Protein Extracts Research Articles

SHAPEv1.0 Scoring curves and peer group benchmarks for dynamic soil health indicators

AbstractThe soil health concept has evolved over the past several decades, recognizing that the response of dynamic soil properties to management is dependent on site‐specific factors. The Soil Health Assessment Protocol and Evaluation (SHAPE) tool provides scores and benchmark values by forming soil peer groups based on site‐specific, climate‐edaphic characteristics. Data for model development were compiled from the Cornell Soil Health Laboratory and the Kellogg Soil Survey Laboratory databases. The SHAPEv1.0 interpretation curves produce scores between 0% and 100% for measured laboratory values that reflect the quantile within a population conditional cumulative distribution function along with measures of uncertainty. The original SHAPE tool was developed for soil organic carbon and has been expanded to include two wet aggregate stability methods, permanganate oxidizable organic carbon, autoclaved citrate extractable protein, and 4‐day microbial respiration. In addition, SHAPE provides site‐specific benchmark values at user‐defined percentiles within a given soil peer group. The difference between the actual measured value and the selected benchmark value represents the soil health opportunity gap. Case studies in Missouri and Texas show that the SHAPE scoring curves are sensitive to land‐use and management practices across multiple soil types and provide a regionally relevant interpretation of key soil health indicators.

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Human Keratinocyte Responses to Woodsmoke Chemicals.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

The impact of incorporating dried cafeteria leftover food on growing APRI rabbits productivity, profitability and meat quality.

This study was conducted to investigate the effect of dried cafeteria leftover food (DCLF) incorporation in growing APRI rabbit diets on nutrient digestibility, live body weight, body weight gain, carcass traits, meat quality, blood parameters, and economic efficiency. Thirty-six APRI rabbits (6 weeks of age), after weaning, were randomly divided into three groups (12 rabbits/group, 613 ± 5 g). During the experiment, rabbits aged 6-14 weeks participated in the experiment. An approximate isocaloric and isonitrogenous composition was achieved in three pelleted experimental diets. For the rabbit groups D1 (as the control), D2, and D3, DCLF was added at doses of 0%, 17.5%, and 35%, respectively. Digestibility trails were carried out at the end of the feeding experiment to evaluate the nutritional and feeding values of the experimental diets. To examine the characteristics of the carcasses, the quality of the meat, the histopathological analysis, and the blood plasma parameters, three rabbits were slaughtered from each group. The results showed that the feeding values (TDN and DE), which had the greatest values, as well as the digesting coefficients for dry matter, organic matter, crude fiber, and ether extract between the D1 and D3 groups were significantly different (p < 0.05). On the contrary, the curde protein, nitrogen-free extract, and digestible crude protein of the examined diets showed no signifcant differences compared with the control diet. The level of DCLF incorporation in diets significantly (p < 0.05) increased the live body weight at 14 weeks; with D3 recording the highest values (2130.9 g) when compared to the D1 and D2 groups. A similar pattern was observed for both daily increase and total body gain (6-14 weeks). Feed conversion recorded insignificant differences among the experimental diets respecting the period 6-14 weeks of age. DCLF levels had a significant effect on dressing and giblet percentages. The chemical composition and color parameters of rabbit meat were similar (p > 0.05), except for fat%, which had the highest value in the D3 group (p < 0.05) compared to the D1 and D2 groups. Economic efficiency was improved by using DLCF up to 35% in terms of gross margin and feed cost/kg gain. In conclusion, using DCLF in the feeding system of rabbits could enhance productive performance, carcass traits, meat quality, and physiological function, as well as improve economic efficiency.

Response surface optimization for deep eutectic solvents extraction of bamboo shoots proteins

To enhance the protein extraction rate of bamboo shoots, the Response Surface Methodology was utilized to optimize the protein extraction process of Chimonobambusa purpurea Hsueh f. & T.P.Yi (syn. Chimonobambusa neopurpurea T.P.Yi) bamboo shoots. Choline chloride and proline were utilized as hydrogen bond acceptors in a total of eight deep eutectic solvents, whereas glucose, sucrose, glycerol, ethylene glycol, acetylpropionic acid, malic acid, and lactic acid were used as hydrogen bond donors. This work examined the protein extraction from bamboo shoots using deep eutectic solvents. Single-factor experiments were conducted to determine the optimal composition of the deep eutectic solvents, along with the ideal gradients of material-liquid ratio gradients, water content, extraction time, and water bath temperature. The impact and interplay of various variables on the protein extraction rate of bamboo shoots were examined using the Box-Behnken Design and Response Surface Methodology. Both the water bath temperature and the extraction time significantly influenced the protein extraction rate of bamboo shoots. Notably, the interaction between extraction time and water bath temperature had the most substantial effect on the protein extraction rate of bamboo shoots. The quadratic response surface regression equation for the protein extraction rate of bamboo shoots relative to the actual values of each factor was obtained as follows: Protein extraction rate = −193.3275 + 3.8395 A - 18.7075 B + 6.3285 C + 0.2125 AB - 0.0089 AC + 0.024 BC - 0.02885 A2 + 0.685 B2 - 0.06455 C2. Finally, this work predicted optimum extraction parameters for bamboo shoots with choline chloride-sucrose (molar ratio 1:2), a material-liquid ratio of 1:50, a water content of 74 %, an extraction time of 4 h, and a water bath temperature of 44 ℃. Under the optimal conditions, the protein extraction yield was 59.39±0.45 %, which aligned with the predicted value from the response surface fitting equation. This indicated that the optimised regression equation may have practical significance. Compared with conventional extraction using a sodium hydroxide (NaOH) solution, deep eutectic solvents extraction of bamboo shoots is more environmentally friendly, non-toxic and harmless. It provides a theoretical and experimental reference for industrial extraction and offers a reference for deep eutectic solvent development in bamboo shoot resource utilization.Key message:To enhance the protein extraction rate of bamboo shoots, the Response Surface Methodology was utilized to optimize the protein extraction process of bamboo shoots.

Evolution of tick vaccinology.

Ticks represent a major concern for society worldwide. Ticks are also difficult to control, and vaccines represent the most efficacious, safe, economically feasible and environmentally sustainable intervention. The evolution of tick vaccinology has been driven by multiple challenges such as (1) Ticks are difficult to control, (2) Vaccines control tick infestations by reducing ectoparasite fitness and reproduction, (3) Vaccine efficacy against multiple tick species, (4) Impact of tick strain genetic diversity on vaccine efficacy, (5) Antigen combination to improve vaccine efficacy, (6) Vaccine formulations and delivery platforms and (7) Combination of vaccines with transgenesis and paratransgenesis. Tick vaccine antigens evolved from organ protein extracts to recombinant proteins to chimera designed by vaccinomics and quantum vaccinomics. Future directions will advance in these areas together with other novel technologies such as multiomics, AI and Big Data, mRNA vaccines, microbiota-driven probiotics and vaccines, and combination of vaccines with other interventions in collaboration with regions with high incidence of tick infestations and tick-borne diseases for a personalized medicine approach.

Quercetin and Kaempferol inhibit HMC-1 activation via SOCE/NFATc2 signaling and suppress hippocampal mast cell activation in lipopolysaccharide-induced depressive mice.

Mast cells (MCs), as the fastest immune responders, play a critical role in the progression of neuroinflammation-related diseases, especially in depression. Quercetin (Que) and kaempferol (Kae), as two major diet-derived flavonoids, inhibit MC activation and exhibit significant antidepressant effect due to their anti-inflammatory capacity. The study aimed to explore the mechanisms of inhibitory effect of Que and Kae on MC activation, and whether Que and Kae suppress hippocampal mast cell activation in LPS-induced depressive mice. In vitro assays, human mast cells (HMC-1) were pretreated with Que or Kae for 1h, then stimulated by phorbol 12-myristate 13-acetate (PMA) and 2,5-di-t-butyl-1,4-benzohydroquinone (tBHQ) for 3h or 12h. In vivo assays, Que or Kae was administered by oral gavage once daily for 14days and then lipopolysaccharide (LPS) intraperitoneally injection to induce depressive behaviors. The secretion and expression of TNF-α were determined by ELISA and Western blotting. The nuclear factor of activated T cells (NFAT) transcriptional activity was measured in HMC-1 stably expressing NFAT luciferase reporter gene. Nuclear translocation of NFATc2 was detected by nuclear protein extraction and also was fluorescently detected in HMC-1 stably expressing eGFP-NFATc2. We used Ca2+ imaging to evaluate changes of store-operated calcium entry (SOCE) in HMC-1 stably expressing fluorescent Ca2+ indicator jGCamP7s. Molecular docking was used to assess interaction between the Que or Kae and calcium release-activated calcium modulator (ORAI). The hippocampal mast cell accumulation and activation were detected by toluidine blue staining and immunohistochemistry with β-tryptase. In vitro assays of HMC-1 activated by PtBHQ (PMA and tBHQ), Que and Kae significantly decreased expression and secretion of TNF-α. Moreover, NFAT transcriptional activity and nuclear translocation of NFATc2 were remarkably inhibited by Que and Kae. In addition, the Ca2+ influx mediated by SOCE was suppressed by Que, Kae and the YM58483 (ORAI inhibitor), respectively. Importantly, the combination of YM58483 with Que or Kae had no additive effect on the inhibition of SOCE. The molecular docking also showed that Que and Kae both exhibit high binding affinities with ORAI at the same binding site as YM58483. In vivo assays, Que and Kae significantly reversed LPS-induced depression-like behaviors in mice, and inhibited hippocampal mast cellactivation in LPS-induced depressive mice. Our results indicated that suppression of SOCE/NFATc2 pathway-mediated by ORAI channels may be the mechanism of inhibitory effect of Que and Kae on MC activation, and also suggested Que and Kae may exert the antidepressant effect through suppressing hippocampal mast cell activation.

Extraction and characterization of R-phycoerythrin from wet and lyophilized macroalgae Solieria filiformis by pressurized water method

The present study evaluated the production of R-phycoerythrin (R-PE) extracted from the macroalgae Solieria filiformis through a green technology (pressurized water method). The extraction process over time and the macroalgae structure were analyzed. Two samples (freeze-dried and wet biomass, with 1 % and 10 % m/v, respectively) were studied at pressures up to 500 bar. R-PE maximum yield was reached for lyophilized seaweed structure after 360 min, obtaining 0.67 mg of R-PE per gram of dry biomass. In contrast, wet biomass reached 0.05 mg of R-PE per gram of wet biomass. Crude and precipitated extracts were characterized using the following techniques: electrophoresis, circular dichroism, and FTIR. The carbohydrate content was also determined. The biomolecule showed R-PE characteristic spectra, fluorescence, and α-helix structure as the main element of its secondary structure (93.28–95.24 %), indicating the presence of other proteins in the precipitated extract. Precipitated extract's cytotoxic activity was assessed through an MTT assay against tumor cell lines of colorectal cancer (HCT116), prostate cancer (PC3), glioblastoma cancer (SNB-19), and HL60 (promyeloblast leukemia). The most prominent in vitro results were in HL-60 and HCT-116 cell lines.

Effects of arbuscular mycorrhizal fungi on the reduction of arsenic accumulation in plants: a meta-analysis.

Arsenic (As) accumulation in plants is a global concern. Although the application of arbuscular mycorrhizal fungi (AMF) has been suggested as a potential solution to decrease As concentration in plants, there is currently a gap in a comprehensive, quantitative assessment of the abiotic and biotic factors influencing As accumulation. A meta-analysis was performed to quantitatively investigate the findings of 76 publications on the impacts of AMF, plant properties, and soil on As accumulation in plants. Results showed a significant dose-dependent As reduction with higher mycorrhizal infection rates, leading to a 19.3% decrease in As concentration. AMF reduced As(V) by 19.4% but increased dimethylarsenic acid (DMA) by 50.8%. AMF significantly decreased grain As concentration by 34.1%. AMF also improved plant P concentration and dry biomass by 33.0% and 62.0%, respectively. The most significant reducing effects of As on AMF properties were seen in single inoculation and experiments with intermediate durations. Additionally, the benefits of AMF were significantly enhanced when soil texture, soil organic carbon (SOC), pH level, Olsen-P, and DTPA-As were sandy soil, 0.8%-1.5%, ≥7.5, ≥9.1 mg/kg, and 30-60 mg/kg, respectively. AMF increased easily extractable glomalin-related soil protein (EE-GRSP) and total glomalin-related soil protein (T-GRSP) by 23.0% and 28.0%, respectively. Overall, the investigated factors had significant implications in developing AMF-based methods for alleviating the negative effects of As stress on plants.

Deciphering proteome changes and meat texture of traditional halal slaughtered spent sheep subjected to low-voltage electrical stimulation and ageing

The present study evaluated the effect of low-voltage electrical stimulation (LVES, 42 V peak, 0.6 Amp, 50 Hz) and ageing in traditional halal slaughtered Nellore crossbred spent-sheep (Ovis aries). The LVES accelerated the post-mortem glycolysis with a significant decline in pH and shear force values and improvement in water-holding capacity, cooking yield, protein extractability, and an early onset of rigor-mortis relative to non-stimulated control. Two-dimensional gel electrophoresis coupled with tandem mass spectrometry identified myosin regulatory light chain 2, NADH dehydrogenase, myoglobin, and glyceraldehyde-3-phosphate dehydrogenase from traditional halal slaughtered sheep meat. PANTHER analysis of differentially expressed proteins indicated their involvement in structural (50%), catalytic (25%), and binding (25%) activities. The current study provides a novel insight into the contribution of different proteins in orchestrating meat texture from traditional halal slaughtered spent sheep meat subjected to LVES and ageing.

Potential use of yeast protein in terms of biorefinery, functionality, and sustainability in food industry.

A growing demand for sustainable, alternative protein sources that are nutrient-dense, such as microorganisms, and insects, has gradually evolved. When paired with effective processing techniques, yeast cells contain substantial substances that could supply the population's needs for food, medicine, and fuel. This review article explores the potential of yeast proteins as a sustainable and viable alternative to animal and plant-based protein sources. It highlights the various yeast protein extraction methods including both mechanical and non-mechanical methods. The application of nanoparticles is one example of the fast-evolving technology used to damage microbial cells. SiO2 or Al2O3 nanoparticles break yeast cell walls and disrupt membranes, releasing intracellular bioactive compounds. Succinylation of yeast protein during extraction can increase yeast protein extraction rate, lower RNA concentration, raise yeast protein solubility, increase amino acid content, and improve yeast protein emulsification and foaming capabilities. Combining physical and enzymatic extraction methods generates the most representative pool of mannose proteins from yeast cell walls. Ethanol or isoelectric precipitation purifies mannose proteins. Mannoproteins can be used as foamy replacement for animal-derived components like egg whites due to their emulsification, stability, and foaming capabilities. Yeast bioactive peptide was separated by ultrafiltration after enzymatic hydrolysis of yeast protein and has shown hypoglycemic, hypotensive, and oxidative action in vitro studies. Additionally, the review delves into the physicochemical properties and stability of yeast-derived peptides as well as their applications in the food industry. The article infers that yeast proteins are among the promising sources of sustainable protein, with a wide range of potential applications in the food industry.

Feasibility of Enzymatic Protein Extraction from a Dehydrated Fish Biomass Obtained from Unsorted Canned Yellowfin Tuna Side Streams: Part II.

The enzymatic extraction of proteins from fish biomasses is being widely investigated. However, little or almost no research has paid attention to the exploitation of unsorted fishery biomasses. This work is part of a larger study, Part I of which has already been published, and focuses on an extensive characterization of two collagenous samples, namely gelatin (G) and hydrolyzed gelatin peptides (HGPs), extracted from a dehydrated fish biomass coming from unsorted canned yellowfin tuna side streams. The results indicate crude protein fractions of 90-93%, pH values between 3 and 5, white-yellow colors, collagen-like FTIR spectra, and 17% in terms of total amino acid content. Viscosity and the study of dynamic viscous-elastic behavior were analyzed. Thermo-gravimetric analysis was performed to assess the residual ashes. Both samples were investigated to determine their molecular weight distribution via size-exclusion chromatography, with a higher total average molecular weight for G compared to HGPs, with values of 17,265.5 Da and 2637.5 Da, respectively. G demonstrated technological properties similar to analogous marine gelatins. HGPs demonstrated antioxidant activity as per FRAP assay. All the results open up new perspectives for the potential use of these substances in biodegradable packaging, dietary supplements, and skin care cosmetics.

Quantification of the aromatase inhibitor letrozole and its carbinol metabolite in mouse plasma by UHPLC-MS/MS

A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 μL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r2 > 0.99) over concentration ranges of 5–1000 ng/mL and 20–2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103–108 % for letrozole and 99.8–107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug.

Complementing Plant and Animal Protein Sources for Broiler Production: Effects on Growth, Carcass Traits, and Economics

This study investigated the effects of complementing plant-based Sesbania grandiflora meal (SGM) and animal-based fermented golden apple snail extract (FGASE) protein sources on the growth performance, carcass characteristics, meat quality, and economic profitability of broiler chickens. A 2x4 factorial experiment was conducted, with SGM levels (0 and 15g kg-1) and FGASE levels in drinking water (0, 10, 20, and 30 mL L-1). SGM diet increased feed and water intake but did not affect final body weight or weight gain. FGASE in water did not influence feed or water intake. The combination of SGM and FGASE improved feed conversion ratio, but higher FGASE levels reduced dressing percentage. Neither SGM nor FGASE significantly impacted most meat cut weights or visceral organ weights, except for a significant interaction on back weight. Economic analysis revealed positive returns on investment for all treatments, with the highest net profit obtained from broilers fed a standard diet supplemented with 10 mL L-1 FGASE. The findings suggest the potential for incorporating SGM and FGASE in broiler diets to improve feed efficiency and profitability, though optimal inclusion levels and processing methods require further investigation.

Study on Antidiabetic Potential of Sessuvium Portulacastrum Aqueous Extract: An In-Silico and In-Vitro Analysis

ABSTRACT Diabetes mellitus is a persistent metabolic condition marked by elevated blood glucose levels due to compromised insulin secretion or functionality. The search for natural antidiabetic agents has gained attention due to their potential effectiveness and safety profiles. Sessuvium portulacastrum, a coastal plant, has been traditionally used for various medicinal purposes. This study investigates the antidiabetic potential of Sessuvium portulacastrum aqueous extract by analyzing its inhibitory effects on key enzymes involved in carbohydrate metabolism and exploring its molecular interactions with critical target proteins. The aqueous extract of Sessuvium portulacastrum was prepared and used for in vitro analysis. The reduced activity of the extract against α-amylase and α-glucosidase enzymes, crucial in glucose absorption and postprandial hyperglycemia, was assessed. Molecular docking techniques were employed to explore the potential interactions between active compounds in the extract and diabetes-related proteins, including BAX, GSK3β, and CADH. The study revealed significant inhibition of both alpha-amylase and alpha-glucosidase enzymes by Sessuvium portulacastrum aqueous extract, indicating its potential to reduce glucose absorption and postprandial hyperglycemia. Moreover, the molecular docking analysis demonstrated strong binding interactions between active compounds in the extract and key proteins involved in diabetes-related pathways, namely apoptotic pathways, glycogen synthesis, and cell adhesion. The findings of this study highlight the promising antidiabetic potential of Sessuvium portulacastrum aqueous extract. Upcoming research should get an attention on isolating and characterizing the active compounds responsible for these effects on antidiabetic therapies from natural sources.

Diverting the Use of Hand-Operated Tablet Press Machines to Bioassays: A Novel Protocol to Test 'Waste' Insoluble Shell Matrices.

To mineralize their shells, molluscs secrete a complex cocktail of proteins-collectively defined as the calcifying shell matrix-that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease inhibitors offer the most tangible perspectives in applied sciences, health, and aquaculture. However, one technical obstacle in testing the activity of shell extracts lies in their high insolubility. In this paper, we present a protocol that circumvents this impediment. After an adapted shell protein extraction and the production of two organic fractions-one soluble, one insoluble-we employ a hand-operated tablet press machine to generate well-calibrated tablets composed of 100% insoluble shell matrix. FT-IR monitoring of the quality of the tablets shows that the pressure used in the press machine does not impair the molecular properties of the insoluble extracts. The produced tablets can be directly tested in different biological assays, such as the bactericidal inhibition zone assay in Petri dish, as illustrated here. Diverting the use of the hand-operated tablet press opens new perspectives in the analysis of insoluble shell matrices, for discovering novel bioactive components.

Extraction of amaranth seed oil using subcritical butane and use of the generated cake for protein extraction

The purpose of this research was to determine the technical feasibility of extracting amaranth seed oil with butane in a subcritical state and to take advantage of the cake generated. To this end, a type of non-germinated grain was characterized, oil was extracted from a germinated grain and the characterized one, the oil obtained was characterized, and the protein was extracted from the defatted cake of the non-germinated one. It was found that the non-germinated grain was made up of 13.33% protein, 7.24% fat, and 9.02% moisture, the optimum yield of this grain was 91%, for the germinated grain, a maximum value of 6.63% for oil mass. By comparing the characteristics of both oils, higher quality was found in the non-germinated oil, and the maximum protein extraction productivity was 5.15%. Thus, it has been concluded that this extraction method is technically feasible.

Biochemical Changes in Embryogenic and Nonembryonic Callus of Glycyrrhiza glabra L. during Somatic Embryogenesis

The continuous extraction of glycyrrhizin from Glycyrrhiza glabra L. has led to the critical endangerment of this therapeutic plant. Glycyrrhizin, a triterpenoid saponin, is a vital secondary metabolite renowned for its diverse pharmacological benefits, including anti-inflammatory, immunomodulatory, antiallergic, antiulcer properties, and even efficacy against HIV. However, the relentless harvesting of roots for high-value glycyrrhizin results in the obliteration of the entire plant, posing a severe threat to its existence and causing a detrimental impact on biodiversity. To counteract this challenge and conserve the species, sustainable approaches are imperative. One such approach involves the utilization of in vitro regeneration through somatic embryogenesis for clonal proliferation and genetic modification. This report presents findings on SE-associated proteins in Glycyrrhiza glabra L., accomplished through the implementation of the discontinuous gel electrophoresis technique. The study focused on a comparative isozyme analysis employing non-embryogenic and embryogenic calli of G. glabra L. Remarkably, these isozymes were exclusively identified and exhibited affiliations with a spectrum of cellular functions. A substantial portion of these proteins are engaged in a diverse range of metabolic and stress-related functions. This study utilized crude enzyme/total protein extracts as a basis to evaluate the genetic stability of in vitro cultivated plant tissue. Acid phosphatase, esterase, and peroxidase were selected as indicators for this assessment. Intriguingly, esterase and acid phosphatase exhibited significant polymorphism between the segregated embryogenic callus (EC) and non-embryogenic callus (NEC) derived from the same G. glabra L. genotype. Comparative analyses encompassing esterase, acid phosphatase, and peroxidase activities, alongside the expression profiles of these isozymes throughout somatic embryogenesis (SE), were conducted using polyacrylamide gel electrophoresis. Notably, the isoenzyme patterns of calluses were assessed. The activity of isozymes within an embryogenic line of callus surpassed that within a non-embryogenic counterpart. However, no conspicuous alterations in quality were observed between the non-embryogenic and embryogenic calluses.

A comprehensive guide to western blotting for tendon research

Tendons are classified as dense fibrous connective tissue. This fibrous composition poses challenges in protein extraction, particularly hindering the application of Western blotting techniques. Because of these challenges, it becomes necessary to implement additional steps and specific solutions to attain success in this methodology with the tissue in question. The objective of this article is to provide a detailed protocol, elucidating each step, and making it easily replicable for researchers. The study focused on the Achilles tendons of Sprague-Dawley rats, emphasizing the need for a tailored approach in working with this tissue. By addressing the nuances of protein extraction from the dense and fibrous tendons, our protocol aims to facilitate the reproducibility of Western blotting experiments, contributing to a better understanding of this tissue.

Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway.

To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 μM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction. Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.