Enzymatic Digestion Method Research Articles

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2mg/ml trypsin + 2.5mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15min, 30min, 45min, and 60min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

Identification and anti-oxidative potential of milk fat globule membrane (MFGM)-derived bioactive peptides released through in vitro gastrointestinal digestion

This study investigated the stability of milk fat globule membrane (MFGM) protein under simulated gastrointestinal conditions using an in vitro enzymatic digestion method. The optimal hydrolysis conditions were determined by monitoring the changes in particle size and zeta-potential of MFGM protein hydrolysates over time. Furthermore, the distribution of small molecular weight peptides with antioxidant activity was explored through DEAE-52 combined with in vitro cell experiments. Two novel antioxidant peptides (TGIIT and IITQ) were identified based on molecular docking technology and evaluated their potential scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+) radicals. TGIIT and IITQ also demonstrated remarkable abilities in promoting mitochondrial biogenesis and activating Keap1/Nrf2 signaling pathway, which can effectively counteract skeletal muscle dysfunction induced by oxidative stress. Thus, MFGM-derived antioxidant peptides have the potential to be employed in food to regulate muscle protein metabolism and alleviate sarcopenia.

Isolation, identification and osteogenic capability analysis of mesenchymal stem cells derived from different layers of human maxillary sinus membrane.

To discover the populations of mesenchymal stem cells (MSCs) derived from different layers of human maxillary sinus membrane (hMSM) and evaluate their osteogenic capability. hMSM was isolated into a monolayer using the combined method of physical separation and enzymatic digestion. The localization of MSCs in hMSM was performed by immunohistological staining and other techniques. Lamina propria layer-derived MSCs (LMSCs) and periosteum layer-derived MSCs (PMSCs) from hMSM were expanded using the explant cell culture method and identified by multilineage differentiation assays, colony formation assay, flow cytometry and so on. The biological characteristics of LMSCs and PMSCs were compared using RNA sequencing, reverse transcription and quantitative polymerase chain reaction, immunofluorescence staining, transwell assay, western blotting and so forth. LMSCs and PMSCs from hMSMs were both CD73-, CD90- and CD105-positive, and CD34-, CD45- and HLA-DR-negative. LMSCs and PMSCs were identified as CD171+ /CD90+ and CD171- /CD90+ , respectively. LMSCs displayed stronger proliferation capability than PMSCs, and PMSCs presented stronger osteogenic differentiation capability than LMSCs. Moreover, PMSCs could recruit and promote osteogenic differentiation of LMSCs. This study identified and isolated two different types of MSCs from hMSMs. Both MSCs served as good potential candidates for bone regeneration.

Human amniotic epithelial cells (HAECS); a potential cell type for the hepatic disorders.

To isolate a homogenous population of human amniotic epithelial cells (hAECs) from the amniotic membrane of the human placenta and differentiate them into hepatic-like cells with the help of small molecules. hAECs were isolated by using the enzymatic digestion method and characterized for the presence of specific stem cell markers. In-vitro, hepatic differentiation of hAECs was carried out by using a combination of small molecules. Differentiated cells were observed under a live cell imaging microscope for morphological changes followed by gene and protein expression analysis by qPCR and immunocytochemistry respectively. The isolated hAECs attained characteristic cuboid epithelial shape and express stem cells marker. The hepatic differentiation method was optimized based on soluble chemical compounds supplied in the culture medium. The differentiated hAECs phenotypically acquire hepatic-like cell features and expressed hepatic markers as well as hepatic protein albumin at immature levels. The isolated population of hAECs is highly proliferative. Moreover, hepatic markers expression in the isolated hAECs makes them an exclusive source for the treatment of chronic liver diseases.

Comparison of preparation methods of rat kidney single-cell suspensions

Preparation of kidney tissue single-cell suspensions is the basis of single-cell sequencing, flow cytometry and primary cell culture, but it is difficult to prepare high quality whole kidney single-cell suspensions because of the complex structure of the kidney. We explored a technique called stepwise enzymatic digestion (StE) method for preparing a single-cell suspension of rat whole kidney tissue which contained three main steps. The first step is to cut the kidney into a homogenate. The second step is the digestion of renal tubules using Multi Tissue Dissociation Kit 2 and the last step is the digestion of glomeruli using type IV collagenase. We also compared it with two previous techniques, mechanical grinding method and simple enzymatic digestion method. The StE method had the advantages of high intrinsic glomerular cells and immune cells harvest rate, high singlets rate and high cell viability compared with the other two techniques. In conclusion, the StE method is feasible, highly efficient, and worthy of further research and development.

Unravelling the Complexity of the +33 C>G [HBB:c.-18C>G] Variant in Beta Thalassemia.

The +33 C>G variant [NM_000518.5(HBB):c.-18C>G] in the 5' untranslated region (UTR) of the β-globin gene is described in the literature as both mild and silent, while it causes a phenotype of thalassemia intermedia in the presence of a severe β-thalassemia allele. Despite its potential clinical significance, the determination of its pathogenicity according to established standards requires a greater number of published cases and co-segregation evidence than what is currently available. The present study provides an extensive phenotypic characterization of +33 C>G using 26 heterozygous and 11 compound heterozygous novel cases detected in Cyprus and employs computational predictors (CADD, RegulomeDB) to better understand its impact on clinical severity. Genotype identification of globin gene variants, including α- and δ-thalassemia determinants, and rs7482144 (XmnI) was carried out using Sanger sequencing, gap-PCR, and restriction enzyme digestion methods. The heterozygous state of +33 C>G had a silent phenotype without apparent microcytosis or hypochromia, while compound heterozygosity with a β+ or β0 allele had a spectrum of clinical phenotypes. Awareness of the +33 C>G is required across Mediterranean populations where β-thalassemia is frequent, particularly in Cyprus, with significant relevance in population screening and fetal diagnostic applications.

[Study on injectable chitosan hydrogel with tendon-derived stem cells for enhancing rotator cuff tendon-to-bone healing].

To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05). TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.

Isolation, identification, and induced differentiation of satellite cells from skeletal muscle of adult tree shrews.

A method for the in vitro isolation, purification, identification, and induced differentiation of satellite cells from adult tree shrew skeletal muscle was established. The mixed enzyme digestion method and differential adhesion method were used to obtain skeletal muscle satellite cells, which were identified and induced to differentiate to verify their pluripotency. The use of a mixture of collagenase II, hyaluronidase IV, and DNase I is an efficient method for isolating adult tree shrew skeletal muscle satellite cells. The P3 generation of cells had good morphology, rapid proliferation, high viability, and an "S"-shaped growth curve. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining indicated that marker genes or proteins were expressed in skeletal muscle satellite cells. After myogenic differentiation was induced, multiple-nucleated myotubes were observed, and the MyHC protein was expressed. The expression of myogenic marker genes changed with the differentiation process. After the induction of adipogenic differentiation, orange-red lipid droplets were observed, and the expression of adipogenic marker genes increased gradually with the differentiation process. In summary, satellite cells from adult tree shrew skeletal muscle were successfully isolated using a mixed enzyme digestion method, and their potential for differentiation into myogenic and adipogenic cells was confirmed, laying a foundation for further in vitro study of tree shrew muscle damage.

Fifty-year study of microplastics ingested by brachyuran and fish larvae in the central English North Sea

Microplastics (MPs) are ubiquitous pollutants in marine environments. Among the many detrimental consequences of microplastic pollution, its consumption by marine biota is of particular relevance for human health, due to exposure through the food web. Long-term time-series biotic samples are overlooked sources of information for microplastics research. These collections are extremely valuable for the detection and monitoring of changes in marine environments. However, there are very few long-term studies (>10 years) of the uptake of microplastics by biota. Here, we used Dove Time Series planktonic samples (from 1971 to 2020) to assess the presence and prevalence of microplastics in the English North Sea coast over time. Fish and brachyuran larvae were selected due to their commercial importance and consequent implications for human health. A custom enzymatic digestion method was used to extract microplastics for FTIR-ATR polymer identification. An increasing cumulative trend in MP ingestion was identified. Cellophane and polyethylene terephthalate were the polymer types found most frequently in both taxa. Although a total higher microplastics uptake was observed in fish, consumption was not significantly different between taxa over time. Equally, results were not clearly related to microplastics shape or polymer type. This work did not find significant long-term evidence on the increasing uptake of microplastic particles by zooplankton over time. However, the results of this report identified additives, plasticisers, and other more complex and hazardous compounds that should not be released to the environment (e.g., bis-(2-hydroxyethyl) dimerate, propylene glycol ricinoleate) inside marine biota. The study detailed herein provides a case study for the use of long-term time-series in providing accurate assessments of microplastic pollution in marine biota.

Effect of electroacupuncture at "Jiaji" (EX-B2) on senescence of nucleus pulposus cells of degenerated lumbar intervertebral disc in rabbits.

To observe the effect of electroacupuncture (EA) at "Jiaji"(EX-B2) on body mass, motor function, expression of caveolin-1 (Cav-1) in nucleus pulposus cells and annulus fibrosus tissue, telomerase activi-ty, relative telomere length and different cell cycle ratio of nucleus pulposus cells in rabbits with intervertebral disc degeneration(IVDD), so as to investigate its mechanism underlying delaying senescence of the degenerated lumbar intervertebral disc nucleus pulposus cells. Twenty-five male New Zealand rabbits with mature bones were divided into control, sham operation, model, EA, and acupuncture groups, with 5 rabbits in each group. The IVDD model was established by inserting kirschner wires to the vertebral bone surface between the lumbar (L)4 and L5 vertebrae, followed by applying continuous axial pressure for 28 d. EA (2 Hz/15 Hz, 1-2 mA) or acupuncture (only insertion of acupuncture needles into bilateral EX-B2, but without electrical stimulation) was applied to bilateral EX-B2 for 20 min, once daily, 6 times a week for 4 weeks. The hindlimb locomotor function (locomotor score) was assessed by using Faden's and colleagues' methods. The general conditions of rabbits in each group were observed, and their body weight changes were measured every week. Nucleus pulposus cells were isolated using enzyme digestion method. After the treatment, the Cav-1 positive cell counts in nucleus pulposus cells and annulus fibrosus tissues were detected by immunohistochemistry, and the telomerase activity of nucleus pulposus cells was detected by PCR-ELISA. The relative telomere length of nucleus pulposus cells was measured by real-time quantitative polymerase chain reaction (real-time qPCR), and the cell cycle of nucleus pulposus was detected by flow cytometry. Compared with the sham operation group, the body mass from 4 to 11 week, locomotor score at 4, 7 and 11 week, telomerase activity, relative telomere length and the proportion of cells in G2/M phase of nucleus pulposus cells were significantly decreased (P<0.01), while Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue, and the proportion of nucleus pulposus cells in the G0/G1 phase considerably increased (P<0.01) in the model group. Relevant to the model group, the EA group rather than the acupuncture group had an increase in the body mass from 8 to 11 week, locomotor score at 11 week, telomerase activity, relative telomere length of nucleus pulposus cells, and the proportion of nucleus pulposus cells in G2/M phase (P<0.01), and a decrease in the Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue and the proportion of cells in G0/G1 phase (P<0.01). No significant differences were found between the model and acupuncture groups in all the indexes mentioned above. EA at EX-B2 has a bene-ficial effect in improving motor function in rabbits with IVDD, which may be related to its functions in reducing the expression of Cav-1 in nucleus pulposus cells and annulus fibrosus, improving cycle arrest, enhancing the telomerase activity and the relative telomere length of nucleus pulposus cells, delaying the senescence of nucleus pulposus cells of the degenerated lumbar intervertebral discs.

A Novel Method for Human Adipose-Derived Stem Cell Isolation and Cryopreservation.

Adipose-derived stem cells (ADSCs) are isolated from abundant adipose tissue and have the capacity to differentiate into multiple cell lineages. ADSCs have raised big interest in therapeutic applications in regenerative medicine and demonstrated to fulfill the criteria for a successful cell therapy. There are several methods for isolation of ADSCs from adipose tissue and cryopreservation of ADSCs. Here, novel methods for the isolation and cryopreservation of ADSCs are presented and focused. Microscopic pieces of adipose tissue were placed on transwell inserts, and the ADSCs were induced to migrate to the lower wells for 1 week. We compared the properties of our ADSCs with those isolated by enzymatic digestion and enzyme-free method of culture plate, and our ADSCs were found to be more stable and healthier. In addition, we proposed a novel cryoprotectant solution (FNCP) containing pectin and L-alanine, which was compared with standard cryoprotectant solution. Overall, our methods proved more useful for ADSCs isolation than other methods and did not require consideration of "minimal manipulation" by the U.S. Food and Drug Administration (FDA). Furthermore, our FNCP did not contain dimethyl sulfoxide and fetal bovine serum, therefore stable storage is possible in xeno-free and animal-free cryopreservation solutions.

Exploring the impact of culture techniques and patient demographics on the success rate of primary culture of human periodontal ligament stem cells

Background/purposePeriodontal ligament stem cells (PDLSCs) have the potential for regenerating periodontal tissue. The study aims to investigate the impact of demographics (ages, gender, disease) and culture techniques (shipping storage time and culture method) on the success of primary culture. Materials and methodsPDLSCs were collected from 51 teeth of 26 patients and cultured via outgrowth (OG) and enzymatic digestion (ED) methods. Cells characteristics were confirmed by flow cytometry, MTT, and ARS. The primary culture success rate was evaluated with a serial chi-square test to determine the relationship with culture technique (ED/OG and ≤4 h/prolonged culture) and patient demographics (Young/Old, Female/Male, and Health/Periodontitis). ResultsThe overall success rate of Health group (69.7%) was higher than Periodontitis (38.9%). Culturing within 4 h possessed a higher success rate (71.8%) than prolonged group (16.7%) regardless of patient demographics, and using OG method (81.5%) revealed more promising. Subgroup analysis of 39 cases (culture within 4 h) found that the success rate of OG was higher than ED in the Old group (87.5%–25.0%) and in the Periodontitis group (83.3%–25.0%). ConclusionPrimary culturing of PDLSCs within 4 h and using the outgrowth method led to higher success rates regardless of patient demographics. It can achieve successful PDLSCs culture of older patients or patients with periodontal disease by appropriate culture technique.

DEVELOPMENT OF A METHOD FOR PRODUCING FAB/F(AB’)2 FRAGMENTS FROM A FULL-LENGTH MONOCLONAL ANTIBODY FOR BIOANALYTICAL ASSAYS

Abstract Background Monoclonal antibodies (mAb) comprise of two Fab fragments and one Fc fragment or one F(ab)’2 fragment and one Fc fragment. While a full-length mAb is frequently used as an assay reagent for bioanalysis, mAb fragments are required in certain cases. For example, to build a sandwich assay for detection of anti-drug antibodies (ADA) for therapeutic antibodies, Fab or F(ab)’2 fragment is used instead of a full-length mAb as capture reagent. This is because therapeutic antibodies, either humanized or fully human, are in many ways indistinguishable from the ADA generated in patients, especially in Fc fragment. When ADA detection methods utilizes anti-human Fc antibodies as the detection reagent, the full-length mAb drug will be directly bound by the detection reagent, causing interference. Preparation of a Fab or F(ab)’2 fragment is therefore needed. Methods and results We are developing a method for enzymatic digestion of therapeutic antibodies to generate monovalent Fab or bivalent F(ab’)2 fragments in this study. With such reagents becoming available, a sandwich ADA assay formats can be expanded to allow anti-human Fc antibodies as detection regents. To standardize the method, we explored various enzymatic conditions, including type of enzymes (i.e., pepsin, papain, and IdeS Protease), digestion-time (1, 2, 4, and 6 h), enzyme to antibody ratio (1:10, 1:20, and 1:40 w/w), IgG species and isotypes (human IgG1-κ, IgG1-λ, and IgG4-κ). The enzymatic hydrolysates were quantified by NanoDrop and purified by dialysis (10K MWCO) and Protein A/G/L magnetic bead methods. The effective recovery of truncated antibodies was &amp;gt; 90%, as assessed by reduced/non-reduced SDS-PAGE and ELISA analysis. Digestion of human IgG1 and IgG4 with pepsin resulted into a complete cleavage into F(ab')2 fragments and degradation of Fc fragments. While IdeS Protease produced an equivalent quantity of F(ab’)2 and Fc fragments with a similar efficiency, removal of the intact Fc fragment was required as an additional step. If the Fab fragments were desired, papain could be used with yield being over 90%. We have subsequently utilized either Fab or F(ab’)2 as a capture reagent for ADA detection. Conclusion We have successfully developed the enzymatic digestion method to prepare Fab or F(ab’)2 fragments. The optimized conditions described here are broadly applicable to different IgG isotypes across many therapeutic antibodies.

Determination of thyroid hormones in lake sturgeon (Acipenser fulvescens) tissues at different developmental stages using a sensitive liquid chromatography-mass spectrometry method

Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid–liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10–50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3−111.2 %) and precision (0.1−4.9 %), matrix effect (75.4−134.1 %), and recovery (41.2−69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.

Human umbilical cord-derived мesenchymal stromal cells mitigate lipopolysaccharide-induced liver injury in rats

Acute Respiratory Distress Syndrome (ARDS) is a severe clinical condition that can cause multi-organ dysfunction, including liver injury. Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) have been shown to possess therapeutic potential for a variety of diseases due to their ability to differentiate into various cell types and their anti-inflammatory and immunomodulatory properties. Purpose: To investigate the potential of hUC-MSCs for treating lipopolysaccharide (LPS)-induced liver injury in rats. Materials and methods: 72 mature male Wistar rats were randomly assigned to nine groups: control, 3 days, 7 days, and 28 days after intranasal administration of LPS, 24 hours of LPS and 2 days of hUC-MSCs, 4 days of LPS and 3 days of hUC-MSCs, 14 days of LPS and 14 days of hUC-MSCs, 21 days of LPS and 7 days of hUC-MSCs injection, and control 3 days after hUC-MSCs injection. The isolation of MSCs from human umbilical cord tissue was performed using an enzymatic digestion method with collagenase I. hUC-MSCs were injected intraperitoneally at a dose of 1∙106 cells/kg body weight. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were measured using the kinetic method. The levels of hepatocellular necrosis, liver structural damage, hepatocyte vacuolation, inflammation and disseminated intravascular coagulation (DIC) were analyzed by histological scoring of sections stained with hematoxylin and eosin. The expression of TGF-β1 in the liver tissue was evaluated by immunohistochemistry. Results: The preclinical study demonstrated that treatment with hUC-MSCs significantly improved liver function and attenuated LPS-induced liver injury in rats. This was evidenced by a reduction in hepatocellular necrosis, liver structural damage, hepatocyte vacuolation, inflammation, signs of DIC, fibrosis and lower levels of serum liver markers ALT, AST and ALP in the hUC-MSCs-treated groups compared to the untreated groups. The study also revealed that the use of hUC-MSCs was more effective at the earlier stage of liver injury. Conclusions: Our findings suggest that hUC-MSCs therapy may hold promise as a potential treatment for LPS-induced liver injury. Further research is needed to better understand the underlying mechanisms and to determine the potential for hUC-MSCs therapy in clinical practice.

Biopanning, Heterologous Expression, and Characterization of a Shark-Derived Single-Domain Antibody Fusion Protein against Pancreatic Lipase.

Nowadays, obesity severely impacts human health and is the fifth leading risk factor that leads to death globally. Pancreatic lipase (PL) inhibitors have attracted extensive attention owing to their role in effective prevention and treatment of obesity. Here, a shark-derived single-domain antibody fusion protein was used to inhibit PL for the first time. After biopanning, the heterologous expression system pET28a-SUMO-D2 was constructed using the method of double restriction enzyme digestion and T4 ligase to achieve the soluble expression of the PL-specific antibody gene D2. According to the calculation of protein concentration, the final expression of fusion protein PL-D2S was 1.183 mg per liter of Luria Bertani broth. The binding ability of the soluble fusion protein PL-D2S to PL was identified. Enzyme-linked immunosorbent assay results showed that the fusion protein PL-D2S exhibited a strong binding affinity to PL. The experimental results of PL inhibition of PL-D2S in vitro showed that the fusion protein could significantly inhibit the activity of PL, with an IC50 of 404 μg/mL. Our study shows that the fusion protein PL-D2S is a potential PL inhibitor to prevent and treat obesity.

A Protocol for Large-Scale Isolation of Rat Primary Osteoblasts Suitable for Exosome Extraction

Objective: This study aims to introduce an optimized method for the large-scale isolation of rat primary osteoblasts, suitable for exosome extraction. Methods: An advancement on the traditional secondary enzyme digestion method, manual bone grinding to clean connective tissue and ophthalmic cutting into fragments have been replaced with oscillating vortex rinsing and a 14 cm straight head shear in a centrifuge tube to cut bone fragments, significantly reducing the experimental time. The experiment was conducted on forty suckling rats. The experimental time of both the optimized and traditional methods were compared. The osteoblasts obtained through the optimized method were observed morphologically, identified by Alkaline phosphatase and alizarin red staining, and their proliferation activity was detected using the EdU method. Sufficient cell supernatant was collected to extract osteoblast-derived exosomes, identified by employing transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Result: The total experimental time of the improved method (5.5±0.17 hours) was significantly shorter than the traditional method (7.25±0.23 hours) with a notable statistical difference (P &lt;0.001); the osteoblasts obtained by the improved method displayed good growth status. On the seventh day, the cells matured and exhibited positive ALP staining. By the 21st day, mineralized nodules were formed, and alizarin red staining was positive. The EdU results showed that osteoblasts demonstrated healthy growth at both 24 and 48 hours, with a proliferation rate of (96.2%±1.3%) at 48 hours, slightly higher than that of (94.6%±2.8%) at 24 hours, with no significant statistical difference (P &gt;0.05); Western blot showed positive results for CD9, CD63, and TSG101, and negative results for Calnexin. Conclusion: The optimized primary osteoblast isolation method can significantly improve experimental efficiency and is suitable for extracting osteoblast-derived exosomes.

Scanning electrochemical microscopy for the stimulation and investigation of human skeletal muscle-derived mesenchymal stem/stromal cells

IntroductionThe human skeletal muscle-derived mesenchymal stem/stromal cells (SM-MSCs) possess myogenic differentiation potential and participate in the muscle regeneration process. However, the question of how to improve human skeletal muscle regeneration remains actual. In this study, the total SM-MSCs population was separated into subpopulations according to the neural cell adhesion molecule (NCAM, CD56), stimulated with an alternating electric field (AC) using scanning electrochemical microscopy (AC-SECM) and the intracellular redox changes, and myogenic differentiation markers were evaluated. MethodsThe total SM-MSC population was isolated from the postoperative human skeletal muscle biopsies by an enzymatic digestion method. The myogenic differentiation of the total SM-MSCs population before and after AC stimulation was evaluated immunohistochemically by the levels of desmin and myogenin. The effect of AC stimulation on the redox capacity of CD56(+) and CD56(-) cell subpopulations as well as on the level of myogenin on indium tin oxide (ITO) surface were also investigated. ResultsThe total SM-MSCs population grown on a glass coverslip weakly responded to AC stimulus, i.e. the level of desmin was slightly increased by the 3rd day of differentiation, while in the not stimulated cells, it increased only by the 7th day. The level of myogenin did not change after AC stimulation. However, the CD56(+) and CD56(-) subpopulations had different redox activities and myogenic differentiation potential: the CD56(+) cells had stronger natural diffusion and were more redox active compared to the CD56(-) cells; the redox activity of CD56(+) cells was more actively stimulated by an alternating electric field than in CD56(-) cells; at control level, the CD56(+) cells had more myogenic differentiation-regulating transcription factor myogenin, which was more intensively stimulated by AC than in CD56(-) cells. Data show that the total population of human SM-MSCs is heterogeneous with different regenerating potential cells that do not equally respond to extracellular stimuli. The SECM can be used in both ways: (i) for extracellular stimulation and (ii) for the investigation of intracellular redox changes of the human SM-MSCs or their subpopulations allowing a deeper understanding of the mechanisms mediating skeletal tissue regeneration both in vitro and in vivo.

A rapid and convenient enzyme digestion method for the analysis of N-glycans using exoglycosidase-impregnated polyacrylamide gels fabricated in an automatic pipette tip.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.

Tracking soluble and nanoparticulated titanium released in vivo from metal dental implant debris using (single-particle)-ICP-MS

BackgroundThis work studies the presence of the Ti, Al and V metal ions and Ti nanoparticles released from the debris produced by the implantoplasty, a surgical procedure used in the clinic, in rat organs. MethodsThe sample preparation for total Ti determination was carefully optimized using microsampling inserts to minimize the dilution during the acid attack of the lyophilized tissues by a microwave-assisted acid digestion method. An enzymatic digestion method was optimized and applied to the different tissue samples in order to extract the titanium nanoparticles for the single-particle ICP-MS analysis. ResultsA statistically significant increase was found for Ti concentrations from control to experimental groups for several of the studied tissues, being and particularly significant in the case of brain and spleen. Al and V concentrations were detected in all tissues but they were not different when comparing control and experimental animals, except for V in brain. The possible presence of Ti-containing nanoparticles mobilized from the implantoplasty debris was tested using enzymatic digestions and SP-ICP-MS. The presence of Ti-containing nanoparticles was observed in all the analyzed tissues, however, differences on the Ti mass per particle were found between the blanks and the digested tissue and between control and experimental animals in some organs. ConclusionThe developed methodologies, both for ionic and nanoparticulated metal contents in rat organs, have shown the possible increase in the levels of Ti both as ions and nanoparticles in rats subjected to implantoplasty.