Enzymatic Digestion of Proteins and HPLC Peptide Isolation

Natural methylxanthines, caffeine, theophylline and theobromine, are widespread biologically active alkaloids in human nutrition, found mainly in beverages (coffee, tea, cocoa, energy drinks, etc.). Their detection is thus of extreme importance, and many studies are devoted to this topic. During the last decade, graphene oxide (GO) and reduced graphene oxide (RGO) gained popularity as constituents of sensors (chemical, electrochemical and biosensors) for methylxanthines. The main advantages of GO and RGO with respect to graphene are the easiness and cheapness of synthesis, the notable higher solubility in polar solvents (water, among others), and the higher reactivity towards these targets (mainly due to – interactions); one of the main disadvantages is the lower electrical conductivity, especially when using them in electrochemical sensors. Nonetheless, their use in sensors is becoming more and more common, with the obtainment of very good results in terms of selectivity and sensitivity (up to 5.4 × 10−10 mol L−1 and 1.8 × 10−9 mol L−1 for caffeine and theophylline, respectively). Moreover, the ability of GO to protect DNA and RNA from enzymatic digestion renders it one of the best candidates for biosensors based on these nucleic acids. This is an up-to-date review of the use of GO and RGO in sensors.

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Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae

Scientific Reports ◽

10.1038/s41598-021-83505-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aiyan Guan ◽

Inge Van Damme ◽

Frank Devlieghere ◽

Sarah Gabriël

Keyword(s):

Enzymatic Digestion ◽

Infected Fish ◽

Effect Of Temperature ◽

Video Recordings ◽

Different Temperatures ◽

Semi Solid ◽

Zoonotic Parasites ◽

The Impact ◽

Phosphate Buffered Saline

AbstractAnisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

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Can keratin scaffolds be used for creating three-dimensional cell cultures?

Open Medicine ◽

10.1515/med-2020-0031 ◽

2020 ◽

Vol 15 (1) ◽

pp. 249-253

Author(s):

Marta Bochynska-Czyz ◽

Patrycja Redkiewicz ◽

Hanna Kozlowska ◽

Joanna Matalinska ◽

Marek Konop ◽

...

Keyword(s):

Cell Cultures ◽

Chemical Activation ◽

Three Dimensional ◽

Enzymatic Digestion ◽

Pepsin Digestion ◽

Cell Culturing ◽

3D Cell Cultures ◽

Associated Proteins ◽

Positive Effect ◽

Dimensional Cell

AbstractThree-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing.

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The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02373-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lydia Bensemmane ◽

Claire Squiban ◽

Christelle Demarquay ◽

Noëlle Mathieu ◽

Marc Benderitter ◽

...

Keyword(s):

Paneth Cell ◽

Stromal Vascular Fraction ◽

Enzymatic Digestion ◽

Cell Compartment ◽

Absorption Function ◽

Radiation Induced ◽

Anti Inflammatory ◽

Abdominal Irradiation ◽

Pro Inflammatory Cytokines ◽

Inflammatory Effect

Abstract Background The intestine is particularly sensitive to moderate-high radiation dose and the development of gastrointestinal syndrome (GIS) leads to the rapid loss of intestinal mucosal integrity, resulting in bacterial infiltration, sepsis that comprise patient survival. There is an urgent need for effective and rapid therapeutic countermeasures. The stromal vascular fraction (SVF) derived from adipose tissue is an easily accessible source of cells with angiogenic, anti-inflammatory and regenerative properties. We studied the therapeutic impact of SVF and its action on the intestinal stem cell compartment. Methods Mice exposed to the abdominal radiation (18 Gy) received a single intravenous injection of stromal vascular fraction (SVF) (2.5 × 106 cells), obtained by enzymatic digestion of inguinal fat tissue, on the day of irradiation. Mortality was evaluated as well as intestinal regeneration by histological analyses and absorption function. Results The SVF treatment limited the weight loss of the mice and inhibited the intestinal permeability and mortality after abdominal irradiation. Histological analyses showed that SVF treatment stimulated the regeneration of the epithelium by promoting numerous enlarged hyperproliferative zones. SVF restored CD24+/lysozyme− and Paneth cell populations in the ISC compartment with the presence of Paneth Ki67+ cells. SVF has an anti-inflammatory effect by repressing pro-inflammatory cytokines, increasing M2 macrophages in the ileum and anti-inflammatory monocyte subtypes CD11b+Ly6clowCX3CR1high in the spleen. Conclusions Through the pleiotropic effects that contribute to limiting radiation-induced lethality, SVF opens up attractive prospects for the treatment of emergency GIS.

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Recent Advances in Chiral Analysis of Proteins and Peptides

Separations ◽

10.3390/separations8080112 ◽

2021 ◽

Vol 8 (8) ◽

pp. 112

Author(s):

Marine Morvan ◽

Ivan Mikšík

Keyword(s):

Amino Acids ◽

Stationary Phases ◽

Enzymatic Digestion ◽

Chiral Separations ◽

Biological Compounds ◽

Health And Disease ◽

Electrophoretic Techniques ◽

A Site ◽

Future Work ◽

Proteins And Peptides

Like many biological compounds, proteins are found primarily in their homochiral form. However, homochirality is not guaranteed throughout life. Determining their chiral proteinogenic sequence is a complex analytical challenge. This is because certain D-amino acids contained in proteins play a role in human health and disease. This is the case, for example, with D-Asp in elastin, β-amyloid and α-crystallin which, respectively, have an action on arteriosclerosis, Alzheimer's disease and cataracts. Sequence-dependent and sequence-independent are the two strategies for detecting the presence and position of D-amino acids in proteins. These methods rely on enzymatic digestion by a site-specific enzyme and acid hydrolysis in a deuterium or tritium environment to limit the natural racemization of amino acids. In this review, chromatographic and electrophoretic techniques, such as LC, SFC, GC and CE, will be recently developed (2018–2020) for the enantioseparation of amino acids and peptides. For future work, the discovery and development of new chiral stationary phases and derivatization reagents could increase the resolution of chiral separations.

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Association of Mast Cell Burden and TIM-3 Expression with Recalcitrant Chronic Rhinosinusitis with Nasal Polyps

Annals of Otology Rhinology & Laryngology ◽

10.1177/0003489421995038 ◽

2021 ◽

pp. 000348942199503

Author(s):

Michael A. Belsky ◽

Erica Corredera ◽

Hridesh Banerjee ◽

John Moore ◽

Li Wang ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Chronic Rhinosinusitis ◽

Nasal Polyps ◽

Cell Activation ◽

Enzymatic Digestion ◽

Sinus Surgery ◽

Mast Cell Activation ◽

Need For Treatment ◽

Inflammatory State

Objectives: Previous work showed that higher polyp mast cell load correlated with worse postoperative endoscopic appearance in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Polyp epithelial mast cells showed increased expression of T-cell/transmembrane immunoglobulin and mucin domain protein 3 (TIM-3), a receptor that promotes mast cell activation and cytokine production. In this study, CRSwNP patients were followed post-operatively to investigate whether mast cell burden or TIM-3 expression among mast cells can predict recalcitrant disease. Methods: Nasal polyp specimens were obtained via functional endoscopic sinus surgery (FESS) and separated into epithelial and stromal layers via enzymatic digestion. Mast cells and TIM-3-expressing mast cells were identified via flow cytometry. Mann-Whitney U tests and Cox proportional hazard models assessed whether mast cell burden and TIM-3 expression were associated with clinical outcomes, including earlier recurrence of polypoid edema and need for treatment with steroids. Results: Twenty-three patients with CRSwNP were studied and followed for 6 months after undergoing FESS. Higher mast cell levels were associated with earlier recurrence of polypoid edema: epithelial HR = 1.283 ( P = .02), stromal HR = 1.103 ( P = .02). Percent of mast cells expressing TIM-3 in epithelial or stromal layers was not significantly associated with earlier recurrence of polypoid edema. Mast cell burden and TIM-3+ expression were not significantly associated with need for future treatment with steroids post-FESS. Conclusions: Mast cell load in polyp epithelium and stroma may predict a more refractory postoperative course for CRSwNP patients. The role of TIM-3 in the chronic inflammatory state seen in CRSwNP remains unclear.

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In vitro assessment of food-derived-glucose bioaccessibility and bioavailability in bicameral cell culture system

Turkish Journal of Biochemistry ◽

10.1515/tjb-2020-0082 ◽

2020 ◽

Vol 45 (5) ◽

pp. 631-637

Author(s):

Cansu Ozel-Tasci ◽

Gozde Pilatin ◽

Ozgur Edeer ◽

Sukru Gulec

Keyword(s):

Cell Culture ◽

Culture System ◽

Metabolic Diseases ◽

Enzymatic Digestion ◽

Cell Culture System ◽

Culture Studies ◽

Experimental Approaches ◽

In Vitro Assessment

AbstractBackgroundFunctional foods can help prevent metabolic diseases, and it is essential to evaluate functional characteristics of foods through in vitro and in vivo experimental approaches.ObjectiveWe aimed to use the bicameral cell culture system combined with the in vitro digestion to evaluate glucose bioavailability.Materials and methodsCake, almond paste, and pudding were modified by adding fiber and replacing sugar with sweeteners and polyols. Digestion process was modeled in test tubes. Rat enterocyte cells (IEC-6) were grown in a bicameral cell culture system to mimic the physiological characteristics of the human intestine. The glucose bioaccessibility and cellular glucose efflux were measured by glucose oxidase assay.Results and discussionThe glucose bioaccessibilities of modified foods were significantly lower (cake: 2.6 fold, almond paste: 9.2 fold, pudding 2.8 fold) than the controls. Cellular glucose effluxes also decreased in the modified cake, almond paste, and pudding by 2.2, 4, and 2 fold respectively compared to their controls.ConclusionOur results suggest that combining in vitro enzymatic digestion with cell culture studies can be a practical way to test in vitro glucose bioaccessibility and bioavailability in functional food development.

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Nano-Scale Modifications of Amniotic Membrane Induced by UV and Antibiotic Treatment: Histological, AFM and FTIR Spectroscopy Evidence

Materials ◽

10.3390/ma14040863 ◽

2021 ◽

Vol 14 (4) ◽

pp. 863

Author(s):

Simona Cavalu ◽

George Roiu ◽

Ovidiu Pop ◽

Denisa A. Petricas Heredea ◽

Traian Octavian Costea ◽

...

Keyword(s):

Ftir Spectroscopy ◽

Antibiotic Treatment ◽

Amniotic Membrane ◽

Ocular Surface ◽

Enzymatic Digestion ◽

Uv Exposure ◽

Amniotic Membrane Transplantation ◽

Corneal Tissue ◽

Nano Scale ◽

Gentamicin Treatment

The efficiency of amniotic membrane (AM) transplantation in different types of ocular surface disorders is due to its outstanding properties such as antifibrotic, antibacterial, anti-inflammatory and antiangiogenic, working as a versatile scaffold to promote corneal tissue epithelialization. A proper preparation, preservation and clinical application are crucial for the best outcomes in the treatment of different severe ocular disorders, taking into account its fragility. In this context, by combining high-sensitivity tools such as atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy with histological and immunohistochemical examination, we aimed to investigate the ultrastructural modifications of the amniotic membrane (AM) upon UV exposure and/or antibiotic treatment, with relevance for clinical applications in ocular surface surgery. From the morphological point of view, we noticed a loss of cuboidal cells in the basal membrane, accompanied by the splitting of collagen fibers upon UV and/or gentamicin treatment, while structural alteration of proteins was evidenced by the FTIR quantitative analysis of the secondary structure. A decrease in α-helix and β-sheet content, accompanied by increased content in less ordered structures (turns, random and side chains), was noticed after all the treatments. At the nano-scale, AFM details showed modifications of collagen fibrils in terms of their thickness and network compaction upon gentamicin and/or UV treatment. The enzymatic digestion assay demonstrated that UV exposure significantly reduces the degradation rate of the AM, while gentamicin treatment promotes an accelerated enzymatic digestion upon UV exposure. In order to highlight the clinical impact of the research, a clinical case is presented showing the relevance of amniotic membrane transplantation in pterygium surgery.

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Assessment of In Vitro Bioaccessibility of Polyphenols from Annurca, Limoncella, Red Delicious, and Golden Delicious Apples Using a Sequential Enzymatic Digestion Model

Antioxidants ◽

10.3390/antiox10040541 ◽

2021 ◽

Vol 10 (4) ◽

pp. 541

Author(s):

Giulia Graziani ◽

Anna Gaspari ◽

Claudio Di Vaio ◽

Aurora Cirillo ◽

Carolina Liana Ronca ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Activities ◽

In Vitro Digestion ◽

Enzymatic Digestion ◽

Beneficial Effects ◽

Golden Delicious ◽

In Vitro Bioaccessibility ◽

Inflammatory Processes ◽

Great Relevance

Four different varieties of apples have been considered (Limoncella, Annurca, Red Delicious, and Golden Delicious) to estimate the extent of colon polyphenolics release after in vitro sequential enzyme digestion. Since several studies report a positive effect of apple polyphenols in colonic damage, we found of interest to investigate the colon release of polyphenols in different varieties of apples in order to assess their prevention of colonic damage. UHPLC-HRMS analysis and antioxidant activity (ABTS, DPPH, and FRAP assays) were carried out on the apple extracts (peel, flesh, and whole fruit) obtained from not digested samples and on bioaccessible fractions (duodenal and colon bioaccessible fractions) after in vitro digestion. Polyphenolic content and antioxidant activities were found to vary significantly among the tested cultivars with Limoncella showing the highest polyphenol content accompanied by an excellent antioxidant activity in both flesh and whole fruit. The overall trend of soluble antioxidant capacity from the soluble duodenal phase (SDP) and soluble colonic phase (SCP) followed the concentrations of flavanols, procyandinis, and hydroxycinnamic acids under the same digestive steps. Our results highlighted that on average 64.2% of the total soluble antioxidant activity was released in the SCP with Limoncella exhibiting the highest values (82.31, 70.05, and 65.5%, respectively for whole fruit, flesh, and peel). This result suggested that enzymatic treatment with pronase E and viscozyme L, to reproduce biochemical conditions occurring in the colon, is effective for breaking the dietary fiber-polyphenols interactions and for the release of polyphenols which can exercise their beneficial effects in the colon. The beneficial effects related to the Limoncella consumption could thus be of potential great relevance to counteract the adverse effects of pro-oxidant and inflammatory processes on intestinal cells.

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