2,3,5-tetrol analogs and evaluated their potential for cancer therapy. The compounds showed promising cytotoxic activity against various cancer cell lines, with 5c, 5f, 5g, and 5j showing the most potent anti-proliferative effects. These compounds induce apoptosis via mitochondrial dysfunction and cell cycle arrest. Further preclinical investigations are needed to establish their therapeutic potential.

Anthracene‐Based Schiff Base Allied Triazolyl Silatranes: A Comparison of Stability and Biological Activity With Open Analog Silane

ABSTRACTIn this article, we have synthesized a series of anthracene‐based triazolyl silatranes (ABSiT 1–7) using their open analogs, that is, anthracene‐based triazolyl tri‐ethoxysilanes via transesterification reaction. All synthesized compounds have been characterized using FTIR, NMR (1H and 13C), DFT techniques, and TGA/DSC analysis to identify the stability and melting temperatures. The prepared silatranes were subsequently subjected to hydrolysis to compare their stability with their open‐chain counterparts and explore their behavior under hydrolytic conditions. The data demonstrate that silatranes are about three times less prone to attack by water than organo‐triethoxysilane and are hydrolytically more stable. Moreover, the thermal stability and pH stability were checked, and it was found that silatranes are stable up to a temperature range of 400°C and at pH 9.4, thus indicating that silatranes are more stable than their corresponding silanes at higher temperatures and large pH range. Moreover, all synthesized silatranes were tested for antioxidant and cytotoxicity activities, and the results disclosed their efficacy. A molecular docking study showed interactions of silatranes with Mutant p53 antitumor protein with binding affinity in the range −8.0 to −9.0 kcal/mol and identified as potent inhibitors.

Structure of a Sulfated Capsular Polysaccharide from the Marine Bacterium Cobetia marina KMM 1449 and a Genomic Insight into Its Biosynthesis

Some marine and extremophilic microorganisms are capable of synthesizing sulfated polysaccharides with a unique structure. A number of studies indicate significant biological properties of individual sulfated polysaccharides, such as antiproliferative activity, which makes them a promising area for further research. In this study, the capsular polysaccharide (CPS) was obtained from the bacterium Cobetia marina KMM 1449, isolated from a marine sediment sample collected along the shore of the Sea of Japan. The CPS was isolated by saline solution, purified by a series of chromatographic procedures, and studied by chemical methods along with 1D and 2D 1H and 13C NMR spectroscopy. The following new structure of the CPS from C. marina KMM 1449 was established and consisted of sulfated and simultaneously phosphorylated disaccharide repeating units: →4)-α-L-Rhap2S-(1→3)-β-D-Manp6PGro-(1→. To elucidate the genetic basis of the CPS biosynthesis, the whole genomic sequence of C. marina KMM 1449 was obtained. The CPS biosynthetic gene cluster (BGC) of about 70 genes composes four regions encoding nucleotide sugar biosynthesis (dTDP-Rha and GDP-Man), assembly (GTs genes), translocation (ABC transporter genes), sulfation (PAPS biosynthesis and sulfotransferase genes) and lipid carrier biosynthesis (wcb operon). Comparative analysis of the CPS BGCs from available Cobetia genomes showed the presence of KMM 1449-like CPS BGC among strains of all three Cobetia species. The study of new natural sulfated polysaccharides, as well as the elucidation of the pathways of their biosynthesis, provides the basis for the development of potential anticancer drugs.

Benzothiazole-triazole hybrids: Novel anticancer agents inducing cell cycle arrest and apoptosis through Bcl-2 inhibition in triple-negative breast cancer.

In this study, we aim to detail the design and synthesis of a series of benzothiazole tethered triazole compounds that incorporate acetamide chains, with the purpose of investigating their potential as anticancer agents. The structural integrity of the compounds was confirmed through characterization using 1H NMR, 13C NMR, mass spectrometry, and IR spectroscopy. The compounds demonstrated notable cytotoxic effects when tested against a range of cancer cell lines, with a specific inhibition observed in triple-negative breast cancer. Among the compounds, the one with trichloro substitution demonstrated the highest potency, as indicated by an IC50 value of 30.49μM. The compounds were found to trigger cell cycle arrest in the G2/M phase and promote apoptosis, as observed in the mechanistic studies. The Bcl-2 protein exhibited significant binding interactions in molecular docking studies, which were then corroborated through molecular dynamics simulations spanning 100ns. The simulations confirmed the stability of the ligand-protein complex, as supported by RMSD, RMSF, and hydrogen bond analyses, reinforcing the proposed mechanism of Bcl-2-mediated apoptosis.

Microwave-Assisted Synthesis of Bis-1,2,3-Triazole Based Benzophenones, In Vitro Antimicrobial Activity and Molecular Docking Studies.

In this work, we have adopted an easy route to synthesizing bis-1,2,3-triazole-based benzophenone compounds via a 1,3-dipolar cycloaddition reaction (Click Chemistry). All the target compounds achieved better yields though the microwave-assisted method than the conventional method. Target compounds structure were confirmed based on the IR, 1H NMR, 13C NMR and HR Mass analysis. Additionally, we have carried out in vitro antibacterial and antifungal activities with Ciprofloxacin and Fluconazole standard drugs, respectively. The compounds 5b, 5f, 5i, 5k and 5n antibacterial, 5a, 5e, 5g, 5i, and 5k showed promising antifungal activity with respect to standard drugs. Further, molecular docking study performed against DNA gyrase and Lanosterol 14-alpha demethylase envisioned promising binding interactions with a good docking score.

Development of agar-alginate marine polysaccharides-based hydrogels for agricultural applications to reduce environmental hazards.

In order to meet global food requirement, innovation in agricultural techniques and pesticide delivery system will be required for sustainable food supply with minimal harmful impact on environment. This article discusses the synthesis of hydrogels for use in controlled release formulations (CRFs) to increase agricultural output while reducing ecotoxicity and health risks. These hydrogels were designed by graft-copolymerization reaction of polyacrylamide and polyvinyl sulfonic acid onto agar-alginate marine polysaccharides. Copolymers were characterized by SEM, AFM, XRD, FTIR and 13C NMR. One gram of copolymeric hydrogels absorbed 14.80 ± 0.53 g of water. The glyphosate herbicide was released in a slow regulated manner over 72 h which is useful to avoid herbicide loss through leaching, evaporation and to reduce environmental hazards. The herbicide released via a non-Fickian diffusion mechanism and release profile was best described by the Korsmeyer-Peppas kinetic model. The release of herbicide from hydrogels occurred slowly and consistently in simulated soil conditions for a prolonged period. A soil adsorption studies of herbicide revealed a reduction in ground water ubiquity score (GUS) for glyphosate encapsulated hydrogels as compared to commercial formulations. Soil water retention was enhanced by the addition of hydrogel in the soil. The degradation of these CRFs can provide micronutrients (N and S) to improve soil quality and minimize the risk of water pollution by reducing the leaching of herbicides.

Tamarind seed gum-based hydrogel for the targeted delivery of imidazobenzothiazole sulfonamide derivative as an anticancer agent.

The current investigation intended to assess the controlled delivery of 7-sulfonamide-2-(4-methylphenyl) imidazo[2,1-b] [1, 3] benzothiazole an anticancer agent (ACA) by tamarind seed gum-based hydrogel; for its potential activity against hepatocellular carcinoma. The FTIR spectra, SEM, 13C NMR, PXRD, and TGA analyses evidenced the successful loading of ACA into the hydrogel system. The rheological testing conveyed the increase in the elastic nature of ACA-loaded hydrogel helping in an effective release. In-vitro delivery of ACA from the hydrogel matrix was maximum at pH 5.5 with controlled and prolonged release of 98.93 ± 1 % over 1680 min. The ACA-release kinetics was well-fitted to the Hill equation model (R2 = 0.9925), leading to a non-Fickian diffusion process (n = 0.5217). The tamarind seed gum-based hydrogel as a potential matrix for the oral administration of the ACA at hepatocellular carcinoma was envisaged and acute oral toxicity assessment on the Drosophila Melanogaster model indicated a high safety profile in-vivo. The ACA-loaded TG-g-poly (AMPS) system showed an enhanced anticancer activity with an IC50 value of 37.27 μg/mL than the ACA (IC50 = 44.75 μg/mL). Studies on the ACA-loaded hydrogel's ability to induce apoptosis in hepatocellular carcinoma cells further supported its anticancer effectiveness in-vitro.

Dinuclear iridium complexes ligated by lithium-ion endohedral fullerene Li+@C60.

The diiridium complexes of lithium-ion endohedral fullerene Li+@C60 were synthesised in high yields. X-ray crystallography revealed the η2:η2-coordination of Li+@C60 and the disorder of the Li+ ion over two sites close to the coordinated carbons. 13C NMR study suggested the presence of dynamic behaviour via haptotropic rearrangements. UV/Vis and CV characteristics were also investigated experimentally and theoretically.

Accurate Molar Mass Estimation of Polyolefin Blends Using High Temperature-size Exclusion Chromatography With Triple Detection: Insight Into Molecular Level Mixing of Polypropylene With Various Application Grades of Polyethylene.

In this study, a commercially available polypropylene homopolymer (H-PP) was blended with blow molding polyethylene (PE) grade via melt mixing using a compounding machine. The resulting blends were subjected to high-temperature size exclusion chromatography (SEC) analysis, coupled with infrared-5 (IR-5), viscometer (VISCO), and multi-angle laser light scattering (MALS) detectors. The molecular weight (MW) and MW distributions were investigated using SEC, and the exact blend compositions were evaluated using 13C nuclear magnetic resonance. The molecular-level mixing of the PP/PE blend composition was assessed by comparing the theoretical and measured weight-average MW (Mw) values obtained using different SEC detection modes. The measured average Mw values of different PP/PE blends obtained using VISCO and multi-angle light-scattering detection were found to be in close agreement with predicted blend ratios, compared to the conventional SEC technique (IR-5), with percentage errors ranging from -4.53 to -5.99 and 1.57 to 7.65, respectively. The linear relationship between the melt flow rate (MFR) and Mw was studied to assess the molecular-level mixing behavior of the prepared blends using different SEC detection modes. Furthermore, the SEC methodology was extended to other application grades of PE with varying MFRs to verify the blend mixing behavior with H-PP, and the obtained results are discussed.