Spin Probe Research Articles

EPR Viscometric Measurements Using a 13C-Labeled Triarylmethyl Radical in Protein-based Biotherapeutics and Human Synovial Fluids.

The viscosity measurements are of clinical significance for evaluation of the potential pathological conditions of biological lubricants such as synovial fluids of joints, and for formulation and characterization of peptide- and protein-based biotherapeutics. Due to inherent potential therapeutic activity, protein drugs have proven to be one of the most efficient therapeutic agents in treatment of several life-threatening disorders, such as diabetes and autoimmune diseases. However, home-use applications for treating chronic inflammatory diseases, such as diabetes and rheumatoid arthritis, necessitate the development of high-concentration insulin and monoclonal antibodies formulations for patient self-administration. High protein concentrations can affect viscosity of the corresponding drug solutions complicating their manufacture and administration. The measurements of the viscosity of new insulin analogs and monoclonal antibodies solutions under development is of practical importance to avoid unwanted highly viscous, and therefore, painful for injection drug formulations. Recently, we have demonstrated capability of the electron paramagnetic resonance (EPR) viscometry using viscosity-sensitive 13C-labeled trityl spin probe (13C1-dFT) to report the viscosity of human blood, and interstitial fluids measured in various organs in mice ex-vivo and in anesthetized mice, in vivo. In the present work, we demonstrate utility of the EPR viscometry using 13C1-dFT to measure microviscosity of commercial insulin samples, antibodies solution, and human synovial fluids using small microliter volume samples (5-50 μL). This viscometry analysis approach provides useful tool to control formulations and administration of new biopharmaceuticals, and for evaluation of the state of synovial fluids of importance for clinical applications.

Изменение физико-химических и органолептических характеристик яблочного пюре при повышенных температурах хранения

Food products change their quality during storage not only under the external impact, but also because they are complex in composition. As a result, food scientists look for new methods to control these internal changes. The research objective was to describe the changes in the physicochemical properties of apple puree during storage at elevated temperatures (40–60°C) and link them with the changes in the sensory profile.
 The study featured homogenized apple puree packaged in composite material and heated up to 40, 50, and 60°C. The colorimetric studies were conducted at 45°/0°, light source D65. The proton relaxation time and the water diffusion coefficient (impulse gradient method) were studied at a frequency of 20 MHz. The analysis of molecular dynamics involved the method of electron paramagnetic resonance of spin probes. The samples were cooled down to –70°C to measure the content of non-crystallizing water by the method of differential scanning calorimetry.
 The color change rate was constant and followed the zero-order kinetic reaction equation with an activation energy of 92 kJ/mol. The changes in color, proton relaxation, and sensory properties correlated when the samples were stored at 50 and 60°C. The analysis of the magnetic relaxation time, the diffusion coefficient of water, and the content of non-crystallizing water indicated that the main changes in the physical structure of the puree during heat treatment occurred as a result of the aggregation of apple cell fragments. These findings were confirmed by the optical microscopy.
 A prolonged exposure to 40–60°C affected the color, the relaxation of water protons, and the size of aggregates of apple cell fragments. It also affected the amount of water that did not crystallize at –70°C. The correspondence between the values of the activation energies was determined by the methods of colorimetry and proton relaxation. Therefore, the coloration and the water changes depended on the same processes. These physical and chemical properties can be used for quantitative assessment of apple puree under thermal treatment.

1H-detected characterization of carbon–carbon networks in highly flexible protonated biomolecules using MAS NMR

In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based 1H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either 13C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond 13C–13C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus Schizophyllum commune using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10mg/kg). At 24h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Disordering of Starch Films as a Factor Influencing the Release Rate of Biologically Active Substances.

The release of a spin probe (nitroxide radical) from polymer films was studied by electron paramagnetic resonance (EPR). The films were fabricated from starch having different crystal structures (A-, B-, and C-types) and disordering degrees. Film morphology (analysis of the scanning electron microscopy (SEM)) depended on the presence of dopant (nitroxide radical) to a larger extent rather than on crystal structure ordering or polymorphic modification. The presence of nitroxide radical led to additional crystal structure disordering and reduced the crystallinity index from the X-ray diffraction (XRD) data. Polymeric films made of amorphized starch powder were able to undergo recrystallization (crystal structure rearrangement), which manifested itself as an increase in crystallinity index and phase transition of the A- and C-type crystal structures to the B-type one. It was demonstrated that nitroxide radical does not form an individual phase during film preparation. According to the EPR data, local permittivity of starch-based films varied from 52.5 to 60.1 F/m, while bulk permittivity did not exceed 17 F/m, which demonstrates that local concentration of water is increased in the regions near the nitroxide radical. The mobility of the spin probe corresponds to small stochastic librations and is indicative of the strongly a mobilized state. The application of kinetic models made it possible to find out that substance release from biodegradable films consists of two stages: matrix swelling and spin probe diffusion through the matrix. Investigation of the release kinetics for nitroxide radical demonstrated that the course of this process depends on the type of crystal structure of native starch.

A novel method for the sterically shielded pyrrolidine nitroxides synthesis using donor–acceptor cyclopropanes

Sterically shielded nitroxides were shown to demonstrate resistance to bioreduction. For this reason, they are attracting much interest as spin labels and probes for in-cell and in vivo EPR and NMR applications. Formal [3 + 2] cycloaddition reaction of donor–acceptor cyclopropanes with propionitrile provided 1-pyrrolines which were converted to the new sterically shielded achiral pyrrolidine nitroxides via several steps.

Detecting nuclear spins in an organosilane monolayer using nitrogen-vacancy centers for analysis of precursor self-assembly on diamond surface

We demonstrated the correlation spectroscopy of organosilane monolayers using an ensemble of shallow nitrogen-vacancy centers as the quantum sensor. Several types of organosilane monolayers were grown directly on the diamond surface by exposing the surface to a silane precursor vapor. The feasibility of detecting 1H and 19F in the monolayer was examined by correlation spectroscopy measurements. The effect of the magnetic dipole–dipole interaction on the peak width was also discussed by comparing the spectrum of the monolayer with that of surface-attached 1H and that of immersion oil. The results highlight the feasibility of nitrogen-vacancy centers as the spin probe for physicochemical analyses of monolayers grown on the diamond surface.

Direct Measurement and Imaging of Redox Status with Electron Paramagnetic Resonance.

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo. Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo, further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

13C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, N-Linked Glycosylated Lipoxygenase.

Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX-substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae, MoLOX complexed with LA, as obtained through the 13C/1H ENDOR-guided MD approach. The catalytically important distance between the hydrogen donor, carbon-11 (C11), and the acceptor, Mn-bound oxygen, (donor-acceptor distance, DAD) for the MoLOX-LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the MoLOX-LA DAD from that of the SLO-LA complex, 3.1 ± 0.1 Å, is functionally important, although is only 0.3 Å, despite the MoLOX complex having a Mn-C11 distance of 5.4 Å and a "carboxylate-out" substrate-binding orientation, whereas the SLO complex has a 4.9 Å Mn-C11 distance and a "carboxylate-in" substrate orientation. The results provide structural insights into reactivity differences across the LOX family, give a foundation for guiding development of MoLOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX-substrate structures.

A comparison of pulse and CW EPR [formula omitted]-relaxation measurements of an inhomogeneously broadened nitroxide spin probe undergoing Heisenberg spin exchange

Nitroxide spin probes are inhomogeneously broadened (IHB) by intramolecular hyperfine interactions with protons (deuterons) producing lines of Voigt shape. Thus, to study T2 relaxation by continuous wave (CW) EPR, the Voigt must be deconvoluted to find the Lorentzian component. For homogeneously broadened lines, T2 is obtained directly from the Lorentzian line widths ΔHppL; however, for IHB lines finding T2 from ΔHppL is more complicated. It has been known for many years that values of ΔHppL of high precision may be obtained from IHB lines; however, direct, accurate comparison of spin exchange frequencies obtained from electron spin echo decay and CW EPR data has been lacking. It is demonstrated here that despite complications in the interpretation of experiments, these two techniques yield the same spin exchange rate constant for spin probes that are the most difficult to treat.

Radical ionic liquid: An efficient self-probe to study heterogeneous structure in glassy state using EPR spectroscopy

Ionic liquids (ILs) exhibit a number of unique properties, including formation of various heterogeneities in their liquid and glassy states. Electron Paramagnetic Resonance (EPR) spectroscopy has proven informative in studies of such heterogeneous nanostructures; however it employs additional small paramagnetic molecules – spin probes – dissolved in ILs as reporters. In this work we synthesized two radical-ILs, which include integrated paramagnetic fragments in their structures, and employed them in continuous wave (CW) and pulse EPR studies of structural peculiarities in IL glasses. In this approach no external-to-IL spin probe is required. We have shown that the trends observed with radical-ILs are similar to those obtained previously using external-to-IL spin probes. In particular, unusual structural anomalies near glass transition temperatures, which were previously found using external-to-IL spin probes, have been now observed using radical-ILs, thus confirming their genuine origin. In general, radical-ILs can be considered as efficient tools to study heterogeneous structure of ILs using EPR.

Impact of Vanadium Complexes with Tetradentate Schiff Base Ligands on the DPPC and EYL Liposome Membranes: EPR Studies

This paper investigates the effect of three vanadium complexes with Schiff base-type tetradentate ligands of general formula N,N′-1,2-cyclohexylenebis(3,5-dichlorosalicylideneiminate) (V1); LVCl2 (L = N,N′-1,2-cyclohexylenebis(5-chlorosalicylideneiminate) (V2); and N,N′-1,3propylenebis (salicylideneiminate) (V3) on the fluidity of liposome membranes obtained by the sonication of natural lecithin (EYL) and synthetic lecithin (DPPC). The study was carried out with TEMPO and 16DOXYL spin probes using the EPR technique. The results show that the effect of the complexes on the fluidity of liposomes whose membranes are in the liquid crystalline phase is much stronger as compared to the liposome membranes in the gel phase.

COMPOSITE MATERIALS BASED ON MOF ZIF-8 AND IONIC LIQUID [BMIm]<sup>+</sup>[BF<sub>4</sub>]<sup>−</sup>: AN EPR STUDY USING NITROXIDE SPIN PROBES

A method for the controlled filling of pores in metal-organic frameworks (MOFs) with ionic liquids (ILs) is proposed, which can be applied for selective gas sorption in MOFs. Using an example of MOF ZIF-8 and IL [BMIm]+[BF4]−, the composites with different IL content were prepared upon control by electron paramagnetic resonance (EPR) of nitroxide spin probes. The effect of IL on the sorption of nitric oxide (II) into these composites was studied using inversion gas chromatography.

Muon studies of the proton conducting polymer Nafion

The fluorinated ionomer Nafion, first discovered by the Du Pont company, is a material that provides efficient proton conducting membranes for application in important technological areas such as hydrogen fuel cells. Although many aspects of the polymer have been studied in relation to these applications, the microscopic mechanisms for proton transport in this polymer are still only poorly understood. We have therefore applied implanted muon techniques to the study of Nafion, aiming to gain information about these mechanisms via the muon acting as a local spin probe. Our results indicate that the muon is highly sensitive to the hydration state of the polymer and to the dynamical processes of the various sub-phases within the material. A three component model is found to describe the data well. This model has one F-µ-F component, reflecting muons in the PTFE-like polymer matrix, a second component representing quasi-static environments dominated by a single close proton and a third component encompassing highly dynamic proton-coupled environments. The properties have been studied within this modelling framework as a function of temperature for both dehydrated and fully hydrated samples.

Enhanced Spin-Nuclei Couplings of Paramagnetic Azametallofullerene Hooped in Cycloparaphenylene

Azametallofullerene Y2@C79N has outstanding electron spin characteristics with potential application in the fields of magnetoreception system and precision measurement as a molecular spin probe. Herein, we report the spin modulation of Y2@C79N by means of host–guest interaction with a carbon nanoring of [12]Cycloparaphenylene ([12]CPP). By means of temperature-dependent electron paramagnetic resonance (EPR) measurements, the adjustable EPR signals of Y2@C79N⊂[12]CPP were observed, revealing the great effect of host–guest interaction on the spin and paramagnetic property of hooped Y2@C79N. The EPR signals of Y2@C79N⊂[12]CPP and pristine Y2@C79N were comparatively investigated in carbon disulfide and in solid state, and it was found that the Y2@C79N⊂[12]CPP complex exhibits enhanced spin-nuclei couplings compared to those of pristine Y2@C79N. We propose that the [12]CPP host can induce the orientation of the Y2@C79N guest, and consequently change the spin-nuclei couplings of Y2@C79N. This study would promote the creation of new molecular spin materials.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Efficacious Reduction of Ferric Ions by Molybdenum Carbide in the Peroxydisulfate Fenton-Like Reaction for Dexamethasone Degradation

The tardy Fe2+ regeneration and the limited pH range confine the practical application of Fe-based homogenous Fenton-like reaction performance. To surmount the abovementioned obstacles, we applied molybdenum carbide (β-Mo2C) as the cocatalyst to boost Fe3+ reduction in the activation of peroxydisulfate (PDS) for the efficient degradation of dexamethasone (DXM). The proposed Fe3+/PDS/β-Mo2C can remove more than 90% DXM in the wide pH range (3.4 to 9.4). Electron spin resonance analysis and probe and quenching tests comprehensively demonstrated that a series of reactive species (•OH, SO4•–, O2•–, 1O2, and Fe(IV)) were formed during the synergistic process, and SO4•– was the leading role. The circulation of Fe3+/Fe2+ was driven by the exposed sites of Mo2+/Mo4+ on the β-Mo2C, which further promoted the PDS activation. The reaction mechanism was clarified by various characterizations and density functional theory calculation, which indicated that the adsorption between Fe3+ and β-Mo2C could form Mo–Fe bonds and lengthen Fe–O and then improve the electron transport from β-Mo2C to Fe3+ and boost Fe3+ reduction. The reactivity and stability of β-Mo2C was higher than those of popular Mo-based materials. This work represents an attempt and possibility for Mo-based inorganic cocatalysis of advanced oxidation processes.

Solanum dulcamara L. Berries: A Convenient Model System to Study Redox Processes in Relation to Fruit Ripening.

The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. Here, we present the EPRI methodology to assess the redox status of fruits and to discriminate between the redox states of different tissues. Interestingly, the intracellular reoxidation of cell-permeable nitroxide probe 3CP was observed for the first time in fruits or any other plant tissue, and its intensity is herein proposed as a reliable indicator of oxidative stress during ripening. The described noninvasive EPRI technique has the potential to have broader application in the study of redox processes associated with the development, senescence, and postharvest storage of fruits, as well as other circumstances in which oxidative stress is implicated.

Carbonyl and defect of metal-free char trigger electron transfer and O2•− in persulfate activation for Aniline aerofloat degradation

Residual flotation reagents in mineral processing wastewater can trigger severe ecological threats to the local groundwater if they are discharged without treatment. Metal-free biochar-induced persulfate-advanced oxidation processes (KCBC/PS) were used in this study to elucidate the degradation of aniline aerofloat (AAF) – a typical flotation reagent. In KCBC/PS system, AAF can be removed at low doses of catalyst (KCBC, 0.05 g/L) and oxidant (PS, 0.3 mM) additions with high efficiency. The analysis revealed the dominance of O2•− among the identified reactive oxygen species (ROS), which achieved deeper mineralization for the AAF degradation in the KCBC/PS system. The role of the electron transfer mechanism was equally important; the importance was corroborated by the chemical quenching experiments, electron spin resonance (ESR) detection, probe experiments, and electrochemical analysis. It benefited from the electron transfer mechanism in the KCBC/PS system and exhibited a wide pH adaptation (3.5–11) and high resistance to inorganic anions for real mining wastewater treatment. Combined with theoretical calculations and other analyses, the carbonyl group was deemed to be the active site of the non-radical pathway of biochar, while the site of the conversion of SO4•− to superoxide radicals by biochar activation represented a defect. These findings revealed a synergistic effect of multiple active sites on PS activation in biochar-based materials. Moreover, the intermediate degradation products of AAF from mass spectrometry indicated a possible pathway through the density functional theory (DFT) method, which was effective in reducing the environmental toxicity of pollutants for the first time according to the T.E.S.T software and seed germination experiments. Overall, our study proposed a novel modification strategy for cost-effective and environmentally friendly biochar-based catalysts, while also deepening our understanding of the mechanism of activation of persulfate by metal-free carbon-based materials.

The possibility of diagnosing the prescription of death in the study of embalmed corpses by the method of spin probes. (Experimental study)

Is to establish the possibility of studying the vitreous body of the eye from embalmed corpses using the spin probe method to develop diagnostic criteria for the prescription of death coming. The article presents data showing the possibility of diagnosing the prescription of death coming by the method of spin probes with the examination of the vitreous body of the eye from embalmed corpses. A total of 510 spectra obtained during incubation of the 1-oxyl-4-oxo-2.2.6.6-tetramethyl-piperidin spin probe in the vitreous body were studied. Measurements of each of the 6 objects were carried out daily for 10 days after embalming. The prospects of studying the vitreous body by spin probes in embalmed corpses for the development of diagnostic criteria for the prescription of death coming up to 4 days from the moment of embalming have been established.