Dehydration Step Research Articles

The Development of a Procedure for the Cryopreservation of the Callus of Anthurium andraeanum by Vitrification

The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used as explants to culture the callus. The cryopreservation procedure of the callus by vitrification was initially established by using the orthogonal experimental method of four factors and three levels in the preculture, loading, and dehydration steps. Furthermore, the vitrification-based cryopreservation was optimized by changing the preculture temperature and loading solution and adding exogenous substances to the plant vitrification solution (PVS2). In this procedure, the callus was precultured at 25 °C for 2 d, and loaded in 50% PVS2 at 25 °C for 60 min. The callus was dehydrated with PVS2 containing 0.08 mM reduced glutathione (GSH) at 0 °C for 60 min. After rapid-cooling in liquid nitrogen for 1 h, it was rapid-warming in a water bath at 40 °C for 90 s and unloaded for 30 min. After 1 d of recovery, the cell relative survival rate of the cryopreserved callus was 64.60%. The results provide a valuable basic and effective method for the long-term conservation of A. andraeanum germplasm resources.

Investigating base-catalyzed aldol condensation reactions on alkali-free hydrotalcites and the role of thermal decomposition

Aldolization is a crucial carbon–carbon coupling reaction used in the conversion of biomass-derived platform molecules into valuable chemicals and fuels. In this work, we combine experiment and theory to explore the role thermal decomposition has in controlling the structure of alkali-free Mg-Al layered double hydroxides (LDHs) and their subsequent performance in the base-catalyzed aldol condensation of acetone, focusing on the constituent aldolization and dehydration steps. LDHs were synthesized via co-precipitation and subjected to calcination at temperatures up to 600 °C to produce mixed metal oxides of the form Mg3AlOz which were further examined by SEM, N2 physisorption, XRD, and CO2-TPD. Aldolization and dehydration behaviors were examined in a batch reactor and fit with kinetic models to capture the sensitivities of both reactions to calcination temperature. Additional experiments involving the selective blocking of base sites by CO2 poisoning and reversal at varying temperatures showed that both aldolization and dehydration are likely driven by the strongest base sites, yielding a site density (0.19 μmol m−2) in agreement with separate propionic acid titration studies. Density functional theory (DFT) simulations of Mg5Al2O8 surfaces were employed to gain microscopic insights into these materials, revealing the importance of Al substitution in creating cation vacancies which can drive aldol condensation through enolate intermediates stabilized in the oxyanion resonance form. High-coverage simulations of the surface show participation of the vacancy in both aldolization and dehydration, rationalizing the coupling of rate trends observed experimentally. Ultimately, this study provides new insights and approaches useful in the design of heterogeneous aldolization catalysts.

A Dual CQD-Catalysis and H-Bond Acceptor for Controlling Product Selectivity and Regioselectivity in Symmetric/Unsymmetric Azoxy Arenes.

Azoxy arenes are valuable compounds in different areas of chemistry, such as organic chemistry, medicinal chemistry, and natural product chemistry. Despite their value, the regioselective synthesis of unsymmetric azoxybenzenes has remained a real challenge in the field. Herein, the product selectivity in oxidative homocoupling of anilines into symmetric azoxybenzenes was first achieved by an asparagine-functionalized CQD catalyst. Subsequently, in the cross-coupling of anilines into the unsymmetric azoxybenzenes via an ortho H-bond acceptor (HBA) on one of the coupling anilines, the regioselectivity was effectively controlled. It was demonstrated that ortho-HBA could mechanistically establish a six-membered intramolecular hydrogen-bonded ring on an N,N'-dihydroxy intermediate. The formed hydrogen bond makes the nearby nitrogen eminently suitable for the slow dehydration step. As a result, the functional oxygen of the azoxy compound is placed far from the HBA. The o-HBA mechanism also controls the regioselectivity ratio in which 1:0 (with an intramolecular H-bonded hexagonal ring), 2:1 (with an intramolecular H-bonded pentagonal ring), and 1:1 (without an ortho-HBA) isomeric mixtures could be achieved. The HBA mechanism was exploited by different substituted anilines, and various unsymmetric azoxybenzenes were synthesized. Finally, with the aid of mechanistic studies, a plausible mechanism for the reaction was proposed.

Kinetic study on the thermal decomposition of iron (II) sulfate using a global optimization approach

The thermal decomposition of sulfates is a major intermediate reaction in oxidizing roasting processes associated with metal sulfides. The optimization of iron (II) sulfate could contribute to lowering processes’ temperatures in complex systems. The present work presents the experimental and modeling results of the thermal decomposition of iron (II) sulfate heptahydrate. The investigation was first based on thermodynamic simulations followed by non-isothermal thermogravimetric analysis. The modeling technique consisted of differential equations formulated to describe each decomposition step individually, with the kinetic parameters estimated using particle swarm optimization (PSO). Using this modeling methodology over a graphical method is advantageous as it can simulate reactions that may occur simultaneously. The modeling succeeded in dehydration and desulfation steps, with an overall R2 value of 0.99. The activation energies and apparent reaction order associated with the dehydration steps were 53.3kJ.mol−1/n=1.65, 88.8kJ.mol−1/n=1.12, and 124kJ.mol−1/n=2.0 for FeSO4.7H2O, FeSO4.4H2O, and FeSO4.H2O, respectively. The desulfation steps were associated with higher activation energy values. In that case, 184kJ.mol−1 and 264kJ.mol−1 for FeSO4 and Fe2(SO4)3, respectively. Regarding the apparent reaction order, both desulfation steps showed to be first-order reactions.

Theoretical Studies on the Catalytic Mechanism and Cyclization Pattern of Fungal Polyketide Synthase Product Template Domain

AbstractAromatic polyketides are pharmaceutically significant natural products. Product template (PT) domains from fungal non‐reducing polyketide synthases (NR‐PKSs) control the regioselective aldol cyclization of highly reactive polyketide intermediates, whose instabilities have hindered experimental studies. The molecular basis of PT‐catalyzed cyclization and aromatization remain ambiguous. Herein, we conducted MD simulations and QM/MM calculations to explore the catalytic mechanHism and the cyclization pattern of PksA PT specific for C4−C9. The cyclization mediated by PksA PT is initiated by the C4 deprotonation followed by the aldol condensation in C4−C9 register. PTs which evolve from ancient PKS dehydratases (DHs) perform dehydration reactions to aromatize the cyclized intermediates. In PksA PT, the catalytic His1345 and Asp1543 are responsible for aldol cyclization and aromatization steps, respectively. A keto‐enol tautomerism at C3 assists in the elimination reaction catalyzed by Asp1543 with a water molecule network, different from most DHs of PKSs. The C2−C7 aldol cyclization in PksA PT can occur, but the subsequent dehydration step is kinetically unfeasible and thermodynamically unfavorable, suggesting that the aromatization steps determine the C4−C9 cyclization mode of PksA PT. Overall, these computational investigations provide detailed mechanistic insights into the regiospecific intramolecular cyclization and aromatization controlled by fungal PksA PT.

Efficient Conversion of Glucose to Hydroxymethylfurfural: One-pot Brønsted Base and Acid Promoted Selective Isomerization and Dehydration.

Development of elegant, selective, and efficient strategies for the production of value-added platform chemicals from renewable feedstocks are in high demand to achieve the future needs and sustainable goals. In this context, an efficient acid-promoted synthesis of highly valuable hydroxymethylfurfural (HMF) has been demonstrated from glucose, a major constituent of lignocellulosic biomass. The major challenge in the conversion of glucose to HMF is the selective isomerization of glucose to ketose, which in the present work has been successfully addressed through the amine-mediated rearrangement of glucose to aminofructose under Amadori rearrangement. Importantly, subsequent dehydration step affords HMF and regenerates the amine employed in the first step, which could be readily recovered. In addition, scale-up and successful integration into one-pot synthesis of HMF proves the efficiency and applicability of the present transformation in large scale application. In addition, the method was also successfully extended to other monosaccharides and disaccharides to produce HMF.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

A new methodology for a rapid and high-throughput comparison of molecular profiles and biological activity of phytoextracts.

To robustly discover and explore phytocompounds, it is necessary to evaluate the interrelationships between the plant species, plant tissue, and the extraction process on the extract composition and to predict its cytotoxicity. The present work evaluated how Fourier Transform InfraRed spectroscopy can acquire the molecular profile of aqueous and ethanol-based extracts obtained from leaves, seeds, and flowers of Cynara Cardunculus, and ethanol-based extracts from Matricaria chamomilla flowers, as well the impact of these extracts on the viability of mammalian cells. The extract molecular profile enabled to predict the extraction yield, and how the plant species, plant tissue, and extraction process affected the extract's relative composition. The molecular profile obtained from the culture media of cells exposed to extracts enabled to capture its impact on cells metabolism, at a higher sensitivity than the conventional assay used to determine the cell viability. Furthermore, it was possible to detect specific impacts on the cell's metabolism according to plant species, plant tissue, and extraction process. Since spectra were acquired on small volumes of samples (25 µL), after a simple dehydration step, and based on a plate with 96 wells, the method can be applied in a rapid, simple, high-throughput, and economic mode, consequently promoting the discovery of phytocompounds.

Unveiling kinetics post rate-determining step in Brønsted acid-catalyzed reactions of fructose: A strategy for 5-hydroxymethylfurfural production from concentrated feedstock

The development of high-yielding catalytic systems is pivotal in biomass upgrading. However, the investigation of kinetics in these complex reaction systems is often highly challenging, especially when reactions leading to byproduct formation are obscured by a preceding, slow rate-determining step. In this work, we introduce a unique approach to kinetic analysis that enables the investigation of reactions occurring post rate-determining step. The technique was applied to the conversion of sugars to 5-hydroxymethylfurfural (HMF), an important platform molecule for sustainable chemical production. First, screening experiments were carried out to identify a catalytic solvent system that achieves a balance between solvent stability, HMF yield, and cost-effectiveness. Our kinetic analysis on the identified system reveals that the fructose reactant undergoes a rate-determining dehydration step to form an unstable reactive intermediate. This intermediate further reacts competitively with other reactive species, such as the hexose reactants or the HMF product, ultimately lowering the HMF selectivity. The calculated relative activation energies of these reactions post rate-determining step provide intuitive insights into the effects of temperature, co-existing reactive species, as well as the previously unexplained effect of feed concentration on HMF selectivity. Additionally, we propose a novel reaction strategy that significantly enhances HMF yield when compared to a traditional batch synthesis. Notably, a high HMF yield of 74 % was achieved with an extreme 55 wt% fructose loading on an aqueous basis (20 wt% loading on a total weight basis) in a low-boiling 80 wt% dioxane/water mixture using an HCl catalyst at 393 K.

Theoretical Studies on the Reaction Mechanism of Schiff Base Formation from Hexoses.

The Maillard reaction is one of the nonenzymatic post-translational modifications of proteins. Products of this reaction are considered to be related to aging diseases and the sensation of taste. In the initial stage of the Maillard reaction, Schiff base formation first occurs by the nucleophilic attack of amine nitrogen in proteins, and then, the reaction proceeds through the formation of 1,2-eminal and Amadori compounds. In this study, we computationally investigated the reaction pathway of Schiff base formation from hexoses. The optimized geometries of energy minima and transition states were calculated by using the density functional theory with the CAM-B3LYP/6-311+G(2d,2p) level of theory. The Schiff base formation progressed through three steps: two steps of carbinolamine formation and one step of dehydration. The dehydration is considered to be the rate-determining step in all hexoses because the activation barrier of the dehydration was higher than that of the carbinolamine formation. Furthermore, the steric configuration of the OH group at positions 2 and 3 affected the activation barrier.

Iodinated lutetium (III) phthalocyanine: Synthesis, photochemical and nonlinear optical properties

New octaiodo-substituted lutetium (III) phthalocyanine was prepared using a six-step synthetic procedure starting from commercially available o-xylene. The amide synthesis and dehydration step were optimized. The influence of the nature of peripheral halogen atoms on optical and photochemical properties was studied. For the complex under study, the possibility of participation in photoreactions of types I and II with the formation of two different forms of reactive oxygen species was investigated: superoxide anion radical and singlet oxygen respectively. Competition between two photoreactions was demonstrated. The Z-scan method revealed a pronounced nonlinear optical response of the iodinated complex, caused by the action of heavy atoms of peripheral iodine groups.

Multistep kinetics in self-induced sol–gel process of amorphous anhydride formation during thermal dehydration of potassium tetraborate tetrahydrate

AbstractThe physico-geometrical mechanism and kinetics of the multistep thermal dehydration of potassium tetraborate tetrahydrate was investigated as a model reaction to produce amorphous anhydrate via the self-induced sol–gel process. The thermal dehydration is composed of three consecutive dehydration steps: (1) a surface reaction in the solid-state accompanied by crack formation; (2) a rapid mass loss process accompanied by liquefaction to form the aggregate of the gel powders; and (3) the dehydration of gel powder aggregate to form a glassy anhydride. The changes in the contribution of the individual dehydration steps to the overall process according to the sample particle size and the heating rate (β) were identified as specific features of the multistep dehydration, which was characterized quantitatively using mathematical deconvolution analysis with log-normal four-parameter functions. The difficulty in determining the apparent activation energy (Ea) of the individual dehydration steps using isoconversional analysis due to the changes in the contribution depending on β values was addressed using modulated temperature thermogravimetry. Using the contributions and the apparent Ea of the individual dehydration steps as initial values, the kinetic description of the multistep thermal dehydration was refined through a kinetic deconvolution analysis using the cumulative kinetic equation. As a result, the individual dehydration steps were kinetically characterized as: (1) a surface reaction described by the first-order rate law with Ea,1 of approximately 68 kJ mol−1; (2) a reaction accompanied by liquefaction controlled by an autocatalytic rate behavior with Ea,2 of approximately 123 kJ mol−1; (3) a diffusion-controlled dehydration of gel powder with Ea,3 of approximately 82 kJ mol−1.

Process intensification for production of dispersants via integration of reaction and separation in a horizontal thin film evaporator

Process intensification can be achieved in specialty chemicals manufacturing via transition from batch-to-continuous processing. However, the absence of headspace in a continuous tubular reactor limits the ability to remove volatile byproducts from the reacting liquid flow. Previous studies showed that the production of succinimide dispersants follows a two-step amidation-dehydration pathway in which water is formed as a by-product. The inability to remove this water imposed a thermodynamic equilibrium limitation on the dehydration step, and hence necessitated a downstream drying step to obtain a dehydrated dispersant product within commercial specifications. In the present study, the use of an agitated, horizontal thin film evaporator (TFE) was investigated as a continuously operated reactive separator which combines reaction and dehydration into a single operating unit, reducing the overall cost and physical footprint of the process. The continuous TFE unit was first experimentally investigated as a stand-alone, integrated reactor-separator. Using this configuration, the obtained imide yield consistently exceeded the maximum thermodynamic yield at each operating temperature, confirming the efficiency of the integrated reaction and separation steps in the TFE. Next, the unit was modeled by integrating reaction kinetics with mass and energy balance equations, using appropriate correlations for heat and mass transfer from the literature which were verified for the experimental set-up. This model provided insights into the system behavior across different operating conditions, including the role of forward and reverse reactions along the TFE unit length. The model was then used to confirm the feasibility of replacing the two-step tubular reactor-evaporator process with a single TFE unit as an intensified reactive separator, further reducing the physical footprint of the process by enabling compact modular process designs.

Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step.

Embryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals. Oocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze-thawing. The survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05). Our study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze-thawing.

Formation, Identification, and Occurrence of the Furan-Containing β-Carboline Flazin Derived from l-Tryptophan and Carbohydrates.

β-Carbolines (βCs) are bioactive indole alkaloids found in foods and in vivo. This work describes the identification, formation, and occurrence in foods of the βC with a furan moiety flazin (1-[5-(hydroxymethyl)furan-2-yl]-9H-pyrido[3,4-b]indole-3-carboxylic acid). Flazin was formed by the reaction of l-tryptophan with 3-deoxyglucosone but not with 5-hydroxymethylfurfural. Its formation was favored in acidic conditions and heating (70-110 °C). The proposed mechanism of formation occurs through the formation of intermediates 3,4-dihydro-β-carboline-3-carboxylic acid (imines), followed by the oxidation to C═O in the carbohydrate chain and aromatization to βC ring with subsequent dehydration steps and cyclization to afford the furan moiety. Flazin is generated in the reactions of tryptophan with carbohydrates. Its formation from fructose was higher than from glucose, whereas sucrose gave flazin under acidic conditions and heating owing to hydrolysis. Flazin was identified in foods by HPLC-MS, and its content was determined by HPLC-fluorescence. It occurred in numerous processed foods, such as tomato products, including crushed tomato puree, fried tomato, ketchup, tomato juices, and jams, but also in soy sauce, beer, balsamic vinegar, fruit juices, dried fruits, fried onions, and honey. Their concentrations ranged from not detected to 22.3 μg/mL, with the highest mean levels found in tomato concentrate (13.9 μg/g) and soy sauce (9.4 μg/mL). Flazin was formed during the heating process, as shown in fresh tomato juice and crushed tomatoes. These results indicate that flazin is widely present in foods and is daily uptaken in the diet.

Elucidating the Dehydration Pathways of K2CO3·1.5H2O.

Potassium carbonate sesquihydrate has previously been identified as a promising material for thermochemical energy storage. The hydration and cyclic behavior have been extensively studied in the literature, but detailed investigation into the different processes occurring during dehydration is lacking. In this work, a systematic investigation into the different dehydration steps is conducted. It is found that at higher temperatures, dehydration of pristine material occurs as a single process since water removal from the pristine crystals is difficult. After a single cycle, due to morphological changes, dehydration now occurs as two processes, starting at lower temperatures. The morphological changes open new pathways for water removal at the newly generated edges, corners, and steps of the crystal surface. The observations from this work may contribute to material design as they elucidate the relation between material structure and behavior.

Catalysis of Indium Ion Electroreduction in the Presence of Acetazolamide in Chlorates(VII) Solutions with Varied Water Activity.

The influence of acetazolamide (ACT) on the kinetics and the mechanism of electroreduction of In(III) ions as a function of changes of the water activity was investigated using electrochemical methods (DC, SWV, CV and EIS, CV). The multi-step mechanism of the electroreduction process should take into account the dehydration step of indium ions and the presence of In-ACT (,,cap-pair" effect) active complexes, mediating electron transfer, located in the adsorption layer. Differences in the electrode mechanism in the presence of ACT were observed for higher chlorates(VII) concentrations (above 4 mol ⋅ dm-3 chlorates(VII)) reflected by a lack of step wise nature of the electrode process. The highest catalytic activity was observed in 4 mol ⋅ dm-3 chlorates(VII).

Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.

Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.

Water vapor effect on the physico-geometrical reaction pathway and kinetics of the multistep thermal dehydration of calcium chloride dihydrate.

This study investigated how water vapor influences the reaction pathway and kinetics of the multistep thermal dehydration of inorganic hydrates, focusing on CaCl2·2H2O (CC-DH) transforming into its anhydride (CC-AH) via an intermediate of its monohydrate (CC-MH). In the presence of atmospheric water vapor, the thermal dehydration of CC-DH stoichiometrically proceeded through two distinct steps, resulting in the formation of CC-AH via CC-MH under isothermal conditions and linear nonisothermal conditions at a lower heating rate (β). Irrespective of atmospheric water vapor pressure (p(H2O)), these reaction steps were kinetically characterized by a physico-geometrical consecutive process involving the surface reaction and phase boundary-controlled reaction, which was accompanied by three-dimensional shrinkage of the reaction interface. In addition, a significant induction period was observed for the second reaction step, that is, the thermal dehydration of CC-MH intermediate to form CC-AH. With increasing p(H2O), a systematic increase in the apparent Arrhenius parameters was observed for the first reaction step, that is, the thermal dehydration of CC-DH to form CC-MH, whereas the second reaction step exhibited unsystematic variations of the Arrhenius parameters. At a larger β in the presence of atmospheric water vapor, the first and second reaction steps partially overlapped; moreover, an alternative reaction step of the thermal dehydration of CC-MH to form CaCl2·0.3H2O was observed between these reaction steps. The physico-geometrical phenomena influencing the reaction pathway and kinetics of the multistep thermal dehydration were elucidated by considering the effects of atmospheric and self-generated water vapor in a geometrically constrained reaction scheme.

Simple Rescue of Opaque Tissue Previously Cleared by iDISCO.

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time. Key features • This simple protocol rescues previously cleared tissue that has turned opaque. • The method does not cause detectable loss of immunofluorescence from previously stained samples. Graphical overview.