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Uterine adenomyosis causing attention to the abnormal smooth muscle layer has recently received increasing attention, which has created the need for a method that can efficiently collect high purity uterine smooth muscle cells (USMC) in a laboratory setting. In this study, we explored the composition ratios of the digestion solution to obtain the optimal digestion solution (DM4). Furthermore, we tested the superiority of the two methods of obtaining adenomyotic uterine smooth muscle by comparing DM4 with the conventional tissue adhesion method by growth rate, single-cell RNA sequencing, and purity of fluorescence identification. The results demonstrated that USMCs produced by the DM4 digestion method exhibited significantly higher rates of proliferation and were more effective in generating mature smooth muscle cells of high purity compared to those obtained using tissue adherence methods, which is more inclined to isolate the progenitor cell population. This study presents a reliable method for isolating USMCs and provides a solid foundation for future studies on the etiology and mechanism of adenomyosis. • A novel dual-enzyme digestion method enhances USMC purity and enhances cell proliferation • The DM4 digestion yields high-purity mature USMCs, while tissue adhesion favors progenitor cells. • CCDC80+ cells undergo a transition from tissue adherence to become fibroblast-like cells that express COLIII. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an in vitro two-step method to simulate the digestive process of donkeys and evaluate the nutritional value of 8 protein feeds using in vivo nylon bag and in vitro two-step methods. Three Xinjiang donkeys of similar weight and age were selected. In vivo and in vitro methods were employed to study dry matter degradation rates and fermentation parameters of the 8 protein feeds. The results showed that: ① comparison between in vivo and in vitro studies revealed that the dry matter degradation rates of fish meal, cottonseed meal, and sunflower meal were significantly higher in the in vitro method than in the in vivo method (P<0.05). ② In enzymatic digestion in vitro, the dry matter degradation rates of fish meal and soybean meal were significantly higher than the other 6 protein feeds (P<0.05). In microbial fermentation in vitro, blood meal and sunflower meal had significantly higher dry matter degradation rates than the other 6 protein feeds (P<0.05). ③ For gas production during fermentation, soybean meal had significantly higher total gas production than the other 7 protein feeds (P<0.05). ④ In vitro fermentation parameters showed that rapeseed meal and meat and bone meal had significantly higher pH values than the other 6 protein feeds (P<0.05). Ammonia nitrogen content was significantly higher in fish meal and blood meal than in the other 6 protein feeds (P<0.05). Acetic acid content in sunflower meal was significantly higher than in the other 7 protein feeds (P<0.05). In conclusion, using enzymatic digestion and microbial inoculation of donkey feces to simulate the digestive process of donkeys can reflect the real situation of protein feed degradation and digestion in donkeys. There are differences among different protein feeds, with soybean meal, blood meal, and fish meal showing higher dry matter degradation rates compared to other protein feeds. [ABSTRACT FROM AUTHOR]

Thailand is globally recognized for its rich biodiversity, a characteristic that extends to the diverse species of indigenous yams within the Dioscorea spp. genus. With the energy crisis, the selection of agricultural materials for ethanol production to replace the use of economic crops is therefore necessary. This research focuses on six specific yam varieties: Amorphophallus konjac, Colocasia esculenta, Dioscorea bulbifera, Dioscorea alata, Dioscorea hispida, and Dioscorea esculenta. The primary objective is to investigate their bioethanol productivity through the implementation of a separate hydrolysis and fermentation process, with the broader aim of contributing to the potential preservation of these invaluable indigenous yam species. Initiating the study, the experimental samples underwent hydrolysis by alpha-amylase enzyme (1% w/winitial starch) for 180 min, followed by glucoamylase enzyme (1% w/winitial starch) for an additional 72 h. Post the initial hydrolysis, the detection of small molecule sugars by high-performance liquid chromatography, revealed a range of 14.49 ± 1.10 to 28.89 ± 0.03 gtotal sugar/L, with maltose emerging as the predominant sugar compound. Subsequent to the second hydrolysis, it was observed that maltose and starch residues across all six samples underwent successful digestion. The peak glucose productivity was attained at the 12-h mark post-glucoamylase hydrolysis. Among the diverse yam varieties under examination, Dioscorea hispida exhibited the highest glucose production, showcasing a hydrolysis efficiency of 62.53 ± 2.08%. Following the fermentation process with S. cerevisiae, the optimal fermentation time was identified as 72 h, at which point all available glucose in the hydrolysate was fully utilized. The bioethanol productivity spanned from 32.31 ± 3.40 to 43.66 ± 0.02 g/L, with Dioscorea hispida demonstrating the highest ethanol productivity at 0.61 ± 0.00 g/L/h, and a yield of 0.42 ± 0.01gEtOH/gGlucose. These findings underscore the potential of Dioscorea hispida as a promising contributor to bioethanol production. [ABSTRACT FROM AUTHOR]

ObjectiveTo apply high performance liquid chromatography-inductively coupled plasma mass spectrometry （HPLC-ICP/MS） to the analysis of five forms of selenium ［Se（Ⅵ）， Se（Ⅳ）， SeCys， MeSeCys， and SeMet］ in livestock meat.MethodsThe selenium species were extracted using the ultrasonic-assisted enzyme method， with C18 reversed-phase column （250 mm×4.6 mm， 5 μm） as the analytical column， 10 mmol/L citric acid and 5 mmol/L sodium hexane sulfonate （pH=4.0， containing 1% methanol） as the mobile phase， and an injection volume of 20 μL.ResultsFour selenium forms were separated in 7 min， with correlation coefficients greater than 0.999， indicating good linearity. The detection limits of Se（Ⅵ）， SeCys， MeSeCys， and SeMet ranged from 0.000 3-0.002 mg/kg. The spiked recoveries of selenium-enriched pork at three concentrations （low， medium， and high） ranged from 80.0% to 103.3%， with a relative standard deviation of less than 6.8%. Certified Reference Material ERM® BC210 revealed values within the standard range.ConclusionThe developed method was suitable for determining Se（Ⅵ） SeCys， MeSeCys， and SeMet in livestock meat. The meat was found to mainly contain SeMet and small amounts of MeSeCys and SeCys.

It is reported herein a new approach to study the orientation and density of mouse antibody grafting on magnetic beads, serving for immunoassays and biosensors with fluorescent detection of biomolecules. This approach is based on selective enzymatic digestion of target grafted antibodies at a specific site below the hinge position to provide F(ab′)2 and Fc fragments, followed by separation and determination of these fragments with size exclusion chromatography (SEC) coupled with fluorescence detection (FLD). The developed method was applied for evaluation of immunoglobulin (IgG2a) grafting capacity on three different biofunctionalized magnetic beads (i.e., Tosyl-activated, carboxylic, protein G). Tosyl-activated and protein G beads at different optimal grafting IgG: bead ratios (i.e., 110 µg: 1000 µg and 240 µg: 1000 µg, respectively) exhibited superior grafting capacity than carboxylic counterparts. Under the optimized conditions, more than 70 % of antibodies were grafted on tosyl-activated and protein G beads in the right orientation. This approach was then demonstrated with different commercially available antibodies specific to amyloid-beta peptide 1–42 (Aβ1–42) for magneto-immunoassays and fluorescent detection of this peptide that is an established biomarker for molecular diagnosis of Alzheimer's disease.

Background: Early food introduction induces tolerance, but epicutaneous exposure, especially via eczema lesions, promotes IgE sensitization. Aiming for safe and effective primary prevention of egg allergy, we examined several protease-digested egg-white (EW) products for three properties: 1) induction of oral tolerance that prevents IgE sensitization, 2) weak IgE binding that can prevent allergic reactions even in IgE-sensitized mice, and 3) minimal epicutaneous IgE sensitization even when in contact with inflamed skin. Methods: Heated EW was digested with several proteases under optimal conditions. First, three-week-old BALB/c female mice were intragastrically administered EW or each protease-digested EW product, followed by intraperitoneal ovalbumin (OVA) or ovomucoid (OVM) injection with alum. Serum OVA- and OVM-specific IgE titers were measured. Second, six-week-old mice were sensitized with OVA/OVM, and the rectal temperature was measured after intraperitoneal administration of EW or each protease-digested EW. Third, EW or each protease-digested EW product was applied to the tape-stripped skin for 3 days/week for 3 weeks. Serum OVA- and OVM-specific IgE titers were measured. Results: Orally administered pepsin-digested EW product (PDEW) and Thermoase PC10F-digested EW product (TDEW) significantly suppressed OVA-/OVM-specific IgE production. Neither product elicited a body temperature decline (anaphylaxis) in OVA-/OVM-sensitized mice. Serum OVA-/OVM-specific IgE levels were significantly lower in mice epicutaneously exposed to PDEW or TDEW than in EW-exposed mice. Conclusions: Two protease-digested EWs showed potential as optimal EW products for early introduction for primary prevention of egg allergy.

Hericium erinaceus, a mushroom species commonly known as Yamabushitake in Japan, is known to have a stimulatory effect on neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Hericenone C, a meroterpenoid with palmitic acid as the fatty acid side chain, is reported to be one such stimulant. However, according to the structure of the compound, the fatty acid side chain seems highly susceptible to lipase decomposition, under in vivo metabolic conditions. To study this phenomenon, hericenone C from the ethanol extract of the fruiting body was subjected to lipase enzyme treatment and observed for changes in the chemical structure. The compound formed after the lipase enzyme digestion was isolated and identified using LC-QTOF-MS combined with 1H-NMR analysis. It was found to be a derivative of hericenone C without its fatty acid side chain and was named deacylhericenone. Interestingly, a comparative investigation of the neuroprotective properties of hericenone C and deacylhericenone showed that the BDNF mRNA expression in human astrocytoma cells (1321N1) and the protection against H2O2-induced oxidative stress was considerably higher in the case of deacylhericenone. These findings suggest that the stronger bioactive form of the hericenone C compound is in fact deacylhericenone. [ABSTRACT FROM AUTHOR]

The latest research progress on separation technology (density separation, elutriation and oil extraction), digestion technology (acid digestion, alkali digestion, oxidation digestion, enzyme digestion and UTS method) and detection technology(visual inspection, spectroscopic analysis and thermal analysis) of microplastics are systematically introduced, and the advantages and disadvantages are compared and analyzed ・ However, the selection of different separation, digestion and detection technologies will lead to differences in the abundance, chemical information and morphology of microplastics・ It is difficult to compare different research results, which affects the research on the environmental behavior, pollution status and temporal and spatial distribution of microplastics・ Therefore, it is proposed that a unified and standardized microplastics pre treatment and detection technology should be established in the future ・ And more attention should be paid to the combined application of image analysis and traditional detection methods・ Meanwhile, machine learning will provide a faster and more accurate detection and classification technology for the research of microplastics. [ABSTRACT FROM AUTHOR]

Several food groups have been reported to contain varying concentrations of plastics. This study was designed to quantitatively investigate for the first time in Australia the presence of plastics in store-bought chicken eggs. Three commonly consumed brands of free-range, free-range organic, barn-laid and backyard (home-laid) chicken egg samples were analyzed for seven common polymers (i.e., polypropylene, polyethylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, poly-(methylmethacrylate) and polycarbonate). Samples were extracted by enzyme digestion and pressurized liquid extraction, followed by quantitative analysis through double-shot microfurnace pyrolysis coupled to gas chromatography-mass spectrometry. No plastics were detected at concentrations > limit of detection (LOD) (from 0.04 μg/g for PS to 0.22 μg/g for PVC) in the egg samples analyzed, regardless of brand and category, suggesting limited exposure of Australians to plastics from consuming eggs This study provides valuable baseline data and underscores the importance of continued monitoring to ensure the safety and integrity of food supplies in the face of rising environmental plastic pollution. [Display omitted] • Plastic contamination in Australian chicken eggs were analyzed. • Concentrations of PP, PS, PET, PVC, PMMA, PC and PE quantified by Pyr-GC/MS. • No plastics were detected at concentrations > LOD in any of the sampled eggs. • Investigating eggs from different regions and other poultry birds can be useful. [ABSTRACT FROM AUTHOR]

Early food introduction induces tolerance, but epicutaneous exposure, especially via eczema lesions, promotes IgE sensitization. Aiming for safe and effective primary prevention of egg allergy, we examined several protease-digested egg-white (EW) products for three properties: 1) induction of oral tolerance that prevents IgE sensitization, 2) weak IgE binding that can prevent allergic reactions even in IgE-sensitized mice, and 3) minimal epicutaneous IgE sensitization even when in contact with inflamed skin. Heated EW was digested with several proteases under optimal conditions. First, three-week-old BALB/c female mice were intragastrically administered EW or each protease-digested EW product, followed by intraperitoneal ovalbumin (OVA) or ovomucoid (OVM) injection with alum. Serum OVA- and OVM-specific IgE titers were measured. Second, six-week-old mice were sensitized with OVA/OVM, and the rectal temperature was measured after intraperitoneal administration of EW or each protease-digested EW. Third, EW or each protease-digested EW product was applied to the tape-stripped skin for 3 days/week for 3 weeks. Serum OVA- and OVM-specific IgE titers were measured. Orally administered pepsin-digested EW product (PDEW) and Thermoase PC10F-digested EW product (TDEW) significantly suppressed OVA-/OVM-specific IgE production. Neither product elicited a body temperature decline (anaphylaxis) in OVA-/OVM-sensitized mice. Serum OVA-/OVM-specific IgE levels were significantly lower in mice epicutaneously exposed to PDEW or TDEW than in EW-exposed mice. Two protease-digested EWs showed potential as optimal EW products for early introduction for primary prevention of egg allergy. [ABSTRACT FROM AUTHOR]

Hericium erinaceus, a mushroom species commonly known as Yamabushitake in Japan, is known to have a stimulatory effect on neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Hericenone C, a meroterpenoid with palmitic acid as the fatty acid side chain, is reported to be one such stimulant. However, according to the structure of the compound, the fatty acid side chain seems highly susceptible to lipase decomposition, under in vivo metabolic conditions. To study this phenomenon, hericenone C from the ethanol extract of the fruiting body was subjected to lipase enzyme treatment and observed for changes in the chemical structure. The compound formed after the lipase enzyme digestion was isolated and identified using LC-QTOF-MS combined with 1H-NMR analysis. It was found to be a derivative of hericenone C without its fatty acid side chain and was named deacylhericenone. Interestingly, a comparative investigation of the neuroprotective properties of hericenone C and deacylhericenone showed that the BDNF mRNA expression in human astrocytoma cells (1321N1) and the protection against H2O2-induced oxidative stress was considerably higher in the case of deacylhericenone. These findings suggest that the stronger bioactive form of the hericenone C compound is in fact deacylhericenone.

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ABSTRACT We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I “F gene” class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

BACKGROUND: Common methods for obtaining chondrocytes include mechanical-enzymatic digestion, sequential digestion of pronase and collagenase, sequential digestion of trypsin and type II collagenase, and simple type II collagenase digestion. OBJECTIVE: To investigate the isolation, extraction and identification of knee joint chondrocytes from Sprague-Dawley rats, and to observe the morphological characteristics of chondrocytes from primary to passages 3. METHODS: We used single-step digestive approach (type II collagenase digestion) to isolate and obtain chondrocytes from the knee joints of SpragueDawley rats in vitro, and then established a culture system in vitro. Cell proliferation assay and inverted phase contrast microscope were used to observe morphohistology of the cells. Toluidine blue staining, type II collagen as well as proteoglycan immunofluorescence staining were used to identify the characteristics of chondrocytes. The experimental protocol was approved by the Ethics Committee of Zhongshan Hospital of Dalian University. RESULTS AND CONCLUSION: Microscopic observation and cell proliferation assay both showed satisfactory cell morphology and proliferation at passages 2-3, and the capacity of the cells decreased gradually. Moreover, toluidine blue staining, type II collagen and proteoglycan immunofluorescence staining revealed superior characteristics of passage 3 chondrocytes cultured in vitro. These findings indicate that chondrocytes can successfully be isolated by the singlestep type II collagenase digestion. Cell proliferation and passage cultivation can also be achieved. And dedifferentiation capacity of chondrocytes is improved gradually after the passages 3 cultured in vitro. Passage 3 cells cultured in vitro have superior characteristics for the use in experimental researches. [ABSTRACT FROM AUTHOR]

Fish gelatin and its hydrolysates exhibit a variety of biological characteristics, which include antihypertensive and antioxidant properties. In this study, fish gelatins were extracted from extrusion-pretreated tilapia scales, and then subjected to analyses to determine the physicochemical properties and antioxidant activity of the extracted gelatins. Our findings indicate that TSG2 (preconditioned with 1.26% citric acid) possessed the greatest extraction yield, as well as higher antioxidant activities compared with the other extracted gelatins. Hence, TSG2 was subjected to further hydrolyzation using different proteases and ultrafiltration conditions, which yielded four gelatin hydrolysates: TSGH1, TSGH2, TSGH3, and TSGH4. The results showed that TSGH4 (Pepsin + Pancreatin and ultrafiltration < 3000 Da) had a higher yield and greater antioxidant activity in comparison with the other gelatin hydrolysates. As such, TSGH4 was subjected to further fractionation using a Superdex peptide column and two-stage reverse-phase column HPLC chromatography, yielding a subfraction TSGH4-6-2-b, which possessed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the other fractions. Further LC-ESI/MS/MS analysis of TSGH4-6-2-b suggested two novel peptides (GYDEY and EPGKSGEQGAPGEAGAP), which could have potential as naturally-occurring peptides with antioxidant properties. These promising results suggest that these antioxidant peptides could have applications in food products, nutraceuticals, and cosmetics.

Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes. [ABSTRACT FROM AUTHOR]

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05–1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from −3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples. [Display omitted] • The LC-MS/MS assay does not require specific antibody reagents and is a powerful tool for quantification of HSA. • The method is selective enough to discriminate co-eluting peptides. • The LC-MS/MS assay is a viable option for a reference method to quantify HSA. [ABSTRACT FROM AUTHOR]

Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91–95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing (scRNA-seq) is a powerful technique that can barcode individual cells and thus used to obtain a gene expression profile for every individual cell within a tissue. This makes scRNA-seq an excellent method for characterizing rare cell populations such as stem cells. We describe how scRNA-seq can be utilized to examine limbal epithelial stem cell population as well as investigate the contribution of autophagy to the function of limbal epithelial stem cells. To accomplish this, we used the Beclin1 heterozygous (Beclin1 het) mouse, a well-established model of autophagy deficiency. We provide a protocol that we developed for the isolation of viable, single-cell suspensions of limbal/corneal tissues, as well as the analysis of scRNA-seq data., (© 2023. Springer Science+Business Media, LLC.)

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides have been successfully investigated for the treatment of different types of diseases. Detection and determination of antisense oligonucleotides and their metabolites are necessary for drug development and evaluation. This review focuses mainly on the first step of the analysis of oligonucleotides i.e. the sample preparation stage, and in particular on the techniques used for liquid chromatography and liquid chromatography coupled with mass spectrometry. Exceptional sample preparation techniques are required as antisense oligonucleotides need to be determined in complex biological matrices. The text discusses general issues in oligonucleotide sample preparation and approaches to their solution. The most popular techniques i.e. protein precipitation, protein enzyme digestion and liquid-liquid extraction are reviewed. Solid phase extraction methods are discussed and the issues connected with the application of each method are highlighted. Other newly reported promising techniques are also described. Finally, there is a summary of actually used techniques and the indication of the direction of future research. [ABSTRACT FROM AUTHOR]

The Tc1 mariner element is widely distributed among organisms and have been already described in different species of fish. The genus Ancistrus (Kner, 1854) has 68 nominal species and is part of an interesting taxonomic and cytogenetic group, as well as presenting a variation of chromosome number, ranging from 2n=34 to 54 chromosomes, and the existence of simple and multiple sex chromosome system and the occurrence of chromosomal polymorphisms involving chromosomes that carry the nucleolus organizer region. In this study, a repetitive element by restriction enzyme, from Ancistrus sp.1 "Flecha" was isolated, which showed similarity with a transposable element Tc1-mariner. Its chromosomal location is distributed in heterochromatic regions and along the chromosomal arms of all specimens covered in this study, confirming the pattern dispersed of this element found in other studies carried out with other species. Thus, this result reinforces the hypothesis that the sequence AnDraI is really a dispersed element isolated. As this isolated sequence showed the same pattern in all species which have different sex chromosomes systems, including in all sex chromosomes, we could know that it is not involved in sex chromosome differentiation. [ABSTRACT FROM AUTHOR]

Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2); extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 (FSG1 hydrolyzed with Alcalase + Flavourzyme), FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 (FSG2 hydrolyzed with Alcalase + Flavourzyme). The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1–FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1–FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products.

Aims: The drive toward more sensitive LC-MS assays has resulted in long, complex methods. We assessed next-generation trypsins to identify a suitable candidate to integrate into protein LC-MS method development strategies, to simplify methods and increase throughput. Materials & methods: The performance of commercially available next-generation trypsins was assessed based on the digestion of protein standards in buffer and complex matrix by LC-high-resolution MS. Results: The performance of all next-generation trypsins assessed exceeded that of an overnight tryptic digest in a fraction of the time. Performing reduction and alkylation prior to digestion with heat-stable trypsins may be beneficial and should be investigated. Conclusion: Promega Rapid-Digestion Trypsin is the best-performing next-generation trypsin, surpassing an overnight tryptic digestion.

Purpose: To compare the effectiveness of two isolation systems for stemness, proliferation and mesenchymal contamination of ex vivo cultured limbal epithelial cells (LECs). Methods: Whole explant and dispase digestion methods were used to isolate LECs. For whole explant isolation, one limbal explant was cultivated up to four consecutive times (through LEC1 to LEC4). The performance of LECs isolated with both systems was evaluated according to the following parameters: immunofluorescent staining for adenosine 5′-triphosphate-binding cassette member 2 (ABCG2), p63, cytokeratin 3 (K3), Ki67, and vimentin, and flow cytometry analysis for ABCG2, Ki67 and vimentin. Results: Twelve LEC cultures were established using whole explant isolation, and nine LEC cultures were established using dispase digestion isolation. In immunofluorescent staining analysis, the ABCG2, p63 and Ki67 expressions were higher in LECs isolated with dispase compared to any LECs isolated with explant. Only the differences in ABCG2 and Ki67 were statistically significant. Further, LEC4 isolated with explant had the highest percentage of cells positive for vimentin, and LEC1 had the highest percentage of cells positive for K3. However, no significant differences were detected. In flow cytometry analysis, the expressions of ABCG2 and Ki67 were statistically higher for LECs isolated with dispase compared to any LECs isolated with explant. Conclusion: Dispase digestion isolation technique was significantly superior to explant isolation techniques in terms of progenitor and proliferative cell contents. [ABSTRACT FROM AUTHOR]

Two distinctive structures were observed on the surface of rice callus treated with cell wall degrading enzymes, pectinase and cellulase by scanning electron microscopy; i.e., membranous, and the fibrillar structures. The membranous layer was digestible with the enzymes, while the fibrillar structures were not. The membranous matrix was mucilaginous, and positive to PAS reaction. Protoplasts were released after the enzyme treatment from the areas covered with the enzyme-digestible mucilaginous layer. The role of the extracellular matrix of the rice callus is discussed from the standpoint of growth and differentiation of the rice callus.

Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [ d = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, d = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.

Brown adipose tissue (BAT) is a specialized fat depot that can dissipate energy through uncoupled respiration and thermogenesis. Various immune cells such as macrophages, eosinophils, type 2 innate lymphoid cells, and T lymphocytes were recently found to have an unexpected involvement in controlling the thermogenic activity of brown adipose tissue. Here, we describe a protocol for isolation and characterization of T cells from brown adipose tissue., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Investigation of the diversity and ecology of endophytic fungi has gained popularity in recent decades. Thereby, culture-independent methods were very instrumental as they revealed greater species richness and challenged the efficiency of isolation methods. However, culture-based methods remain the only possibility to provide strains for future studies. To improve the efficiency of endophyte isolation, we compared seven different artificial media that included a variety of plant extracts. Furthermore, we developed a method based on enzymatic digestion of plant tissue to improve the isolation efficiency in terms of species richness. The effect of additional plant extract in media that contained yeast and malt extract was of minor significance, whereas isolations using enzymatic digestion of plant leaf tissue combined with a 1:10 dilution of nutrients revealed a much higher diversity of species. Using this protocol, we isolated three times as many species from the same leaves than with previously described methods. This is reflected by the large number of singletons obtained in culture-independent approaches to study the diversity of endophytic fungi. [ABSTRACT FROM AUTHOR]

Abstract: A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome. [Copyright &y& Elsevier]

This study is aimed at detecting diatom in lung, liver and kidney tissues using PCR - DHPLC technology after different periods of enzyme digestion to assess the effect of enzyme digestion on the detection of diatom species. Twenty Randomly selected experimental rabbits were drowned at the same place. Their liver, kidney, and lung tissues were removed for sampling. After the extraction of DNA from the samples, amplification was conducted with specific primers of the SSU gene of diatom. Then, an analysis was performed with agarose gel electrophoresis and DHPLC. Within 2 h-8 h, the amount of the diatom species found in the lung gradually increased over time and was statistically significant (P<0.05). After 8 h, with enzyme digestion, the amount of the diatom species found in lung showed no significant increase (P>0.05). However, as for the liver and kidney, within 2h-6h, the amount of the diatom species gradually increased over time and was statistically significant (P<0.05). After 6h, the fig. did not present significant growth (P>0.05). The amount of the diatom species found in the organs after different periods of digestion time had significant differences, which provides a reference for the detection of diatoms and also, has a good application prospect in the forensic identification of drowning. [ABSTRACT FROM AUTHOR]

Natural rubber (NR) is an essential biomaterial the supply of which is insecure. Taraxacum kok-saghyz (TK), a rubber-producing dandelion, is an alternative source of NR that has excellent potential for commercial development. Scalable methods for rubber extraction at high yield, quality and purity are needed to meet this potential. We previously developed an aqueous, enzymatic method for the extraction and purification of NR from TK roots. Alkaline pretreatment significantly improved the yield of TK NR using this method, but its effects on NR yield, purity and quality at a range of base concentrations and temperatures was not tested. In this study, TK roots (after inulin extraction) were pretreated at sodium hydroxide loading rates of 0, 33, 66 and 132 mg NaOH/g and temperatures of 25, 70, 125 and 160 °C. Treatment with pectinase and cellulase enzymes was used to solubilize root structural carbohydrates and liberate NR. Rubber quality and impurities were then characterized. Results showed that increasing NaOH loading rate and temperature significantly improved rubber yield, but reduced rubber molecular weight and rubber gel content. Rubber purity and thermal properties were largely unaffected by pretreatment conditions. FTIR and SEM analysis revealed that alkaline pretreatment primarily altered protein and other secondary components of TK NR. [Display omitted] • Alkaline pretreatment at several conditions were optimized for TK rubber extraction. • Elevated temperatures and NaOH loadings increased TK rubber yield. • Pretreatment and enzyme digestion recovered 80% of the rubber yield with 97% purity. • High temperature (>120 °C) and NaOH loading negatively impacted rubber quality. • Non-rubber components of TK rubber are drastically affected by alkaline pretreatment. [ABSTRACT FROM AUTHOR]

Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/ MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/ MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10 cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents the alkanethiol-functionalized environmental effects of individual DNA molecules in nanopores on enzyme digestion at the single-molecule level. A template consisting of gold deposited within a solid-state nanoporous polycarbonate membrane was used to trap individual λ-DNA and enzyme molecules. The gold surfaces were modified with various functional groups (–OH, –COOH, –NH3). The enzyme digestion rates of single DNA molecules increased with decreasing nanopore diameters. Surprisingly, the digestion rates in the l-cysteine chemisorbed nanopores were 2.1–2.6 times faster than in the mercaptoethanol chemisorbed gold nanopores, even though these nanopores had equivalent interspacial areas. In addition, the membrane of chemisorbed cysteamine with ionized functional groups of H3N+ at pH 8.2 had a greater positive influence on the enzyme digestion rate than the membrane of chemisorbed mercaptoproponic acid with ionized carboxyl groups (COO-). These results suggest that the three-dimensional environment effect is strongly correlated with the functional group in confined nanopores and can significantly change the enzyme digestion rates for nanopores with different internal areas. [Copyright &y& Elsevier]