Chemical Digestion Research Articles

Preparation of a Orexin Precursor Protein by Chemical Digestion

Endogenous peptide hormones are often produced in vivo in the form of precursor proteins that contain pre- and pro-leader sequences, which are subsequently processed into the biologically active mature peptide. Therefore, it is important to study the folding of precursor proteins to understand the mechanisms of post-translational modifications and the formation of the active conformation of the mature peptide. However, it is generally difficult to obtain precursor proteins from intact organs because only small amounts are produced in vivo. To overcome this issue, an E. coli expression system is employed to prepare recombinant proteins.Pro-orexin consists of orexin A, B, and a C-terminal peptide. To investigate the mechanism responsible for the processing and post-translational modifications of orexin peptides, we tried to express the recombinant pro-orexin in E. coli cells. However, recombinant pro-orexin was not expressed in E. coli cells. In addition, it was also difficult to obtain pro-orexin, even though Trx-fused pro-orexin was readily produced in E. coli cells because the digestion of Trx-fused pro-orexin with an enzyme yielded significant amounts of non-specific digests. Therefore, to solve this issue, we employed a chemical method to release pro-orexin from fusion proteins.K. Pane et al. recently reported a chemical method to obtain a target protein from an onconase-fused (ONC-fused) protein. They applied a chemical cleavage of the fusion protein at the Asp-Pro sequence with acetic acid. We employed this chemical method to produce recombinant pro-orexin. ONC-fused pro-orexin was successfully over-expressed as an inclusion body in E. coli BL21(DE3) cells and treated with acids under several sets of conditions to cleave the Asp-Pro sequence of ONC-fused pro-orexin. The results will be discussed in this paper.

Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen.

Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and neurosurgery for glioblastoma treatment. This protocol describes the details for preparing high purity primary astrocytes from human GBM. Tumor tissue is disrupted using mechanical dissociation and chemical digestion in this protocol. 2 weeks after plating the cell suspension in culture, primary astrocytes are available for further subculturing and immunocytochemistry of S100-beta antigen.

On-line prediction of sodium content in vacuum packed dry-cured ham slices by non-invasive near infrared spectroscopy

In the present study, non-invasive near infrared spectroscopy (NIRS) was evaluated as a potential on-line analytical technique to predict the sodium content in dry-cured ham slices. Samples of 310 packages were scanned by applying a remote fibre-optic probe to the surface of the slices, at different temperatures, with no previous manipulation. The sodium content of the meat samples was determined by a reference method based on Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES) after chemical digestion. Partial least squares (PLS) regression was used as a chemometrics method to perform the calibrations. The models yielded acceptable results with cross validation correlation coefficients (R2CV) determined 86.2–90.2%. The prediction capacity reached in the external validation was 3.63, with a standard prediction error of 0.12% Na. These results show that NIR measurements could be implemented on the packaging line of dry-cured ham slices to provide accurate and relevant information about the sodium content of each packaged products.

Leaching of electrodic powders from lithium ion batteries: Optimization of operating conditions and effect of physical pretreatment for waste fraction retrieval

Experimental results of leaching tests using waste fractions obtained by mechanical pretreatment of lithium ion batteries (LIB) were reported. Two physical pretreatments were performed at pilot scale in order to recover electrodic powders: the first including crushing, milling, and sieving and the second granulation, and sieving. Recovery yield of electrodic powder was significantly influenced by the type of pretreatment. About 50% of initial LIB wastes was recovered by the first treatment (as electrodic powder with size <0.5mm, Sample 1), while only 37% of powder with size <1mm (Sample 2) can be recovered by the second treatment. Chemical digestion put in evidence the heterogeneity of recovered powders denoting different amounts of Co, Mn, and Ni. Leaching tests of both powders were performed in order to determine optimized conditions for metal extraction. Solid/liquid ratios and sulfuric acid concentrations were changed according to factorial designs at constant temperature (80°C). Optimized conditions for quantitative extraction (>99%) of Co and Li from Sample 1 are 1/10g/mL as solid/liquid ratio and +50% stoichiometric excess of acid (1.1M). Using the same solid/liquid ratio, +100% acid excess (1.2M) is necessary to extract 96% of Co and 86% of Li from Sample 2. Best conditions for leaching of Sample 2 using glucose are +200% acid excess (1.7M) and 0.05M glucose concentration. Optimized conditions found in this work are among the most effective reported in the literature in term of Co extraction and reagent consumption.

Comparative proteomic assessment of matrisome enrichment methodologies.

The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome.

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Extraction of microplastic from biota: recommended acidic digestion destroys common plastic polymers

The chemical digestion of tissue from marine biota for microplastic analysis is currently conducted following a variety of protocols published in scientific literature. Often there is a lack of information on whether and to which degree the applied chemicals are destructive to microplastic particles of various polymer types. In the present study we report that a digestion protocol recently recommended by ICES using nitric and perchloric acid has strong detrimental effects on several common plastic polymers, in particular polyamide and polyurethane and to a lesser degree acrylonitrile butadiene styrene, polymethyl methacrylate and polyvinylchloride. Raman spectroscopic measurements revealed changes in peak occurrence and intensity for several polymers that did not otherwise show visual macroscopic changes. We developed and tested an alkaline digestion protocol in order to preserve small microplastic particles while removing organic tissue material. We recommend this method for the development of guidelines for plastic microplastic monitoring in biota.

Speciation and potential long-term behaviour of chromium in urban sediment particulates

PurposeChromium, a potentially harmful element, occurs commonly within the urban sediment cascade as a result of abundant industrial and transport-related sources. The risks that Cr-bearing particles pose to ecosystems and humans depend on the solid-phase chemical speciation of Cr and its environmental mobility. In this study, we adopt an integrated geochemical approach to investigate and determine the long-term fate of Cr in the urban sediment cascade.Materials and methodsWe use bulk chemical digests, sequential chemical extraction analysis, electron microscopy, electron microprobe and microfocus XANES analysis to describe the solid-phase speciation, geochemical characteristics and potential long-term behaviour of Cr in urban particulate matter from both aquatic sediment and road dust sediment (RDS) in Manchester, UK.Results and discussionCr-bearing grains within RDS and aquatic sediment are predominantly iron oxides and alumino-silicate glass grains. Electron microprobe analysis indicates Cr concentrations up to 3300 and 133,400 μg g−1 in the RDS and aquatic grains, respectively. XANES analysis indicates that Cr(III) is the dominant oxidation state, with only trace amounts of Cr(VI). Importantly, Cr speciation does not appear to have changed between sedimentary environments and the dominance of Cr(III) suggests limited bioavailability or toxicity under predominant environmental (anoxic and neutral pH) conditions in the aquatic sediment sink. Furthermore, geochemical analyses suggest the environmental mobility of Cr in the aquatic sediment sink is low (compared to other toxic metals) due to its association mainly with alumino-silicate glass grains and its inclusion as an integral part of the glass structure.ConclusionsIndustrial glass grains are a major component of urban sediment worldwide. The speciation and geochemical investigations performed in this study suggest most Cr within the urban sediment cascade may be resistant to environmental processes that could mobilise other toxic metals.

Direction-dependent mechanical characterization of cellulose-based composite vulcanized fiber

Abstract Vulcanized fiber is a macromolecular cellulose-based composite material manufactured using the parchmentizing process. The cellulose is produced from the chemical digestion of plant-based raw materials (wood, cotton) or textile waste. Chemical additives used during manufacturing are completely removed. After the process, vulcanized fiber possesses improved properties concerning mechanical strength and abrasion as well as corrosion resistance in comparison to its raw materials. Concerning its economic life cycle assessment, low density, electrical insulating capability and balanced properties, vulcanized fiber has a potential, up to now unused, as a light and renewable structural material for applications in automotive or civil engineering industries. Research activities concerning the mechanical properties are insufficient and existing standards are out-of-date. In this work, for the first time a direction-dependent characterization of the process-related anisotropic mechanical properties of the material is realized with the aim to formulate an adequate material model for numerical simulations in the next step.

A heterogeneity of the pheasant (Phasianus colchicus L.) erythrocyte histone H1 subtype H5

In a previous work (Górnicka-Michalska et al. (1998)), an occurrence of genetic variants in the chicken erythrocyte histone H5 has been presented. Here, the pheasant histone H5 heterogeneity is characterized to verify if the interspecies variability of this protein is caused by the analogous determinants. During screening histone H1 preparations isolated from the pheasant erythrocytes, histone H5 was identified as differently located in the electrophoretic gels. According to the rate of electrophoretic migration, two histone H5 phenotypes (H5a and H5b) possessing similar quantitative proportion (P>0.05) were distinguished. A rare phenotype H5a (frequency 0.26) migrating faster in the SDS-PAGE was low mobile in the AU-PAGE, in contrast to the frequent phenotype H5b (frequency 0.74) that moved slowly in the SDS-PAGE and roamed faster in the AU-PAGE. The electrophoretic properties of histone H5 phenotypes may reflect disparities in their net charge and molecular weight. Peptide maps of histone H5 phenotypes, obtained by partial chemical cleavage (NBS) and limited enzymatic digestion (α-chymotrypsin), revealed their C-peptides possessing the same electrophoretic mobility and the N-peptides having variable rate of the electrophoretic migration. Based on this, the identified phenotypic variation seems to be determined by a histone H5 phenotype-specific amino acid sequence region situated in the N-terminal portion of its molecule. According to the identified varied sequence stretches, histone H5 phenotype may induce specific effects related to the organization and/or function of the pheasant chromatin.

Anthropogenic microfibres pollution in marine biota. A new and simple methodology to minimize airborne contamination

Research studies on the effects of microlitter on marine biota have become more and more frequent the last few years. However, there is strong evidence that scientific results based on microlitter analyses can be biased by contamination from air transported fibres. This study demonstrates a low cost and easy to apply methodology to minimize the background contamination and thus to increase results validity. The contamination during the gastrointestinal content analysis of 400 fishes was tested for several sample processing steps of high risk airborne contamination (e.g. dissection, stereomicroscopic analysis, and chemical digestion treatment for microlitter extraction). It was demonstrated that, using our methodology based on hermetic enclosure devices, isolating the working areas during the various processing steps, airborne contamination reduced by 95.3%. The simplicity and low cost of this methodology provide the benefit that it could be applied not only to laboratory but also to field or on board work.

Trace element-mineral associations in modern and ancient iron terraces in acid drainage environments

Iron-rich sediments commonly cover riverbeds that have been affected by acid drainage associated with sulfide-mineral oxidation. Freshly-formed precipitates correspond to poorly-crystalline oxyhydroxysulfates that recrystallize over time. This study examined the distribution and mineral association of trace elements (e.g., As, Cu, Zn) in modern and ancient (~6Ma) Fe terraces in the Tinto river basin, Spain. The mineral composition of the terraces was determined by Raman μ-spectroscopy. Chemical digestions, electron probe microanalyses, and synchrotron-based μ-X-ray fluorescence mapping were used to examine As, Cu, and Zn distribution and corresponding mineral associations. Fresh precipitates at modern terrace surfaces were dominated by schwertmannite, which contained high As, Cu, Mn, and Zn concentrations. However, schwertmannite transforms into goethite over days to weeks in the deeper part of the current terraces and into hematite over centuries. Affinity for trace elements was generally highest for schwertmannite and lowest for hematite, which suggests that their retention by Fe terraces decreases during mineral transformation. Hence, schwertmannite acts as temporary sink for contaminants, which are again released over long time periods. These findings should be considered for management and treatment of possible water resources affected by acid mine drainage.

Cutting food in terrestrial carnivores and herbivores.

Insects and mammals cut their food up into small pieces to facilitate ingestion and chemical digestion. Teeth and jaws act as cutting tools, but, unlike engineering tools designed for a specific purpose, must generally cope with substantial variation in food properties and work at many scales. Knowing how teeth and jaws work effectively requires an understanding of the cutting on the edges and the mechanisms that remove cut material. Variability and heterogeneity of diet properties are not well known, and, for example, may be higher and overlap more in the browsing and grazing categories of plant diets. A reinterpretation of tooth function in large mammal browsers and grazers is proposed.

Chemical distribution of hazardous natural radionuclides during monazite mineral processing

It is very important to calculate the radioactivity concentration for low-grade monazite ore (50%) and different other materials produced as results of chemical processing stages to avoid the risk to workers. Chemical processing of low-grade monazite pass through different stages, washing by hydrochloric acid and digested with sulfuric acid and influence of pH on the precipitation of rare earth elements has been studied. The radioactivity concentrations of 238U(226Ra) and 232Th as well as 40K were calculated in crude low-grade ore and found to be 54,435 ± 3138, 442,105 ± 29,200 and 5841 ± 345 Bq/kg, respectively. These values are greatly higher than the exempt levels 25 Bq/kg. After chemical digestion of the ore, the results demonstrated that un-reacted material contains significant radioactivity reached to approximately 8, 13 and 23% for 238U, 232Th and 40K, respectively. The results show that 60% of 232Th are located in the digested white slurry with small portions of 238U and 40K. Most of 238U radioactivity is extracted in the green phosphoric acid which produced from conversion of P2O5 by H2SO4 into phosphoric acid. The average values of the Raeq for monazite ore, un-reacted black precipitate, white precipitate, brown precipitate and crystalline material samples were calculated and found to be 687,095 ± 44,921, 85,068 ± 5339, 388,381 ± 22,088, 313,046 ± 17,923 and 4531 ± 338 Bq/kg, respectively. The calculated values of Raeq are higher than the average world value (it must be less than 370 Bq/kg). Finally the external hazardous, internal hazardous and Iγr must be less than unity. This means that specific radiation protection program must be applied and implemented during monazite processing.

Thermal treatment of electronic waste in a fluidised bed and chemical digestion of solid products.

The article presents the results of e-waste thermal treatment in a fluidised bed reactor and solid products digestion under acidic conditions. During the processes, measurements of carbon monoxide, carbon dioxide, volatile organic compounds, nitrogen oxides, sulphur dioxide, hydrogen chloride, hydrogen bromide, hydrogen cyanide, ammonia, phenol, aliphatic and aromatic hydrocarbons, hydrogen fluoride and phosgene were carried out. Several digestion tests of the solid residue in sulphuric acid (VI) at 25 °C-65 °C, for 55 min-24 h were conducted. In each case, the dilution method was used, i.e. preliminary digestion in concentrated sulphuric acid (VI) (95%) for 40 min, and then dilution to expected concentrations (30%-50%). Most preferred results were obtained using sulphuric acid (VI) with a target concentration of 40% at 65 °C, where the leaching degrees were 76.56% for copper, 71.67% for iron, 91.89% for zinc and 97.40% for tin. The time necessary to effectively carry out the digestion process was 220 min.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

Microscopic anthropogenic litter in terrestrial birds from Shanghai, China: Not only plastics but also natural fibers

The level of contamination by microscopic anthropogenic litter (0.5–5mm) in terrestrial ecosystems is not well understood. After chemical digestion in 10% KOH, microscopic anthropogenic litter from the gastrointestinal tracts of 17 terrestrial birds was identified and categorized under a stereomicroscope based on its physical properties and melting tests. In total, 364 items from 16 birds were identified as microscopic anthropogenic litter, ranging in size from 0.5 to 8.5mm. No relationship between plastic load and body condition was found. Natural fibers, plastic fibers and fragmented plastics represented, respectively, 37.4% (136 items), 54.9% (200 items) and 7.7% (28 items) of total litter items. Small sample sizes limited our ability to draw strong conclusions about the metabolism of natural fibers, but the decline in the proportion of natural fibers from the esophagus to stomach to intestine suggested that they may be digestible. Particles smaller than 5mm represented more than 90% of the total number of pollutant items. Particles with colors in the mid-tones and fibrous shapes were overwhelmingly common particles. The results reflect pollution by microscopic anthropogenic litter in the terrestrial ecosystem of the study area. Microscopic natural fibers, which may disperse and adsorb chemical pollutants differently from microplastic and may pose an even greater risk, are in urgent need of further research.

Characterization of Glasses in One Type of Alumina Rich Fly Ash by Chemical Digestion Methods: Implications for Alumina Extraction

In recent years, one type of alumina rich fly ash (ARFA) with about 50 wt% of alumina has been extensively investigated for alumina extraction in China. Due to the silica in ARFA, alumina extraction would have to generate a huge amount of solid waste. There is a growing interest in the glasses in ARFA, because they are composed mainly of silica and could be removed prior to alumina extraction. In this work, the glasses in ARFA have been investigated by chemical methods, that is, acid and base digestions. The chemical compositions have been measured by XRF for ARFA from the digestion processes. The K2O standard, XRD, and FTIR spectroscopies were successfully used to define the digestions processes, and size analysis and SEM-EDX provided rich information on particle transformations. As a result, acid and base digestion methods were found to produce very similar results for the glasses in ARFA. The K2O standard was attributed to the formation of glasses by illites, and TiO2and Fe2O3were proposed to originate from ilmenite in alumina rich coals (ARC). Some implications of the results were also discussed for the alumina extraction from ARFA.

Accurate determination of low-level chemical oxygen demand using a multistep chemical oxidation digestion process for treating drinking water samples

A multistep wet-chemical oxidation digestion technique was developed for determination of low-level COD in drinking water by using a potentiometric titration method.

The chemical digestion of Ti6Al7Nb scaffolds produced by Selective Laser Melting reduces significantly ability of Pseudomonas aeruginosa to form biofilm.

In our previous work we reported the impact of hydrofluoric and nitric acid used for chemical polishing of Ti-6Al-7Nb scaffolds on decrease of the number of Staphylococcus aureus biofilm forming cells. Herein, we tested impact of the aforementioned substances on biofilm of Gram-negative microorganism, Pseudomonas aeruginosa, dangerous pathogen responsible for plethora of implant-related infections. The Ti-6Al-7Nb scaffolds were manufactured using Selective Laser Melting method. Scaffolds were subjected to chemical polishing using a mixture of nitric acid and fluoride or left intact (control group). Pseudomonal biofilm was allowed to form on scaffolds for 24 hours and was removed by mechanical vortex shaking. The number of pseudomonal cells was estimated by means of quantitative culture and Scanning Electron Microscopy. The presence of nitric acid and fluoride on scaffold surfaces was assessed by means of IR and rentgen spetorscopy. Quantitative data were analysed using the Mann-Whitney test (P ≤ 0.05). Our results indicate that application of chemical polishing correlates with significant drop of biofilm-forming pseudomonal cells on the manufactured Ti-6Al-7Nb scaffolds ( p = 0.0133, Mann-Whitney test) compared to the number of biofilm-forming cells on non-polished scaffolds. As X-ray photoelectron spectroscopy revealed the presence of fluoride and nitrogen on the surface of scaffold, we speculate that drop of biofilm forming cells may be caused by biofilm-supressing activity of these two elements.