Browsing by Subject "NMR"

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  • Niemelä, Miska Aleksanteri (Helsingin yliopisto, 2022)
    Master's thesis project includes the backbone assignment of the human activity-regulated cytoskeleton-associated protein C-lobe (hArc, Uniprot ID: Q7LC44), 7-fluoroindole-based tryptophan-labeling method, and comparing that with the 100% double-labeled and 20%(13C) fractionally labeled samples. The project focuses on the effects of 7-fluoroindole-based fluorotryptophan-labeling. hArc C-lobe has only one tryptophan, which makes the analysis easier. Typically fluorotryptophan-labeling is a costly method – fluorotryptophan itself is very expensive and attaching the fluorine to the tryptophan while expressing is expensive and complicated. Fluoroindolebased labeling circles around the problem, as indole and serine are used in procaryotic systems for tryptophan biosynthesis – meaning that fluoroindole, which is cheap, could be used as an alternative for previous methods. Fluoro-labeled tryptophan is used in protein NMR; for example, in binding studies – fluorine-probes are sensitive, and binding of ligand or protein would move these peaks, indicating binding. This project aims to get an insight into the application of this labeling method. The goal is to see if one could utilize one sample with both (1H, 15N, 13C) labeling and 7-fluorotryptophan labeling for binding and structural studies. However, fluorine is very electronegative, affecting surrounding structures and possibly sequentially nearby amino acids. This possible effect will be observed and determined by comparing the 1H15N-chemical shifts between well-established labeling methods and fluoroindolebased labeling. To determine what amino acids in the protein are affected, if they are affected, will be determined by using the backbone assignment results and the results from the sample comparisons.
  • Delepierre, Gwenn; Heise, Katje; Malinen, Kiia; Koso, Tetyana; Pitkänen, Leena; Cranston, Emily; Kilpeläinen, Ilkka; Kostiainen, Mauri A.; Kontturi, Eero; Weder, Christoph; Zoppe, Justin; King, Alistair (2021)
    When cellulose nanocrystals (CNCs) are isolated from cellulose microfibrils, the parallel arrangement of the cellulose chains in the crystalline domains is retained so that all reducing end-groups (REGs) point to one crystallite end. This permits the selective chemical modification of one end of the CNCs. In this study, two reaction pathways are compared to selectively attach atom-transfer radical polymerization (ATRP) initiators to the REGs of CNCs, using reductive amination. This modification further enabled the site-specific grafting of the anionic polyelectrolyte poly(sodium 4-styrenesulfonate) (PSS) from the CNCs. Different analytical methods, including colorimetry and solutionstate NMR analysis, were combined to confirm the REG-modification with ATRP-initiators and PSS. The achieved grafting yield was low due to either a limited conversion of the CNC REGs or side reactions on the polymerization initiator during the reductive amination. The end-tethered CNCs were easy to redisperse in water after freeze-drying, and the shear birefringence of colloidal suspensions is maintained after this process.
  • van der Lee, Sven J.; Teunissen, Charlotte E.; Pool, Rene; Shipley, Martin J.; Teumer, Alexander; Chouraki, Vincent; van Lent, Debora Melo; Tynkkynen, Juho; Fischer, Krista; Hernesniemi, Jussi; Haller, Toomas; Singh-Manoux, Archana; Verhoeven, Aswin; Willemsen, Gonneke; de Leeuw, Francisca A.; Wagner, Holger; van Dongen, Jenny; Hertel, Johannes; Budde, Kathrin; van Dijk, Ko Willems; Weinhold, Leonie; Ikram, M. Arfan; Pietzner, Maik; Perola, Markus; Wagner, Michael; Friedrich, Nele; Slagboom, P. Eline; Scheltens, Philip; Yang, Qiong; Gertzen, Robert E.; Egert, Sarah; Li, Shuo; Hankemeier, Thomas; van Beijsterveldt, Catharina E. M.; Vasan, Ramachandran S.; Maier, Wolfgang; Peeters, Carel F. W.; Grabe, Hans Joergen; Ramirez, Alfredo; Seshadri, Sudha; Metspalu, Andres; Kivimäki, Mika; Salomaa, Veikko; Demirkan, Ayse; Boomsma, Dorret I.; van der Flier, Wiesje M.; Amin, Najaf; van Duijn, Cornelia M. (2018)
    Introduction: Identifying circulating metabolites that are associated with cognition and dementia may improve our understanding of the pathogenesis of dementia and provide crucial readouts for preventive and therapeutic interventions. Methods: We studied 299 metabolites in relation to cognition (general cognitive ability) in two discovery cohorts (N total = 5658). Metabolites significantly associated with cognition after adjusting for multiple testing were replicated in four independent cohorts (N total = 6652), and the associations with dementia and Alzheimer's disease (N = 25,872) and lifestyle factors (N = 5168) were examined. Results: We discovered and replicated 15 metabolites associated with cognition including subfractions of high-density lipoprotein, docosahexaenoic acid, ornithine, glutamine, and glycoprotein acetyls. These associations were independent of classical risk factors including high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, and apolipoprotein E (APOE) genotypes. Six of the cognition-associated metabolites were related to the risk of dementia and lifestyle factors. Discussion: Circulating metabolites were consistently associated with cognition, dementia, and lifestyle factors, opening new avenues for prevention of cognitive decline and dementia. (C) 2018 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer's Association.
  • Ahvenainen, Patrik; Kontro, Inkeri; Svedström, Kirsi (2016)
    Cellulose crystallinity assessment is important for optimizing the yield of cellulose products, such as bioethanol. X-ray diffraction is often used for this purpose for its perceived robustness and availability. In this work, the five most common analysis methods (the Segal peak height method and those based on peak fitting and/or amorphous standards) are critically reviewed and compared to two-dimensional Rietveld refinement. A larger () and more varied collection of samples than previous studies have presented is used. In particular, samples () with low crystallinity and small crystallite sizes are included. A good linear correlation () between the five most common methods suggests that they agree on large-scale crystallinity differences between samples. For small crystallinity differences, however, correlation was not seen for samples that were from distinct sample sets. The least-squares fitting using an amorphous standard shows the smallest crystallite size dependence and this method combined with perpendicular transmission geometry also yielded values closest to independently obtained cellulose crystallinity values. On the other hand, these values are too low according to the Rietveld refinement. All analysis methods have weaknesses that should be considered when assessing differences in sample crystallinity.
  • Tanaka, Atsushi; Khakalo, Alexey; Hauru, Lauri; Korpela, Antti; Orelma, Hannes (2018)
    In this study, we investigate the “chemical welding” of paper with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) using a two-step process. First, the IL is transported into the structure of the paper as a water solution. Then, partial dissolution is achieved by activation with heat (80–95 °C), where the water evaporates and the surfaces of the fibres partially dissolve. The activated paper is washed with water to remove IL, and dried to fuse fibre surfaces into each other. The “chemically welded” paper structure has both elevated dry and wet strength. The treatment conditions can be adjusted to produce both paper-like materials and films. The most severe treatment conditions produce films that are fully transparent and their oxygen and grease barrier properties are excellent. As an all-cellulose material, the “chemically welded” paper is fully biodegradable and is a potential alternative to fossil fuel-based plastics.
  • Hendrik, Nathaniel James (Helsingin yliopisto, 2017)
    Cocoa butter (CB) is the predominant continuous phase in chocolate systems and has a significant impact on the macroscopic properties of the end product. Conventional methods such as differential scanning calorimetry (DSC), pulsed nuclear magnetic resonance (pNMR), X-ray diffraction (XRD) and polarized light microscopy (PLM) have been used to study CB crystallization primarily in bulk. Potential of alternative techniques to study crystallization such as Raman spectroscopy and Fourier Transform infrared spectroscopy (FTIR) has been explored. The main objective of this thesis research was to study the feasibility of both conventional and alternative techniques to study CB crystallization in different matrices and in tempered conditions. Bulk fat (CB with 1%, 5% or without lecithin), suspensions (CB with 1% lecithin (on fat basis) and sucrose or inulin) and chocolates were sampled as such (non-tempered systems) subjected to a laboratory scale tempering procedure to produce tempered systems. Both non-tempered and tempered products were subjected to DSC, NMR, XRD, PLM, Raman spectroscopy, FTIR and diffusing wave spectroscopy (DWS), in which primary crystallization was monitored or long-term storage was assessed. A toolbox was developed comprising feasibility of complementary techniques and, moreover, the toolbox was used to study the effect of lecithin and bulking materials on the CB crystallization behavior. The tempering procedure was successfully validated for every sample, as proven by the melting profile at 6 hours through DSC. The determination of the solid fat content (SFC) from the raw free induction decay signal by NMR showed to be more useful than the scripted SFC, especially for bulk fat systems. XRD showed its feasibility to study fat polymorphism for both bulk matrices and suspensions, except when sucrose is present, due to its interference in short spacings. PLM could only be used for non-tempered bulk fat systems since in other systems sample preparation cannot be standardized to measure crystallinity. FTIR and Raman spectroscopy seemed to be useful complementary techniques and capable of differentiating polymorphic forms, as is also possible using XRD. DWS showed to be comparable with DSC with an additional improved deconvolution of crystallization peaks. This study resulted in a feasibility toolbox and was used to study the effect of lecithin concentration and bulking materials, where the addition of 1% lecithin concentration in bulk fat and usage of inulin in model suspensions improves the crystallization of the CB matrix.
  • Figueiredo, Patricia; Lahtinen, Maarit; Agustin, Melissa; Morais de Carvalho, Danila; Hirvonen, Sami-Pekka; Penttilä, Paavo A.; Mikkonen, Kirsi S. (2021)
    The production of lignin nanoparticles (LNPs) has emerged as a way to overcome the highly variable and complex molecular structure of lignin. It can offer morphological control of the lignin polymer, allowing the formation of stable LNP dispersions in aqueous media, while increasing the potential of lignin for high-value applications. However, the polydispersity and morphology of LNPs varies depending on the lignin grade and preparation method, and a systematic comparison using different technical lignins is lacking. In this study, it was attempted to find a green fabrication method with a distinct solvent fractionation of lignin to prepare LNPs using three different technical lignins as starting polymers: BLN birch lignin (hardwood, BB), alkali Protobind 1000 (grass, PB), and kraft LignoBoost (softwood, LB). For that, three anti-solvent precipitation approaches to prepare LNPs were systematically compared: 70 % aqueous ethanol, acetone/water (3 : 1) and NaOH as the lignin solvent, and water/aqueous HCl as the anti-solvent. Among all these methods, the acetone/water (3 : 1) approach allowed production of homogeneous and monodisperse LNPs with a negative surface charge and also spherical and smooth surfaces. Overall, the results revealed that the acetone/water (3 : 1) method was the most effective approach tested to obtain homogenous, small, and spherical LNPs from the three technical lignins. These LNPs exhibited an improved stability at different ionic strengths and a wider pH range compared to the other preparation methods, which can greatly increase their application in many fields, such as pharmaceutical and food sciences.
  • Sokka, Iris K.; Imlimthan, Surachet; Sarparanta, Mirkka; Maaheimo, Hannu; Johansson, Mikael P.; Ekholm, Filip S. (2021)
    Halogenation can be utilized for the purposes of labeling and molecular imaging, providing a means to, e.g., follow drug distribution in an organism through positron emission tomography (PET) or study the molecular recognition events unfolding by nuclear magnetic resonance (NMR) spectroscopy. For cancer therapeutics, where often highly toxic substances are employed, it is of importance to be able to track the distribution of the drugs and their metabolites in order to ensure minimal side effects. Labeling should ideally have a negligible disruptive effect on the efficacy of a given drug. Using a combination of NMR spectroscopy and cytotoxicity assays, we identify a site susceptible to halogenation in monomethyl auristatin F (MMAF), a widely used cytotoxic agent in the antibody-drug conjugate (ADC) family of cancer drugs, and study the effects of fluorination and chlorination on the physiological solution structure of the auristatins and their cytotoxicity. We find that the cytotoxicity of the parent drug is retained, while the conformational equilibrium is shifted significantly toward the biologically active trans isomer, simultaneously decreasing the concentration of the inactive and potentially disruptive cis isomer by up to 50%. Our results may serve as a base for the future assembly of a multifunctional toolkit for the assessment of linker technologies and exploring bystander effects from the warhead perspective in auristatin-derived ADCs.
  • Ottka, Claudia; Vapalahti, Katariina; Määttä, Ann-Marie; Huuskonen, Nanna; Sarpanen, Sinikka; Jalkanen, Liisa; Lohi, Hannes (2021)
    Background The kidneys have many essential metabolic functions, and metabolic disturbances during decreased renal function have not been studied extensively. Objectives To identify metabolic changes in blood samples with increased serum creatinine concentration, indicating decreased glomerular filtration. Animals Clinical samples analyzed using a nuclear magnetic resonance (NMR) based metabolomics platform. The case group consisted of 23 samples with serum creatinine concentration >125 mu mol/L, and the control group of 873 samples with serum creatinine concentration within the reference interval. Methods Biomarker association with increased serum creatinine concentration was evaluated utilizing 3 statistical approaches: Wilcoxon rank-sum test, logistic regression analysis (false discovery rate (FDR)-corrected P-values), and random forest classification. Medians of the biomarkers were compared to reference intervals. A heatmap and box plots were used to represent the differences. Results All 3 statistical approaches identified similar analytes associated with increased serum creatinine concentrations. The percentages of citrate, tyrosine, branched-chain amino acids, valine, leucine, albumin, linoleic acid and the ratio of phenylalanine to tyrosine differed significantly using all statistical approaches, acetate differed using the Wilcoxon test and random forest, docosapentaenoic acid percentage only using logistic regression (P <.05), and alanine only using random forest. Conclusions and Clinical Importance We identified several metabolic changes associated with increased serum creatinine concentrations, including prospective diagnostic markers and therapeutic targets. Further research is needed to verify the association of these changes with the clinical state of the dog. The NMR metabolomics test is a promising tool for improving diagnostic testing and management of renal diseases in dogs.
  • Kakko, Tia; King, Alistair W. T.; Kilpeläinen, Ilkka (2017)
    Cellulose acetate is widely used in films, filters, textiles, lacquer and cosmetic products. Herein we demonstrate the production of cellulose esters under homogeneous conditions using 1,5-diazabicyclo[4.3.0]non-5-ene acetate ([DBNH][OAc]) as solvent. The reagents have been chosen such that the system is recyclable, i.e. by-products are low boiling and easy to remove. It is demonstrated that cellulose acetate can be synthesized with different degree of substitution (DS) values, and that some commonly used acylation regents, like vinyl carboxylates react well without additional base catalyst. Low to high DS values are possible with good recovery of high purity ionic liquid (IL). A linear correlation method of two separate methods, IR and P-31 NMR, is proposed to reliably assess the DS of the products. The recyclability of the solvent is demonstrated by acetylating cellulose with isopropenyl acetate to high degree and regeneration into water. After regeneration of cellulose acetate from the IL with addition of water, the residual water was entrained using n-butanol to minimize hydrolysis of [DBNH][OAc], to allow for high recovery and high purity of the ionic liquid. Thus, an overall scheme for batch cellulose acetylation and recovery of [DBNH][OAc] from aqueous solutions is proposed.
  • Toukola, Peppi (Helsingin yliopisto, 2021)
    In this thesis the suitability of Nuclear Magnetic Resonance (NMR) spectroscopy in the identification of rubbers in museum collections is discussed through a literature review and experimental work where samples from the rubber collection of Tampere Museums were analysed with different NMR techniques. The literature part of this thesis focuses on recent (2011-2020) scientific publications on analytical instrumental techniques used in the identification of cultural heritage plastics. Vibrational spectroscopy methods utilizing hand-held or portable devices have been the most prominent methods used in characterization of historical plastics materials. Bench-top devices and analytical techniques requiring sampling were used to acquire more detailed analysis results. However, NMR spectroscopy was not used as the main analysis technique in the reviewed publications. In the experimental part altogether 21 rubber object samples and 8 reference samples were identified using 1D and 2D NMR techniques in solution state. Three samples were additionally analysed with solid-state High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy. The chemical structures of the samples were confirmed with these methods. To further explore fast and more automated identification of the rubber samples a statistical classification model utilizing acquired solution-state 1H NMR data was developed. Three rubber types were chosen for the analysis. The model was created using analysis data from the museum object samples and validated using the reference sample data. Identification rate of 100 % was achieved.
  • Ghini, Veronica; Abuja, Peter M.; Polasek, Ozren; Kozera, Lukasz; Laiho, Päivi; Anton, Gabriele; Zins, Marie; Klovins, Janis; Metspalu, Andres; Wichmann, H-Erich; Gieger, Christian; Luchinat, Claudio; Zatloukal, Kurt; Turano, Paola (2022)
    The development of metabolomics in clinical applications has been limited by the lack of validation in large multicenter studies. Large population cohorts and their biobanks are a valuable resource for acquiring insights into molecular disease mechanisms. Nevertheless, most of their collections are not tailored for metabolomics and have been created without specific attention to the pre-analytical requirements for high-quality metabolome assessment. Thus, comparing samples obtained by different pre-analytical procedures remains a major challenge. Here, H-1 NMR-based analyses are used to demonstrate how human serum and plasma samples collected with different operating procedures within several large European cohort studies from the Biobanking and Biomolecular Resources Infrastructure - Large Prospective Cohorts (BBMRI-LPC) consortium can be easily revealed by supervised multivariate statistical analyses at the initial stages of the process, to avoid biases in the downstream analysis. The inter-biobank differences are discussed in terms of deviations from the validated CEN/TS 16945:2016 / ISO 23118:2021 norms. It clearly emerges that biobanks must adhere to the evidence-based guidelines in order to support wider-scale application of metabolomics in biomedicine, and that NMR spectroscopy is informative in comparing the quality of different sample sources in multi cohort/center studies.
  • Farrar, Zoe May (Helsingin yliopisto, 2020)
    Mycosporine-like Amino Acids (MAAs) are small, secondary metabolites, with the ability to absorb UV light. They are produced by cyanobacteria to act as a sunscreen. The aim of this study was to catalogue MAA genetic and chemical diversity in strains of the cyanobacterial genus Nostoc. MAAs were detected in 21 of the 68 Nostoc strains using LC/MS. Fifty four different MAAs were detected across the Nostoc strains. Glycosylated MAAs were detected in 17 of the 21 strains with hexose being the most commonly occurring sugar. Surprisingly, two structurally distinct MAAs were detected from a lichen symbiont strain, Nostoc sp. UHCC 0926. Chemical analysis detected a theoretical methylated and glycosylated variant (m/z 583, C23H39N2O15), and a suspected tri-core variant (m/z 757, C34H53N4O15) with three chromophore rings as opposed to one which is typically found. The glycosylated MAA was predicted to have a hexenimine core which was methylated and had two hexose moieties. The tri-core consisted of 2 aminohexenone cores, one on either side of a central aminohexenimine core. An 8.3 Mb draft genome sequence was obtained to identify the MAA biosynthetic gene cluster responsible for the biosynthesis of these two unusual MAAs. This resulted in the detection of two gene clusters mysA-B-C1 and mysD-C2-C3. This gene cluster organisation was compared with those of other Nostoc strains. The gene cluster organization in Nostoc sp. UHCC 0926 was unique because it was the only strain to have two gene clusters and three mysC genes despite one of the other Nostocs having the ability to produce a tri-core MAA. The strain was cultured and harvested to allow for the extraction and purification of the target MAAs. The tri-core MAA structure was confirmed by NMR. However only a putative structure for the glycosylated MAA was made. The UV absorption spectrum of the tri-core MAA had an absorption maximum at 312 nm while the glycosylated and methylated MAA had an absorption maximum at 336 nm. The investigation into the MAA production of UHCC strains expands the known chemical and genetic diversity of MAAs produced by strains of the Nostoc genus.
  • Wang, Ying; Yang, Yang; He, Jun; Cao, Jinxuan; Wang, Hongfei; Ertbjerg, Per (2020)
    The objective was to characterize the effect of wooden breast (WB) myodegeneration on the metabolite profile of chicken meat by H-1 NMR and multivariate data analysis. The results displayed that the metabonome of chicken breast consisted predominantly of 30 metabolites, including amino acids, organic acids, carbohydrates, alkaloids, nucleosides and their derivatives. WB-affected samples showed higher leucine, valine, alanine, glutamate, lysine, lactate, succinate, taurine, glucose, and 5'-IMP levels, but lower histidine, beta-alanine, acetate, creatine, creatinine, anserine and nicotinamide adenine dinucleotide levels compared to normal fillets (p <0.05). In conclusion, results indicated that WB-affected fillets possessed a unique biochemical signature. This unique profile could identify candidate biomarkers for diagnostic utilization and provide mechanistic insight into biochemical processes leading to WB myopathy in commercial broiler chickens.
  • Kohl, Lukas; Myers-Pigg, Allison; Edwards, Kate A.; Billings, Sharon A.; Warren, Jamie; Podrebarac, Frances; Ziegler, Susan E. (2021)
    Plant litter chemistry is altered during decomposition but it remains unknown if these alterations, and thus the composition of residual litter, will change in response to climate. Selective microbial mineralization of litter components and the accumulation of microbial necromass can drive litter compositional change, but the extent to which these mechanisms respond to climate remains poorly understood. We addressed this knowledge gap by studying needle litter decomposition along a boreal forest climate transect. Specifically, we investigated how the composition and/or metabolism of the decomposer community varies with climate, and if that variation is associated with distinct modifications of litter chemistry during decomposition. We analyzed the composition of microbial phospholipid fatty acids (PLFA) in the litter layer and measured natural abundance δ13C-PLFA values as an integrated measure of microbial metabolisms. Changes in litter chemistry and δ13C values were measured in litterbag experiments conducted at each transect site. A warmer climate was associated with higher litter nitrogen concentrations as well as altered microbial community structure (lower fungi:bacteria ratios) and microbial metabolism (higher δ13C-PLFA). Litter in warmer transect regions accumulated less aliphatic-C (lipids, waxes) and retained more O-alkyl-C (carbohydrates), consistent with enhanced 13C-enrichment in residual litter, than in colder regions. These results suggest that chemical changes during litter decomposition will change with climate, driven primarily by indirect climate effects (e.g. greater nitrogen availability and decreased fungi:bacteria ratios) rather than direct temperature effects. A positive correlation between microbial biomass δ13C values and 13C-enrichment during decomposition suggests that change in litter chemistry is driven more by distinct microbial necromass inputs than differences in the selective removal of litter components. Our study highlights the role that microbial inputs during early litter decomposition can play in shaping surface litter contribution to soil organic matter as it responds to climate warming effects such as greater nitrogen availability.
  • Vainio, Mika (Helsingfors universitet, 2016)
    The literature review dealed with fat replacers in frankfurters. Manufacturing, properties and usages of microcrystalline cellulose were described. Also other fat replacers that are at the moment in use are described, such as hydrocolloids and proteins from vegetable. The literature review also described different methods that have been used to measure properties of frankfurters. The aim of the experimental work was to find out the influence of two different types of microcrystalline cellulose, Vivapur® 105(i) and Arbocel® M80(ii), on the properties of frankfurters, and can they been used as a fat replacer. Three different concentrations of microcrystalline cellulose (MCC) (1 %, 3 % and 5 %) Were studied and compared to the control sample. Measured properties of frankfurters were pH, water holding, cooking loss, firmness and bite force. Also amount of free water was measured with NMR. Although adding MCC decreased pH of frankfurter closer to the isoelectric point, it did not affect as lowering water holding. Both MCC(i) and MCC(ii) increased significantly (p<0,05) firmness of the frankfurters when measuring was made with warm samples, excluding MCC(i) 3 % concentration. Effect was not as visible in the measurements of bite force.
  • Koivunotko, Elle; Merivaara, Arto; Niemela, Akseli; Valkonen, Sami; Manninen, Kalle; Makinen, Henrik; Viljanen, Mira; Svedstrom, Kirsi; Diaz, Ana; Holler, Mirko; Zini, Jacopo; Paasonen, Lauri; Korhonen, Ossi; Huotari, Simo; Koivuniemi, Artturi; Yliperttula, Marjo (2021)
    The diversity and safety of nanofibrillated cellulose (NFC) hydrogels have gained a vast amount of interest at the pharmaceutical site in recent years. Moreover, this biomaterial has a high potential to be utilized as a protective matrix during the freeze-drying of heat-sensitive pharmaceuticals and biologics to increase their properties for long-term storing at room temperature and transportation. Since freeze-drying and subsequent reconstitution have not been optimized for this biomaterial, we must find a wider understanding of the process itself as well as the molecular level interactions between the NFC hydrogel and the most suitable lyoprotectants. Herein we optimized the reconstitution of the freeze-dried NFC hydrogel by considering critical quality attributes required to ensure the success of the process and gained insights of the obtained experimental data by simulating the effects of the used lyoprotectants on water and NFC. We discovered the correlation between the measured characteristics and molecular dynamics simulations and obtained successful freeze-drying and subsequent reconstitution of NFC hydrogel with the presence of 300 mM of sucrose. These findings demonstrated the possibility of using the simulations together with the experimental measurements to obtain a more comprehensive way to design a successful freeze-drying process, which could be utilized in future pharmaceutical applications.
  • Aly, Ashraf A.; El-Sheref, Essmat M.; Brown, Alan B.; Braese, Stefan; Nieger, Martin; Abdelhafez, El-Shinnaa M. N. (2019)
    A new one pot reaction of substituted thiosemicarbazides with 2-bromoacetophenone and carbonyl compounds gave 2-hydrazonothiazoles in good yields. The structures of the isolated compounds were corroborated by NMR, IR, mass spectra and elemental analyses in addition to X-ray structure determination. [GRAPHICS] .
  • Aly, Ashraf A.; Bräse, Stefan; Hassan, Alaa A.; Mohamed, Nasr K.; Abd El-Haleem, Lamiaa E.; Nieger, Martin; Morsy, Nesrin M.; Abdelhafez, Elshimaa M. N. (2020)
    A new series of methyl 2-(2-(4 '-[2.2]paracyclophanyl)-hydrazinylidene)-3-substituted-4-oxothiazolidin-5-ylidene)acetates3a-fwere synthesized from the reaction of paracyclophanyl-acylthiosemicarbazides2a-fwith dimethyl acetylenedicarboxylate. Based upon nuclear magnetic resonance (NMR), infrared (IR), and mass spectra (HRMS), the structure of the obtained products was elucidated. X-ray structure analysis was also used as unambiguous tool to elucidate the structure of the products. The target compounds3a-fwere screened against 60 cancer cell lines. They displayed anticancer activity against a leukemia subpanel, namely, RPMI-8226 and SR cell lines. The activity of compound3awas found as the most cytotoxic potency against 60 cancer cell lines. Consequently, it was selected for further five doses analysis according to National Cancer Institute (NCI) protocol. The cytotoxic effect showed selectivity ratios ranging between 0.63 and 1.28 and between 0.58 and 5.89 at the GI(50)and total growth inhibition (TGI) levels, respectively. Accordingly, compound3aunderwent further mechanistic study against the most sensitive leukemia RPMI-8226 and SR cell lines. It showed antiproliferation with IC50 = 1.61 +/- 0.04 and 1.11 +/- 0.03 mu M against RPMI-8226 and SR cell lines, respectively. It also revealed a remarkable tubulin inhibitory activity, compared to colchicine with IC50 = 4.97 mu M/mL. Caspase-3, BAX, and Bcl-2 assays for3ausing annexin V-FITC staining revealed significant pro-apoptotic activity. Furthermore, multidrug-resistant leukemia SR cells were used to show better resistance indices (1.285 ng/mL, 1.15-fold) than the reference. Docking studies with beta-tubulin indicate that most of the tested compounds illustrated good binding at the colchicine binding site of the enzyme, especially for compound3a, which made several interactions better than that of the reference colchicine.
  • Heikkinen, Harri A.; Backlund, Sofia M.; Iwai, Hideo (2021)
    Uniformly C-13- and N-15-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of C-13-labeled glucose by a factor of five using a fractional 20% C-13- and 100% N-15-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [C-13, N-15]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional C-13-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% C-13, 100% N-15]-labeled sample for small proteins (