Trypsin Solution Research Articles

Improvement of protocol for detachment assays in B16F10 cells

B16F10 (B16) murine melanoma cells have the characteristic of strong adhesion to culture surfaces. The use of trypsin solutions in high doses to release this type of cell is not suitable. When B16F10 cells are released with high concentrations of trypsin they do not form a precipitate after centrifugation and consequently the cells will be lost when discarding the supernatant. To solve this problem, we established a solution with phosphate buffered saline (PBS) and ethylenediaminetetraacetic acid (EDTA) at low trypsin concentrations. Finally, we verified the efficiency of this solution on the quantity of cells recovered after centrifugation.

Chloroform/Methanol Protein Extraction and In-solution Trypsin Digestion Protocol for Bottom-up Proteomics Analysis.

Bottom-up proteomics utilizes sample preparation techniques to enzymatically digest proteins, thereby generating identifiable and quantifiable peptides. Proteomics integrates with other omics methodologies, such as genomics and transcriptomics, to elucidate biomarkers associated with diseases and responses to drug or biologics treatment. The methodologies employed for preparing proteomic samples for mass spectrometry analysis exhibit variability across several factors, including the composition of lysis buffer detergents, homogenization techniques, protein extraction and precipitation methodologies, alkylation strategies, and the selection of digestion enzymes. The general workflow for bottom-up proteomics consists of sample preparation, mass spectrometric data acquisition (LC-MS/MS analysis), and subsequent downstream data analysis including protein quantification and differential expression analysis. Sample preparation poses a persistent challenge due to issues such as low reproducibility and inherent procedure complexities. Herein, we have developed a validated chloroform/methanol sample preparation protocol to obtain reproducible peptide mixtures from both rodent tissue and human cell line samples for bottom-up proteomics analysis. The protocol we established may facilitate the standardization of bottom-up proteomics workflows, thereby enhancing the acquisition of reliable biologically and/or clinically relevant proteomic data. Key features • Tissue/cell pellet sample preparation for bottom-up proteomics. • Chloroform/methanol protein extraction from murine tissue samples. • In-solution trypsin digestion proteomics workflow.

Assessment of the Effect of 10% Trypsin and 25% Bromelain on the Micro-Shear Bond Strength of Etch-and-Rinse Adhesive Systems

Background and Objective: The removal of collagen by chemical methods can increase the micro-shear band strength of composite restorations. The present study aimed to investigate the effect of bromelain 25% and trypsin 10% on the micro-shear bond strength of Etch-and-rinse adhesives. Materials and Methods: After cutting the chewing surface and exposing the dentin to a diameter of 5mm, the samples were randomly assigned to three equal groups (n=45): in the control group, after etching the adhesive surface, a Single Bond was used. In the other group, after etching, the surface was prepared with bromelain enzyme. In the last group, the trypsin enzyme was used to prepare the surface before applying the adhesive. Thereafter, the cylinders of ES Flow composite were placed on the bond surface. Following that, the micro-shear strength of the samples was calculated in megapascals. Results: There was a significant difference between the mean of the trypsin group and that of the control group (P=0.001), as well as the bromelain group and the control group (P<0.001). Conclusion: The preparation of etched dentin by trypsin and bromelain enzyme solutions increased the micro-shear bond strength between composite materials and dental dentin in Etch-and-rinse adhesives.

RELEVANCE OF THE USE OF POSTMORTEM BIOMATERIAL OF DOMESTICATED YAK (BOS GRUNNIENS) TO OBTAIN STEM CELLS FROM BONE MARROW

The development of new technology methods for cryopreservation of animal cells has contributed to the introduction of stem cell banks for clinical use, including transplantation and regenerative veterinary medicine, and their further use to avoid problems of donors’ shortage. The research aims to determine the possibility of using the bone marrow of a domesticated yak as a source of stem cells after slaughter. Moreover, we should determine the suitability of post-mortem biological material for obtaining stem cells based on the index of proliferation and viability of cultured cells. Therefore, we used the following materials and methods: bone marrow, obtained from the femur of a domesticated yak in the post-slaughter period. Bone marrow samples were taken in compliance with the rules of asepsis in a sterile test tube. Thus, we added 0.25% trypsin solution to the biomass (the ratio of bone marrow to the solution is 10:1) and placed it in a refrigerator (t +40 °C) for 24 hours for enzymatic disaggregation. Furthermore, we carried out the culturing of the obtained cells in a CO2 incubator according to the standard method with passivation after the formation of a monolayer by 90%–100%. When culturing a suspension of cells obtained from post-slaughter bone marrow material, we noted the appearance of cell colonies six days after sowing. Periodic passivation of stem cells contributed to an increase in the biomass of actively proliferating cells. In addition, we found that stem cells isolated from post-slaughter bone marrow material of domesticated yak have significant proliferative potential, as evidenced by proliferation indices in the range from the first to the third passages and high cell viability. Thus, one can use the obtained post-mortem material in the form of the bone marrow of a domesticated yak as an additional source of stem cells. This post-mortem biological material is suitable for the isolation of stem cells 72 hours after the slaughter of an animal, which opens up the possibility of its transportation over long distances.
 The scientific novelty of the research is in the context of the international Convention on Biological Diversity, which means the variability of living organisms from all sources, including ecosystems and ecological complexes they are a part of. Within the strategy and action plan for the conservation of biodiversity of the Russian Federation, the ecosystem service of livestock production is significant for preserving the traditional way of life of the indigenous peoples of Siberia and the Russian Far East (it has regional and local significance). This global problem can be solved by (1) developing cell technologies for species not yet involved in it and (2) improving existing ones.

Anchovy’s protein as a potential precursor of Angiotensin-I Converting Enzyme (ACE) inhibitory peptide and Dipeptidyl Peptidase-IV (DPP-IV) inhibitory peptide by an in silico approach

Protein from fish is known as the precursor of biologically active peptides that exert various health benefits such as antihypertensive, antitumor, and immunomodulatory properties. Hence, this study aimed to perform an extraction of anchovy, LC-MS/MS analysis and in silico evaluation of the major proteins in anchovies as potential precursors of biologically active peptides in addition to determining whether such peptides can be released by selected proteolytic enzymes. Anchovy was subjected to protein extraction followed by total soluble protein concentration determination using Bradford assay. The sample was subjected to in-solution trypsin digestion, which was then analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A bioinformatic approach by PEAKS Studio was used to identify the protein. A total of four anchovy’s proteins which are myosin light chain 1, V(D)J recombination-activating protein, 60 kDa chaperonin and heat shock protein 90AA1 were identified. Then, the identified proteins were subjected to in silico approach using the BIOPEP database. The biological potential of the theoretically released angiotensin-I converting enzyme (ACE) inhibitory peptides and dipeptidyl peptidase (DPP)-IV inhibitory peptides were predicted by determining the frequency of occurrence of fragments with a given activity. 60 kDa chaperonin and heat shock protein 90AA1 predicted the highest number of biological activities for ACE inhibitory peptides (284 and 264 fragments) and DPP-IV inhibitory peptides (395 and 409 fragments). The most promising enzyme for the generation of bioactive peptides from anchovy protein was anticipated to be pepsin (pH > 2), which theoretically released a high number of DPP-IV inhibitory peptides and ACE inhibitory peptides through the action of in silico proteolysis. Overall, this work highlighted that anchovy protein could be a promising precursor of bioactive peptides that have ACE and DPP-IV inhibitory activities for developing functional food or nutraceutical products.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Study of the effect of accelerated electrons on the structural characteristics of the bovine serum albumin using liquid chromatography-mass spectrometry and high-resolution tandem mass spectrometry

A method for quantification of the dose effect of ionizing radiation on the structural characteristics of bovine serum albumin (BSA) in aqueous solution through identification of unique peptides of protein domain structures using high-resolution liquid chromatography-mass spectrometry is proposed. BSA with the initial concentration of 500 mg/liter in a physiological solution was exposed to irradiation at a dose rate of 18. 5 Gy/sec using an accelerated electron beam with the maximum energy of 1 MeV at an average beam current of 1 uA. The absorbed dose in the sample volume was estimated using a Fricke (ferrous sulphate) dosimeter. After irradiation of BSA solution at 0.3, 0.6,1.8, and 20 kGy we analyzed the structural integrity of the protein native form and then quantified the content. For this, masses more than 30 kDa were removed using centrifugation. Then BSA was subjected to enzymatic hydrolysis with the addition of trypsin solution, and the resulting peptides with a mass of more than 10 kDa were repeatedly removed. The resultant samples were then examined using liquid chromatography mass spectrometry (LC-MS) and high-resolution tandem mass spectrometry (HRMS-MS/MS). The content of intact protein molecules was assessed by determining the concentrations of unique peptides corresponding to each of the three domains into which the amino acid sequence of BSA was divided. Using the developed methodology, a change in the natural conformation of bovine serum albumin (denaturation) in water samples induced by ionizing radiation at a dose ranging from 0.3 to 20 kGy was revealed on average in 71% of protein molecules exposed to doses up to 1 kGy in 79% of molecules exposed to doses of 4 kGy and in 99 % to 100% of molecules exposed to doses of 8 and 20 kGy.

Biomimetic vascular tissue engineering by decellularized scaffold and concurrent cyclic tensile and shear stresses

Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at −20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress–strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues.Graphical

A Facile Surface-Imprinting Strategy for Trypsin-Imprinted Polymeric Chemosensors Using Two-Step Spin-Coating

Surface imprinting used for protein recognition in functional cavities is highly effective in imprinting biomacromolecules to avoid template encapsulation during the formation of a molecularly imprinted polymer (MIP) matrix. Herein, we introduce a facile surface-imprinting method based on two-step spin-coating and photopolymerization to design highly efficient imprinted sites on polymeric films to detect trypsin (TRY). Well-distributed template imprinting is successfully achieved for maximized sensing responses by controlling the composition of functional monomers and crosslinkers in the precursor solution and the concentration of TRY in the imprinting solution. The MIP film exhibits higher sensitivity (−841 ± 65 Hz/(μg/mL)) with a coefficient of determination of 0.970 and a higher imprinting factor of 4.5 in a 0.24 µg/mL TRY solution compared to the nonimprinted polymer (NIP) film. Moreover, the limit of detection and limit of quantification are calculated to be 25.33 and 84.42 ng/mL, respectively. Finally, the selectivity coefficient is within the range of 3.90–6.78 for TRY against other proteins. These sensing properties are superior to those of the corresponding nonimprinted polymer matrix. Thus, the proposed facile surface-imprinting method is highly effective for protein imprinting with high sensitivity and selectivity.

Prospecting of Anti-Cancer Peptides (ACPs) from proteome of muscle tissue from Indian walking catfish, Clarias magur (Hamilton 1822) by Mass spectrometry and in silico approaches

The freshwater walking catfish Clarias magur (Hamilton, 1822) is one of the most popular aquaculture fishes in Indian and the Asian subcontinent because of its nutritional value and great taste. Muscle proteome of C. magur was studied. Muscle tissue is of significance in fishes due to presence of high protein content and poly unsaturated fatty acids (PUFA) proved to have medicinal and therapeutic value. Fishes procured were acclimatised and muscle tissue was studied. Briefly, muscle protein extract prepared from C. magur, by homogenizing muscle tissue in 50 mMTris buffer with protease inhibitor for downstream processing, digested with trypsin in solution, reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAA) for LC/MS analysis. The peptides were separated on Waters Synapt G2 Q-TOF equipped with Electro-Spray ionisation (ESI) for DATA independent acquisition for MS analysis. The raw data was processed by Protein Lynx Global Server (PLGS) software. Peptide tolerance limit was set at 50 ppm with minimum fragment match of 2 peptides for proteins. In silico approach was used to retrieve ACPs from muscle proteome derived from LC/MS by BIOPEP, Anti-CP and iDACP online servers. Out of a total of 468 peptides, 60 peptides showed anti-cancer peptide (ACPs) activity. Out of 19 non-allergenic peptides as analysed by AlerPred software, one peptide was toxic as revealed by ToxinPred software. The peptides were ranked with 0.9884 being highest and 0.0341 being the lowest by Peptide Ranker tool. This study reports C. magur derived anti-cancer bioactive peptides as a natural, less toxic anti-cancer therapeutic source exhibiting anti-cancer activity by activating apoptosis in mitochondria slaying cancer cells.

Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (kcat/km) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

The effect of leukocyte admixture on the wound healing effect of a platelet clot

The objective of the presented study was to find out whether the leukocytes in the platelet clot affect the healing of a complicated wound. The study was conducted on Wistar rats. Before the wound was inflicted, the animals were immersed in sevoflurane anesthesia. Wounds were inflicted in the interscapular area with a scalpel preheated in the flame of a gas burner to about 300.00 C. Three days after the wound was inflicted, in order to reduce the amount of cellular detritus, enzymatic necroectomy was performed first by 60-minute applications of trypsin solution (10.0 mg /ml), and then surgical necroectomy. The animals were divided into 4 groups: control group No. 1 (wounds were not sutured), control group No. 2 (wounds were sutured, but without the introduction of a fibrin clot), experimental group No. 1 (wounds were sutured after placing a platelet clot in them), experimental group No. 2 (wounds were sutured after placing a platelet clot in themleukocyte clot). According to the results of the study, it was shown that in both experimental groups, the healing of the wound defect occurred faster than in the control. However, when comparing the healing effects of pure platelet and plateletleukocyte clots, the result was ambiguous. A platelet-leukocyte clot, in comparison with a purely platelet clot, somewhat slowed down wound healing by inducing early rejection of sutures and slowing down scar remodeling. According to the results of the study, it can be concluded that the leukocytes in the clot can actually clear the wound. But in the case of application of such a clot to a wound, the surface of which was previously subjected to intensive necroectomy, its leukocyte component can direct its pro-inflammatory effect not on pathogenic microflora and necrotic detritus, but on healthy tissues. Thereby inducing hyperergic inflammation and slowing healing. The obtained result suggests that the study should be expanded, using wounds of varying degrees of infection and with varying degrees of necrosis in the wound canal area as models.

Analysis and identification of suspected snake venom samples using nano-ultra-high performance liquid chromatography-high resolution mass spectrometry

Snake venom is a complex mixture secreted from the glands of poisonous snakes， which contains proteins， peptides， lipids， nucleosides， sugars， amino acids， amines， metal ions， and other components. According to the toxicological classification， snake venoms can be classified as neurotoxins， anticoagulants and procoagulant toxins， cardiac toxins， other toxin proteins， and enzymes. Proteins and peptides are the key components of snake venom. The establishment of rapid， accurate analysis and identification methods for proteins in snake venom is a prerequisite for snake venom-related forensic identification， intoxication events， and pharmaceutical development. Until now， the classical analysis and identification methods have mainly been biochemical or immunoassays for DNA or proteins， such as polymerase chain reaction， agglutination test， enzyme-linked immunosorbent assay， fluorescent immunoassay， and various biosensing approaches. These methods have some limitations such as a high false-positive ratio， low sensitivity， poor anti-interference ability， and limited species discrimination capability. In recent years， with the rapid development of mass spectrometry （MS） techniques， the proteomics of snake venom has also attracted much attention and has contributed to the identification of snake species， in which non-targeted and targeted proteomics represent two main divisions. However， species identification via proteomics is in its infancy in forensic science. First， the tandem MS spectra of peptide sequences are highly complex， which poses a great challenge for the strict and accurate matching of peptides based on the rational speculation of MS fragmentation rules and theoretical calculations in non-targeted proteomics. Second， for the confirmation and identification of unknown substances， reference substances are commonly needed， but those for snake venom are lacking. Proteomics in snake venom identification is still in progress to improve the identification confidence and clarify the identification rules. In this work， a method based on nano-ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry （Nano LC-MS/HRMS） and size exclusion chromatography （SEC） was developed for identifying proteins and their source species， with strict rules for five suspected snake venom samples and their contamination in one case. Three SEC elution peaks were obtained from each of the five samples， which were lyophilized and treated with trypsin in solution， and then separated and analyzed by Nano LC-MS/HRMS. First， the Full MS/dd MS2 mode was used for the non-targeted acquisition of peptide information in the samples， and after submission to the Swiss-Prot database， the protein databases of Serpentes， Colubroidea， Elapidae， Elapinae， and Naja were contracted stepwise and compared. A total of 32 proteins from Naja atra were identified under the conditions of both peptide spectrum match and false discovery rate less than 1%， and number of characteristic peptides greater than or equal to two. All of these were derived from ten families of Naja atra， mainly three-finger toxins， metalloproteinases， and phospholipase A2. Proteins D3TTC2， D5LMJ3， Q7T1K6， Q9DEQ3， and Q9YGI4 were the most common among the five samples. Finally， the parallel reaction monitoring mode was adopted to select two unique peptides for each protein for targeted verification. It was considered that a protein in the samples was truly identified when it met the strict standard "the Δm/z of at least 75% y+ and b+ ions of each unique peptide was less than 5 ppm". After these consequently procedures， we identified that all five samples contained the venom of the Naja atra. Our identification method is a systematic and strict example that can provide effective technical support for the forensic identification of snake venom poisoning， as well as for pharmaceutical development toward snake venoms.

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Comparative Analysis of the Proteomic Profile of HaCaT Keratinocytes Using a 1DE Concentrating Gel

Using tandem mass spectrometry with electrospray ionization, a comparative analysis of HaCaT keratinocyte proteins was carried out before and after exposure of cells to sodium dodecyl sulfate (25 mg/ml) for 48 hours; proteins encoded by human chromosome 18 genes were chosen as the comparison proteins. A total of 2418 proteins were detected in the HaCaT immortalized human keratinocytes, 70% of these proteins were identified by two or more unique peptides. Panoramic mass spectrometry analysis identified 38 proteins encoded by chromosome 18 genes, 27 proteins were common to control HaCaT cells and HaCaT cells exposed to SDS. Using the Metascape database (https://metascape.org), an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of HaCaT keratinocytes was performed before and after the SDS exposure. The SDS exposure resulted in a slight enrichment of the GO term "response to stimulus" (GO:0050896) and the related GO term "negative regulation of biological process" (GO:0048519). We found decreased expression levels of membrane proteins encoded by chromosome 18 genes related to cell-cell adhesion (GO:0098609), such as DSC1, DSC3, and DSG1. A decrease in the expression level of desmosomal cadherins is characteristic of malignant neoplasms developing from epithelial tissue cells of various internal organs, mucous membranes, and skin. The method of preparation of HaCaT keratinocyte samples used in this work increased the sensitivity of proteomic analysis of cell culture and made it possible to identify twice as many proteins in one gel strip as compared to the number of proteins (1284) in HaCaT samples subjected to osmotic shock and cleavage by trypsin in solution.

Cross-Linked Gamma Polyglutamic Acid/Human Hair Keratin Electrospun Nanofibrous Scaffolds with Excellent Biocompatibility and Biodegradability.

Recently, human hair keratin has been widely studied and applied in clinical fields due to its good histocompatibility, biocompatibility, and biodegradability. However, the regenerated keratin from human hair cannot be electrospun alone because of its low molecular weight. Herein, gamma polyglutamic acid (γ-PGA) was first selected to fabricate smooth and uniform γ-PGA/keratin composite scaffolds with excellent biocompatibility and biodegradability by electrospinning technology and a chemical cross-linking method in this study. The effect of electrospinning parameters on the structure and morphology, the mechanism of chemical cross-linking, biocompatibility in vitro cell culture experiments, and biodegradability in phosphate-buffered saline buffer solution and trypsin solution of the γ-PGA/keratin electrospun nanofibrous scaffolds (ENS) was studied. The results show that the cross-linked γ-PGA/keratin ENSs had excellent water stability and biodegradability. The γ-PGA/keratin ENSs showed better biocompatibility in promoting cell adhesion and cell growth compared with the γ-PGA ENSs. It is expected that γ-PGA/keratin ENSs will be easily and significantly used in tissue engineering to repair or regenerate materials.

Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide

蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer 2.2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI+模式进行了多反应监测(MRM)定性定量分析。结果显示,特征肽在2.5~30 ng/mL范围内线性关系良好,相关系数(r)大于0.9996,多水平加标回收率范围为95.5%~101.9%,各水平平行测定结果的相对标准偏差(RSD)为1.1%~3.2%,完全能够满足实际样品检测需求。方法简便快捷,灵敏度高,专属性强,可用于矛头蝮蛇蛇毒种属鉴别及svTLE含量测定,从源头保证血凝酶类产品的质量,并可为其他蛇毒类产品的质量控制提供参考。

Preparation of laser microporous porcine acellular dermal matrix and observation of wound transplantation

To prepare a new type of porcine acellular dermis matrix (PADM) with the new laser microporous technique and verify its safety and feasibility. A novel porcine acellular dermis matrix (ADM) was prepared by using sequential combined decellularization of trypsin, neutral protease and SDS solution method and fully rinsed with ultrasonic wave. Specific laser microporous technology was used to prepare the laser micropore porcine acellular dermal matrix (LPADM). SD rats were chose as the animal models and autologous skin was transplanted by one-step method to observe and detect the graft activity, immunogenicity and vascularization degree of the novel PADM. A porcelain white, shiny, soft and elastic dermal matrix was prepared in this study, the results showed low DNA residue and low cytotoxicity. HE staining and SEM observation revealed that the PADM had neither residual cells nor cell fragments, while the collagen bundles were intact and orderly arranged. All the SD rats survived. No infection or skin allergy was found after surgery. None of the animals lost weight. Histological examination showed that the LPADM was fully vascularized with little tissue destruction in the experiment group. Immunohistochemical staining for CD31 showed ideal vascularization in the experiment group, and immunohistochemical staining for TNF-α showed there were no statistical significance of inflammatory reaction in both groups. This study demonstrated that the novel PADM prepared by sequential combined decellularization of trypsin, neutral protease and SDS solution method and new laser microporous technique was effective and safe in animal transplantation.

Enzymatic Digestion of Fresh-Frozen Human Cornea After Riboflavin/Ultraviolet-A Collagen Crosslinking

Objective: Crosslinking (CXL) of the cornea by using riboflavin (RBF)/ultraviolet-A (UVA) has been developed for stiffening the collagenous matrix of the cornea. Collagenases and other metalloproteinases are known to play an important role in the pathogenesis of corneal ulceration. Our aim in this study was to show and compare the morphological and histopathological changes of the CXL human corneas against enzymatic digestion by collagenase, trypsin and pepsin solutions. Materials and Methods: Eighteen corneas from nine fresh frozen cadavers were included in this study. The crosslinked group (n=12) was treated with the combined riboflavin (RBF) and ultraviolet-A (UVA) and the control group (n=6) was only treated with RBF. After treatment corneas were exposed to collagenase, trypsin and pepsin enzymes. For each enzyme group one cornea was evaluated morphologically and the other one was evaluated microscopically. Results: The collagenase treated control group, digestion of the cornea was noted in the first days and completed on the 5th day. The control cornea treated with pepsin was fully digested after 14 days. In the trypsin treated CXL cornea complete digestion was noted after 18 days. In both groups, enzyme activity was observed to be parallel to each other morphologically, but CXL cornea was evaluated to be more resistant to enzymatic digestion. Conclusion: In conclusion, CXL increases resistance of the cornea against enzymatic digestion. In addition to the biomechanical support, this study was also showing the histopathological changes of CXL procedure and the results of enzymatic digestion, supporting new treatment options in the corneal diseases.

Automated-Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies.

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.