Trypsin Solution Research Articles

The Efficiency of Decellularization of Bovine Pericardium by Different Concentrations of Sodium Dodecyl Sulfate

Background. In modern cardiovasclar surgery, it is a promising method to use xenotissues, which in their properties are close to human tissues, in order to restore the integrity of the heart chambers, its walls or valves. Decellularization of extracellular matrix (EMC) is applied in the process of creation of such bioprotheses. In EMC, the elastin and collagen components are preserved, and antigenic molecules are eliminated resulting in reduction the risk of rejection. The study is devoted to assessment of the histological, molecular-genetic and cytotoxic properties of decellularized bovine pericardium, processed with various concentrations of trypsin enzyme.Objective. The aim of the work is evaluation of efficiency of bovine pericardial decellularization, based on the use of trypsin enzyme with 1% Sodium Dodecyl Sulfate (SDS) and 0.1% SDS.Methods. Bovine pericardium was used as a biomaterial for decellularization. Decellularization protocol 1 envisages processing of samples with 0.25% Trypsin solution at 24 °C with constant shaking (200 rpm) along with processing with 1% ionic SDS detergent. The samples, prepared according to protocol 2, were processed with a low concentration of 0.1% SDS. Histological and morphological properties along with detection of nucleic acids concentration in the samples were studied. Matrix samples were cultured in a human fibroblasts cell culture suspension in order to determine cytotoxicity.Results. Histological examination has not revealed any presence of cells in tissues, decellularized in accordance with both protocols. More than 99% of the nucleic acids was removed from decellularized bovine matrix. During our study, we have not observed cytotoxic effect in vitro for protocol 2 matrix samples, decellularized with only 0.1% SDS. Focal destruction of fibroblasts was observed in conditions of long-term cultivation in protocol 1 samples (Trypsin + 1% SDS). Cells formed abnormal morphological aggregates. Samples of this group have also demonstated sructural changes in collagen and elastin fibers.Conclusions. Studies have shown that pericardial matrix tissue, decellularized with low-concentration of 0.1% SDS, has the same biological properties as the native pericardium. Decellularization of bovine pericardium, using trypsin enzyme with 1% SDS, has a cytotoxic effect on human cells.

Shotgun Proteomics and Mass Spectrometry as a Tool for Protein Identification and Profiling of Bio-Carrier-Based Therapeutics on Human Cancer Cells.

Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis, proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data on a protein search engine software such as PEAKS studio loaded with protein database for the species of interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified will be obtained and can be used to determine proteins identified with high confidence. This method can be applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro. In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied to investigate the proteome dynamics.

Fabrication of Decellularized Engineered Extracellular Matrix through Bioreactor-Based Environment for Bone Tissue Engineering

Extracellular matrix (ECM)-contained grafts can be achieved by decellularization of native bones or synthetic scaffolds. Limitations associated with harvesting the native bone has raised interest in preparing in vitro ECM bioscaffold for bone tissue engineering. Here, we intend to develop an ECM-contained construct via decellularizing an engineered gelatin-coated β-tricalcium phosphate (gTCP) scaffold. In order to find an optimal protocol for decellularization of cell-loaded gTCP scaffolds, they were seeded with buccal fat pad-derived stem cells. Then, four decellularization protocols including sodium dodecyl sulfate, trypsin, Triton X-100, and combined solution methods were compared for the amounts of residual cells and remnant collagen and alteration of scaffold structure. Then, the efficacy of the selected protocol in removing cells from gTCP scaffolds incubated in a rotating and perfusion bioreactor for 24 days was evaluated and compared with static condition using histological analysis. Finally, decellularized scaffolds, reloaded with cells, and their cytotoxicity and osteoinductive capability were evaluated. Complete removal of cells from gTCP scaffolds was achieved from all protocols. However, treatment with Triton X-100 showed significantly higher amount of remnant ECM. Bioreactor-incubated scaffolds possessed greater magnitude of ECM proteins including collagen and glycosaminoglycans. Reseeding the decellularized scaffolds also represented higher osteoinductivity of bioreactor-based scaffolds. Application of Triton X-100 as decellularization protocol and usage of bioreactors are suggested as a suitable technique for designing ECM-contained grafts for bone tissue engineering.

Biocompatiblibility analysis of the decellularized bovine pericardium

The decellularized bovine pericardium and its potential use as a natural scaffold is a promising approach in the field of tissue engineering and regenerative medicine. The reaction of the host toward decellularized scaffolds depends on their biocompatibility, which should be satisfied being before applied in clinical use. Purpose: to evaluate the biocompatibility of the extracellular matrices, which were decellularized by trypsin enzyme and anionic sodium dodecyl sulfate (SDS) detergent. Material and methods. Pericardial sacs were acquired from 12-18 months’ age bulls. Tissue decellularization was performed by using 0.25 % Trypsin solution and 1 % ionic SDS for group I and 0.1 % SDS for group II samples. The implantation was performed on Wistar rats. The tissue samples were stained with hematoxylin & eosin, Congo red and Masson's Trichrome for histological analysis. Results. In group 1 in 3 months after subcutaneous implantation in rats we noticed the inflammation in surrounding tissue and degradation of the implant. Under the same conditions in animals of group 2 implant replacement with growing immature connective tissue was noted. Bio-implant of this group did not degrade, moreover it's integrated to the tissues of experimental rats. Conclusion. Our results showed that decellularized bovine pericardium by 0.1 % SDS can become an alternative material for tissue engineering and has the potential for further use in human surgery.

Self-assembly synthes is of trypsin-immobilized monolithic microreactor for fast and efficient proteolysis

Fast and highly efficient digestion of proteins is essential for high-throughput proteomic analysis. Herein, a facile approach was developed for self-assembly preparation of trypsin-immobilized capillary monolithic column and its application as an immobilized enzyme microreactor (IMER) for fast and highly efficient proteolysis was described. The performance of the trypsin-immobilized monolithic enzyme microreactor was evaluated by in-situ digestion of model proteins. The results showed that the trypsin-immobilized monolithic enzyme microreactor had much higher tryptic digestion efficiency than the free trypsin in solution, where the coverage of peptide sequences by mass spectrometry (MS)-based analysis could bear comparison with the free one, while the digestion time was dramatically shortened from 12 h to 16 s. Furthermore, the trypsin-immobilized monolithic enzyme microreactor also exhibited good practicability to complex human serum sample, in which the total of 45 peptides from human serum albumin (HSA) matched with sequence coverage of 75% were precisely identified. The successful application demonstrated the promising potential of the trypsin-immobilized capillary monolithic column as the IMER in high-throughput proteomic analysis.

A Syringe-Filter-based Portable Microreactor for Size-selective Proteolysis of Low Molecular-weight Proteins

Abstract Portable microreactors (PMRs) have valuable applications in the field of proteomics. However, no PMRs have been developed for the size-selective proteolysis of low molecular-weight proteins (LWPs) with mass less than 30 kDa, which are of great importance in biomarkers, biomolecular signaling and the field of pathology. In this study, a novel syringe-filter-based PMR using core-shell mesoporous silica (CSMS) microspheres as an enzyme support was reported. Because of the unique properties of CSMS microspheres, the PMR was easily fabricated by on-site physical adsorption even without any incubation, which was accomplished by simply pushing trypsin solution through a syringe filter containing the microspheres. Efficient immobilization of trypsin was demonstrated by characterizing the PMR using various spectroscopic techniques. The PMR loading amount was approximately 2.9 mg/g trypsin/CSMS microspheres with negligible leaching. The capability of the proposed PMR for size-selective proteolysis was assessed by investigating the digestion of proteins with different molecular weights, i.e., cytochrome C (12.38 kDa), lysozyme (14.39 kDa), myoglobin (17.00 kDa) and ferritin (440.00 kDa). The results of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) analysis showed that, by using the proposed PMR, the peptide numbers and sequence coverage of LWPs in the sample were enhanced owing to the size-exclusion interaction of the mesopores against large proteins. The proposed PMRs had essential features of easy fabrication, low cost, reusability and efficient size-selective proteolysis, and performing multiple functions including size-exclusion of LWPs, trypsin digestion and filtration in a single injection step of protein samples. This study provided a robust approach for fabricating PMRs that could perform rapid size-selective digestion of LWPs.

A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method.The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion.The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

Phosphoproteome Analysis in Immune Cell Signaling.

Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). © 2020 Wiley Periodicals LLC. Basic Protocol 1: SILAC labeling of mouse macrophages Basic Protocol 2: Stimulation, cell lysis and Western Blotting Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment Basic Protocol 4: Quantitative mass spectrometry Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Protein preparation and prospects for its use in meat technology

The article considers the specifics of using the growing lupine protein formulation developed by the authors in the meat products production. The formulation technology includes: removing the shell from lupine seeds; grinding the seeds to obtain flour; diluting lupine flour in drinking water; heating the solution; fermenting with glucoamylase; centrifuging; autoclaving; cooling; fermenting with trypsin activated by the blue light; centrifuging; ultrafiltrating; drying to a dry matter content of 80.0–90.0 %. Trypsin solution irradiation with blue light with a wavelength of 435–470 nm allows to increase its activity by 3 times. When using this technology, the yield of protein concentrate is 24.3 % by weight of the raw materials. The resulting protein formulation is a fine powder of light-yellow color without a pronounced smell and taste, with a protein content of 64.1 %. The protein formulation has high functional and technological properties: moisture-retaining ability – at the level of 517.0 %; fat-retaining and emulsifying – at the level of 208.0 and 98.0%, respectively. To justify the use of protein preparation in the meat products production, the researchers studied its functional and technological properties by introducing it into minced poultry (chicken) in the ratio of white and red meat 1:1 and replacing salted meat raw materials in an amount of 30.0% with a step of 10.0 %. With an increase in the amount of hydrated protein formulation in minced meat to 30.0 %, the indicators of moisture-retaining and fat-retaining capacity increase by 117.0 and 54.0 %, respectively. The authors concluded that there was a possibility of developed protein preparation use in the meat products recipe without reducing its biological value.

Differential Biodegradation Kinetics of Collagen Membranes for Bone Regeneration.

Native collagen-based membranes are used to guide bone regeneration; but due to their rapid biodegradation, this treatment is often unpredictable. The purpose of this study was to investigate the biodegradability of natural collagen membranes. Three non-cross-linked resorbable collagen barrier membranes were tested: Derma Fina (porcine dermis), Evolution Standard (equine pericardium) and Duo-Teck (equine lyophilized collagen felt). 10 × 10 mm2 pieces of membranes were submitted to three different degradation procedures: (1) hydrolytic degradation in phosphate buffer solution, (2) enzyme resistance, using a 0.25% porcine trypsin solution, and (3) bacterial (Clostridium histolyticum) collagenase resistance test. Weight measurements were performed with an analytic microbalance. Thickness was measured with a digital caliper. Membranes were analyzed at different time-points, up to 21 d of immersion. A stereomicroscope was used to obtain membranes’ images. ANOVA and Student Newman Keuls were used for mean comparisons (p < 0.05), except when analyzing differences between time-points within the same membrane and solution where pair-wise comparisons were applied (p < 0.001). Derma Fina attained the highest resistance to all degradation challenges. Duo-Teck was the most susceptible membrane to degradation, complete degradation occurred as soon as 8 h. The bacterial collagenase solution performed as the most aggressive test as all membranes presented 100% degradation before 21 d.

P0659MULTIMODAL IMAGING OF MALDI MSI DATA

Abstract Background and Aims MALDI mass spectrometric imaging (MALDI MSI) is a powerful histologic tool for the analysis of biomolecules in tissue samples. MALDI MSI measurements results in a high sensitivity and accuracy of spatial distribution of biomolecules in tissue samples The resolution information of MALDI MSI is in the range of 5-10 µm in the spatial distribution and has the ability to identify proteins, peptides, lipids and small biomolecules directly in tissue samples in one analytical step..For a more detailed analysis of MALDI MSI data and a correlation between the molecular and microscopic level, a combination of MALDI MSI data and histological staining is essential. By combining MALDI MSI data and histological data, much more information are obtained than from a single analysis of both methods. Therefore, MALDI MSI data sets and histological staining were fused to a 3D model presenting a biomolecule distribution of the whole organ and provide more information than a single tissue section. We developed, established and validate an algorithm for an automatic registration of MALDI data with different histological image data for the cross-process evaluation of multimodal data sets for creating 3D models. This multimodal image approach simplifies and improves molecular analyses of tissue samples clinical research and diagnosis. Method The data sets for the fusion and creating of a 3D model consist of mass spectrometric data as well as histological and Immunohistochemical staining methods. Histological tissue sections of a whole mice kidney were prepared. For MALDI MSI data the organ sections were coated and incubated with a trypsin solution were performed by using a sprayer for MALDI imaging. As matrix, α-cyano-4-hydroxycinnamic acid was used. MALDI MSI was performed using the Rapiflex. For histological staining the hematoxylin-eosin and Gomori staining were chosen. For Immunohistochemical double staining and immunofluorescence, were used for the detection of Collagen type I, smooth muscle actin and the cell nuclei. Results By using a mathematical registration, a perfect superposition of the individual histological sections mass spectrometric data was achieved. It is possible to combine mass spectrometric data, histological and Immunohistochemical data sets in a high number and to reconstruct the measured mice kidney. By using different imaging methods, a variety of information about tissue structure as well as tissue changes and protein distribution can be obtained. The fusion of the data also offers a virtual incision of the organ from any angle and level. The algorithms are adapted to take the data fusion automatically offering a high-throughput approach for clinical diagnostics and the possibility to involved artificial intelligence in its interpretation in research. Conclusion There is a successful fusion of MALDI MSI data and different histological and Immunohistochemical staining data sets of a whole organ

Fragmentation of Proteins in the Corona of Gold Nanoparticles As Observed in Live Cell Surface-Enhanced Raman Scattering

Surface-enhanced Raman scattering (SERS) can provide information on the structure, composition, and interaction of molecules in the proximity of gold nanoparticles, thereby enabling studies of adsorbed biomolecules in vivo. Here, the processing of the protein corona and the corresponding protein-nanoparticle interactions in live J774 cells incubated with gold nanoparticles was characterized by SERS. Samples of isolated cytoplasm, devoid of active processing, of the same cell line were used as references. The occurrence of the most important SERS signals was compared in both types of samples. The comparison of signal abundances, supported by multivariate assessment, suggests a decreased nanoparticle-peptide backbone interaction and an increased contribution of denatured proteins in endolysosomal compartments, indicating an interaction of protein fragments with the gold nanoparticles in the endolysosome of the living cells. To study the protein fragmentation in a model and to confirm the assignment of specific spectral signatures in the live cell spectra, SERS data were collected from a solution of bovine serum albumin (BSA) digested by trypsin as an enzymatic model and from solutions of intact BSA and trypsin. The spectra from the enzymatic model confirm the strong interaction of protein fragments with the gold nanoparticles in the endolysosomal compartments. By proteomic analysis, using combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry of the extracted hard corona, we directly identified protein fragments, some originating from the culture medium. The results illustrate the use of appropriate models for the validation of SERS spectra and have potential implications for further developments of SERS as an in vivo analytical and biomedical tool.

Evaluation of cholesterol lowering property and antibacterial activity of two potential lactic acid bacteria isolated from the intestine of snakehead fish (Channa argus)

The use of lactic acid bacteria (LAB) is a strategy employed to improve aquatic animal health status and to prevent aquaculture diseases. In the present study, two potential LAB were isolated from the intestines of Channa argus (C. argus) towards the assessment of their probiotic potential. Based on the preliminary screening on 178 isolates obtained from the intestines of C. argus, only ten LAB isolates were selected by using LAB selective media (MRS-CHOL medium) for their high cholesterol removal capacity. Five potential LAB isolates (W1, W24, C11, L1 and L19) were all had high inhibitory effects on a variety of aquatic pathogenic bacteria and all able to survive in acidic and alkaline conditions, higher tolerance to bile salt, high surface hydrophobicity, high auto-aggregation property and they were found to tolerate in in pepsin and trypsin solution environmental condition. Based on the presently observed biological characteristics, it is concluded that the W24 and L19 isolates were more effective than the W1, C11 and L1 isolates. Two potential LAB strains, W24 and L19 were identified as Enterococcus faecalis (Genbank: MT102745.1) and Lactococcus lactis (Genbank: MT102746.1) respectively by 16S rRNA sequencing. The identification of physiological and biochemical properties results showed that the W24 and L19 isolates could ferment a variety of sugars, which was in line with the characteristics of LAB. The two potential LAB isolates (W24 and L19) were susceptible to the vast majorityantibiotics and were all respectively exhibited γ-haemolysis (i.e. no haemolysis) activity. The biosafety assay confirmed that the two potential LAB isolates were not pathogenic to the host fish. Therefore, the E. faecalis W24 and L. lactis L19 may be a promising candidate for the development of functional micro-ecological feed. This study has provided insight into how optimal LAB should be isolated and screened from the intestines of C. argus.

Human Colonoids as a Model for Studying Host‐Pathogen Interactions with Campylobacter jejuni

BackgroundCampylobacter is a leading cause of bacterial enterocolitis in the U.S. and around the world. In addition to the acute morbidity, it is associated with delayed sequelae such as environmental enteropathy and post‐infection irritable bowel syndrome. Host responses to Campylobacter infection are poorly studied due to colonization resistance in mice thereby restricting virulence studies to immortalized cell lines. These models lack the complexity of the host environment. Recent studies have highlighted the use of organoids for studying enteric pathogens. We propose that human organoids provide a unique platform for studying pathogenic mechanisms incorporating unique host (human) and infection (strain) characteristics.ObjectiveTo demonstrate human colonic organoids (colonoids) as a model for studying C. jejuni infection.MethodsSigmoid colonic biopsies were collected from healthy volunteers between 18–35 years of age (n=3). Intestinal crypts were isolated, washed and plated in matrigel domes for colonoid formation. Mature colonoids were harvested after 7 days, and dissociated using a 0.05% trypsin solution. These were plated on collagen IV coated plates, generating 2D‐monolayers for infection experiments. Three different C. jejuni strains (Sequence Types 45, 22, 8) were grown to an OD600 of 1 and used to infect the colonoid monolayers. To assess adhesion and internalization, colonoids were infected for a total of 3 hours after which monolayers were either washed and subsequently lysed (to determine attached plus invaded bacteria) or treated with gentamycin and then lysed (to determine invaded bacteria). Bacterial quantification was done by plating on Mueller Hinton agar. Additional colonoid monolayers were grown on permeable transwell inserts. Baseline transepithelial resistance (TER) of confluent monolayers was recorded and then continuously assessed every hour after the addition of the C. jejuni strains (using impedance spectroscopy in cellZscope) to evaluate changes in barrier integrity.ResultsAll C. jejuni strains attached and invaded colonoid monolayers derived from all three volunteers. Two strains (ST 45 and 22) caused greater adhesion and invasion in all three colonoids compared to the third strain (Adhesion mean (SEM): 4.32 x 10−2 (2.33 x 10−3), 9.36 x 10−3 (4.82 x 10−4) vs 7.79 x 10−3 (3.51 x 10−3); Invasion mean (SEM) 3.47 x 10−4 (2.13 x 10−5), 2.33 x 10−4 (1.36 x 10−4) vs 4.57 x 10−5 (1.73 x 10−5) (ANOVA, p&lt;0.05, n=3/group for both analyses). Additionally, greater drop in TER was seen in colonoid monolayers exposed to the two strains with higher adhesion and invasion (Mean % of baseline TER at 30 hr post‐infection 2.58% (1.21), 14.14% (3.39) vs 42.29% (18.49), p&lt;0.01). These results correlated with virulence of these strains in conventional T84 monolayers. Transcriptomic and proteomic characterization of the C. jejuni infection in these colonoids is in progress.ConclusionsHuman colonoids are useful to assess the role of the host in infectivity and epithelial barrier responses to C. jejuni infection. Future work involves determining responses in colonoids derived from volunteers with specific characteristics to create unique host‐bacterial infection models.Support or Funding InformationSupported by funding from Department of Defense W81XWH‐18‐1‐0074

Improved in-solution trypsin digestion method for methanol-chloroform precipitated cellular proteomics sample.

Methanol-chloroform based protein precipitation is an essential step in many liquid chromatography-tandem mass spectrometry-based cellular proteomics applications. However, re-solubilization of the total protein precipitate is difficult using regular in-solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re-solubilization of membrane fractions. In this study, we demonstrated an application combining methanol-chloroform based protein precipitations and deoxycholic acid assisted re-solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry-based bottom-up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in-solution trypsin digestion studies were presented on methanol-chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid-treated samples compared to other established methods. Furthermore, liquid chromatography-tandem mass spectrometry analysis of an equal amount of proteins from methanol-chloroform precipitated, and methanol-chloroform/deoxycholic acid-treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.

Urine Proteomics: Evaluation of Different Sample Preparation Workflows for Quantitative, Reproducible, and Improved Depth of Analysis.

The growing field of urinary proteomics shows promise to expand the number of biomarkers for the diagnosis and prognosis of a number of human diseases. With the rapid developments in mass spectrometry methods for proteome quantification, there exists an opportunity for improved sample processing and separation workflows to make important contributions to urine proteomic analyses. Here we evaluate the performance of four sample preparation methods: MStern, PreOmics in-StageTip (iST), suspension-trapping (S-Trap), and conventional urea In-Solution trypsin hydrolysis for nondepleted urine samples. Data-dependent acquisition (DDA) mode on a QExactive HF mass spectrometer was used for single-shot label-free data acquisition. Our results demonstrate a high degree of reproducibility within each workflow. PreOmics iST yields the best digestion efficiency, whereas the S-Trap workflow gives the greatest number of peptide and protein identifications. Using the S-Trap method and starting with ∼0.5 mL, we identify ∼1500 protein groups and ∼17 700 peptides from DDA analysis with a single injection on the mass spectrometer.

The tolerance of human epidermal cells to trypsinization in vitro.

To characterize the tolerance of different types of human epidermal cells to trypsinization in vitro and develop a new method to separate and purify melanocytes according to their tolerance to trypsinization. Epidermal cells were obtained by separating the epidermis from human foreskins. Some of those cells were used for routine culture, and then were subjected to differential trypsin digestion. The remaining epidermal cells were resuspended in a 0.25% trypsin solution and then were neutralized by the addition of bovine serum at different time points. Immunofluorescence staining of HMB45, K15 and vimentin was used to identify melanocytes, keratinocytes and fibroblasts, respectively. We found that Keratinocytes, melanocytes and fibroblasts are primary cells obtained from conventional cultures of human skin. Purified keratinocytes and melanocytes can be obtained by conventional differential trypsin digestion, but fibroblasts in the melanocyte population quickly gain a survival advantage after passage. With longer trypsin digestion times, the number of adherent cells decreased, the time required for cell attachment increased, and the proportion of melanocytes increased. There were no obvious keratinocytes in cell populations obtained after 12h of trypsinization of epidermal cells, and more short spindle-shaped melanocytes appeared, all of which were HMB45-positive. In conclusion, the tolerance of human epidermal melanocytes to trypsinization in vitro was better than epidermal keratinocytes, and that property can be used to purify melanocytes and was better than traditional differential trypsin digestion. The morphology of cells that survived the long-term trypsin digestion changed and they had good proliferative activity, but seemed to be more immature.

Післязабійний кістковий мозок великої рогатої худоби – перспективне джерело стовбурових клітин

The development of modern methods of cryopreservation of animal cells has promoted to introduce the concept of creating of stem cell banks for their future use, to avoid problems with donor shortages. Large-scale cryopreservation of stem cells began in the 1990s with the establishment of human cord blood banks. The issue of long-term storage of stem cells is also acute in veterinary medicine. To obtain of cellular material, is needed a source suitable for cell isolation. As such a source it is advisable to consider a slaughter material from productive animals. The purpose of this study is to establish the possibility of cattle bone marrow use after slaughter as source of stem cells, on the basis of the proliferation index and the viability of the cultured cells to determine the suitability of this biological material for the obtaining of stem cells 72 hours after slaughter of the animal. Materials and methods. The bone marrow was obtained from the femur of a cow, 3 years old, who was slaughtered in a slaughterhouse. The bone marrow was collected with sterile tweezer into a sterile tube, filled with 0.25% trypsin solution (ratio of bone marrow volume to trypsin solution – 10: 1) and placed in a refrigerator (t + 4 0C) for 24 hours for enzymatic disaggregation. After bone marrow disaggregation, cultivation of cells was performed in a CO2-incubator using of disposable plastic Petri dishes (d = 30 mm) according to the standard procedure by periodically passaging them after forming a monolayer by 95–100%. Results. Cultivation of a suspension of cells, derived from the post-mortemal bone marrow of a cow, revealed that colonies of cells began to emerge 6–7 days after sowing. Passaging the cells with 0,25% trypsin-Versen solution and sowing them in new cultural plates contributed to increase the mass of cells that actively proliferated. It was established that stem cells, isolated from the post-mortemal bone marrow of cattle, have significant proliferative potential, as evidenced by proliferation indices&nbsp; from I to III passages and high viability of cells. Thus, the post-mortemal bone marrow of the cattle can be used as an alternative source of stem cells. This biological material is suitable for the isolation of stem cells, even 72 hours after slaughtering the animal, which opens the prospects of its transportation over long distances.

Выделение эндогенных антимикробных пептидов из клеток крови

A technique for isolating the endogenous antimicrobial peptides (AMP) has been developed. Leukocyte-erythrocyte-platelet blood mass of donors was used as the material for preparing endogenous AMP. The hydrolysate was obtained with the trypsin solution from this mass, and then the components were fractionated using the filter chromatography column (Simax CSN ISO 3585, Russia). For gel filtration, 1.5 g of Sephadex G-25 was applied to the filter surface followed by sterilizing filtration of the obtained substance through bactericidal filters having a pore diameter of 0.2 μm. The antimicrobial peptide fractions were isolated using high performance liquid chromatography. Subsequently, AMP were encapsulated into the organosilicon niosomes. Key words: antimicrobial peptides, defensins, niosomes, antibiotic-resistant microorganisms