Effect of enzymatic hydrolysis with Alcalase or Protamex on technological and antioxidant properties of whey protein hydrolysates.

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18-20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies.

Insights into dynamic evolution of glucuronofucogalactoglucan from water extract of Agrocybe cylindracea during maturation.

This study explored the physicochemical properties and structural characteristics of Agrocybe cylindracea polysaccharides at four developmental stages, as well as their dynamic evolution during maturation. Results showed that the polysaccharides from A. cylindracea water extract exhibited similar structural characteristics across all four maturity stages, despite a significant reduction in yields. Four water-soluble heteroglycans, including one high molecular weight (ACPM-Et50-I) and three low molecular weight (ACPM-Et50-II, ACPM-Et60, ACPM-Et80), were isolated from A. cylindracea at each maturity stage. ACPM-Et50-I was identified as branched heterogalactans, while ACPM-Et60 and ACPM-Et80 were branched heteroglucans. However, ACPM-Et50-II was characterized as a branched glucuronofucogalactoglucan at the tide-turning stage but a glucuronofucoglucogalactan at the pileus expansion stage due to the increase of its α-(1 â†’ 6)-D-Galp. In general, although the structural skeletons of most A. cylindracea heteroglycans were similar during maturation as shown by their highly consistent glycosyl linkages, there were still differences in the distribution of some heteroglucans. This work has for the first time reported a glucuronofucogalactoglucan in A. cylindracea and its dynamic evolution during maturation, which may facilitate the potential application of A. cylindracea in food and biomedicine industries.

The study of galactomannans with different molecular weights and their ability to form microparticles suitable for pulmonary delivery.

The influence of locust bean gum (LBG) galactomannans (GMs) molecular weight (Mw) to assemble microparticulate systems was evaluated, and carriers for deep lung delivery were developed. A commercial batch of LBG with a mannose/galactose (M/G) ratio of 2.4 (batch 1) was used to study the influence of different microwave partial acid hydrolysis conditions on carbohydrate composition, glycosidic linkages, and aqueous solutions viscosity. The microwave treatment did not affect the composition, presenting 4-Man (36-42 %), 4,6-Man (27-35 %), and T-Gal (24-25 %) as the main glycosidic linkages. Depolymerization led to a viscosity reduction (≤0.005 Pa·s) with no major impact on polysaccharide debranching. The structural composition of the LBG galactomannans were further elucidated with sequence-specific proteins using carbohydrate microarray technologies. A second batch of LBG (M/G 3.3) was used to study the impact of GMs with different Mw on microparticle assembling, characteristics, and insulin release kinetics. The low-Mw GMs microparticles led to a faster release (20 min) than the higher-Mw (40 min) ones, impacting the release kinetics. All microparticles exhibited a safety profile to cells of the respiratory tract. However, only the higher-Mw GMs allowed the assembly of microparticles with sizes suitable for this type of administration.

Glycerol-driven adaptive evolution for the production of low-molecular-weight Welan gum: Characterization and activity evaluation.

Through adaptive laboratory evolution (ALE) of Sphingomonas sp. ATCC 31555, fermentation for production of low-molecular-weight welan gum (LMW-WG) was performed using glycerol as sole carbon source. During ALE, GPC-MALS analysis revealed a gradual decrease in WG molecular weight with the increase of adaptation cycles, accompanied by changes in solution conformation. LMW-WG was purified and structurally analyzed using GPC-MALS, monosaccharide composition analysis, infrared spectroscopy, NMR analysis, atomic force microscopy, and scanning electron microscopy. Subsequently, LMW-WG obtains hydration, transparency, antioxidant activity, and rheological properties. Finally, an in vitro simulation colon reactor was used to evaluate potential prebiotic properties of LMW-WG as dietary fiber. Compared with WG produced using sucrose as substrate, LMW-WG exhibited a fourfold reduction in molecular weight while maintaining moderate viscosity. Structurally, L-Rha nearly completely replaced L-Man. Furthermore, LMW-WG demonstrated excellent hydration, antioxidant activity, and high transparency. It also exhibited resistance to saliva and gastrointestinal digestion, showcasing a favorable colonization effect on Bifidobacterium, making it a promising symbiotic agent.

Effectiveness of topical hyaluronic acid of different molecular weights in xerosis cutis treatment in elderly: a double-blind, randomized controlled trial.

Dry skin is a common dermatological condition that frequently affects the elderly. A contributing cause to dry skin is a reduced concentration of hyaluronic acid (HA) in both the epidermis and dermis. The effectiveness of moisturizer containing HA as a therapy for dry skin is impacted by its specific molecular weight. Low molecular weight HA (LMWHA) is believed to be more effective in replenishing skin hydration in aging skin compared to High Molecular Weight HA (HMWHA) due to its ability to penetrate the stratum corneum. However, there is a lack of clinical research supporting this claim. A double-blind, randomized controlled trial was conducted on 36 residents of a nursing home in Jakarta. The participants, aged between 60 and 80 years, had been diagnosed with dry skin. Each test subject was administered three distinct, randomized moisturizing lotions (LMWHA, HMWHA, or vehicle), to be topically applied to three separate sites on the leg. Skin capacitance (SCap), transepidermal water loss (TEWL), and specified symptom sum score (SRRC) were measured at weeks 0, 2, and 4. After four weeks of therapy, area that was treated with LMWHA showed greater SCap values compared to the area treated with HMWHA (56.37 AU vs. 52.37 AU, p = 0.004) and vehicle (56.37 AU vs. 49.01 AU, p < 0.001). All groups did not show any significant differences in TEWL and SRRC scores. No side effects were found in all groups. The application of a moisturizer containing LMWHA to the dry skin of elderly resulted in significant improvements in skin hydration compared to moisturizers containing HMWHA and vehicle. Furthermore, these moisturizers demonstrated similar safety in treating dry skin in the elderly. ClinicalTrials.gov Identifier NCT06178367, https://clinicaltrials.gov/study/NCT06178367 .

Novel structure of secreted small molecular weight antigen Mtb12 from Mycobacterium tuberculosis.

Mtb12, a small protein secreted by Mycobacterium tuberculosis, is known to elicit immune responses in individuals infected with the pathogen. It serves as an antigen recognized by the host's immune system. Due to its immunogenic properties and pivotal role in tuberculosis (TB) pathogenesis, Mtb12 is considered a promising candidate for TB diagnosis and vaccine development. However, the structural and functional properties of Mtb12 are largely unexplored, representing a significant gap in our understanding of M. tuberculosis biology. In this study, we present the first structure of Mtb12, which features a unique tertiary configuration consisting of four beta strands and four alpha helices. Structural analysis reveals that Mtb12 has a surface adorned with a negatively charged pocket adjacent to a central cavity. The features of these structural elements and their potential effects on the function of Mtb12 warrant further exploration. These findings offer valuable insights for vaccine design and the development of diagnostic tools.

Characterization, Antioxidant Capacity and Protective Effect of Peptides from Cordyceps militaris Cultivated with Tussah Pupa on Oxidative Injured HepG2 Cells.

The antioxidant capacity and protective effect of peptides from protein hydrolysate of Cordyceps militaris cultivated with tussah pupa (ECPs) on H2O2-injured HepG2 cells were studied. Results indicated ECP1 (<3 kDa) presented the strongest antioxidant activity compared with other molecular weight peptides. Pretreated with ECPs observably enhanced survival rates and reduced apoptosis rates of HepG2 cells. ECPs treatment decreased the ROS level, MDA content and increased CAT and GSH-Px activities of HepG2 cells. Besides, the morphologies of natural peptides from C. militaris cultivated with tussah pupa (NCP1) and ECP1 were observed by scanning electron microscopy (SEM). Characterization results suggested the structure of NCP1 was changed by enzymatic hydrolysis treatment. Most of hydrophobic and acidic amino acids contents (ACC) in ECP1 were also observably improved by enzymatic hydrolysis. In conclusion, low molecular weight peptides had potential value in the development of cosmetics and health food.

Ultrafiltration fractionation of potentially inhibitory substances of hydrothermal liquefaction aqueous phase derived from municipal sludge.

Hydrothermal liquefaction (HTL) is a promising thermo-chemical technology for municipal sludge treatment due to its potential for biocrude oil recovery and minimizing biosolids management costs. However, the process generates a high volume of an aqueous byproduct that needs to be treated due to its high chemical oxygen demand (COD) and various organic and inorganic compounds. Although the aqueous phase is known to contain recalcitrant and potentially inhibitory substances that may affect its biological treatment, their molecular weight distribution (MwD) and its impact on anaerobic biodegradability are poorly understood. Ultrafiltration (UF) was conducted to fractionate HTL aqueous into different molecular weight (Mw) fractions using 300, 100, 10, and 1 kDa membranes. Mesophilic biochemical methane potential (BMP) assays were conducted to assess the anaerobic biodegradability of each fraction, and the first-order model was used to calculate the degradation kinetics of potential inhibitory compounds. The highest percentage of organics (65 %) was found in the Mw<1 kDa range, whereas the 10>Mw>1 kDa had the lowest percentage (8 %). There was no significant difference in the cumulative specific methane produced from various Mw fractions (p>0.05). The Mw<1 kDa fraction had the highest first-order specific methane production rate (0.53 day-1), whereas the unfiltered HTL had the lowest (0.38 day-1). Although UF fractionation increased the rate of anaerobic degradation of HTL aqueous for the Mw<1 kDa fraction, the observed methane potential was only 55 % of the theoretical value. This implies that 45 % of COD remains undegraded even after permeation through the lowest Mw cut-off membrane. Therefore, further characterization of HTL aqueous is needed for compounds with molecular weights below 1 kDa to fully understand the nature of inhibitory organics and their impact on anaerobic digestion. Furthermore, pretreatments utilizing techniques such as adsorption and advanced oxidation may be necessary to enhance the specific methane yields from various HTL aqueous fractions, thereby bringing them closer to the theoretical yield.

Glucan polysaccharides isolated from Lactarius hatsudake Tanaka mushroom: Structural characterization and in vitro bioactivities.

Commercially available mushroom polysaccharides have found widespread use as adjuvant tumor treatments. However, the bioactivity of polysaccharides in Lactarius hatsudake Tanaka (L. hatsudake), a mushroom with both edible and medicinal uses, remains relatively unexplored. To address this gap, five L. hatsudake polysaccharides with varying molecular weights were isolated, named LHP-1 (898 kDa), LHP-2 (677 kDa), LHP-3 (385 kDa), LHP-4 (20 kDa), and LHP-5 (4.9 kDa). Gas chromatography-mass spectrometry, nuclear magnetic resonance, and atomic force microscopy, etc., were employed to determine their structural characteristics. The results confirmed that spherical aggregates with amorphous flexible fiber chains dominated the conformation of the LHP. LHP-1 and LHP-2 were identified as glucans with α-(1,4)-Glcp as the main chain; LHP-3 and LHP-4 were classified as galactans with varying molecular weights but with α-(1,6)-Galp as the main chain; LHP-5 was a glucan with ß-(1,3)-Glcp as the main chain and ß-(1,6)-Glcp connecting to the side chains. Significant differences were observed in inhibiting tumor cell cytotoxicity and the antioxidant activity of the LHPs, with LHP-5 and LHP-4 identified as the principal bioactive components. These findings provide a theoretical foundation for the valuable use of L. hatsudake and emphasize the potential application of LHPs in therapeutic tumor treatments.

The quantitative molecular weight-antimicrobial activity relationship for chitosan polymers, oligomers, and derivatives.

Chitosan and chitosan derivatives can kill pathogenic microorganisms including bacteria and fungi. The antimicrobial activity is dependent on the degree of acetylation, substituent structure, and molecular weight. Over the past four decades, numerous studies have endeavored to elucidate the relationship between molecular weight and the activity against microorganisms. However, investigators have reported divergent and, at times, conflicting conclusions. Here a bilinear equation is proposed, delineating the relationship between antimicrobial activity, defined as log (1/MIC), and the molecular weight of chitosan and chitosan derivatives. Three constants AMin, AMax, and CMW govern the shape of the curve determined by the equation. The constant AMin denotes the minimal activity expected as the molecular weight tends towards zero while AMax represents the maximal activity observed for molecular weights exceeding CMW, the critical molecular weight required for max activity. This equation was applied to analyze data from seven studies conducted between 1984 and 2019, which reported MIC (Minimum Inhibitory Concentration) values against bacteria and fungi for various molecular weights of chitosan and its derivatives. All the 29 datasets exhibited a good fit (R2 ≥ 0.5) and half excellent (R2 ≥ 0.95) fit to the equation. The CMW generally ranged from 4 to 10 KD for datasets with an excellent fit to the equation.

Extraction and Isolation of Two Polysaccharides from Chloranthus japonicus Sieb. and Evaluation of Their Anti-Gastric Cancer Activities.

Two unreported heteropolysaccharides, denoted as YCJP-1 and YCJP-2, were isolated from the herbs of Chloranthus japonicus. YCJP-1 was a heteropolysaccharide composed of glucose, galactose, arabinose, mannose, rhamnose, and a minor proportion of uronic acids, with the molecular weight mainly distributed in the 74,475-228,443 Da range. YCJP-2 was mainly composed of glucose, mannose, and galactose, with the molecular weights ranging from 848 to 5810 Da. To further evaluate the anti-gastric cancer effects of C. japonicus, the inhibitory effects of the crude polysaccharide (YCJP) and the purified polysaccharides (YCJP-1 and YCJP-2) were determined using a CCK-8 assay and colon-forming assay on MGC-803 and AGS gastric cancer cell lines. Our results showed that YCJP, YCJP-1, and YCJP-2 possess prominent inhibitory effects on the proliferation of MGC-803 and AGS cells, and the AGS cell was more sensitive to YCJP, YCJP-1, and YCJP-2. Moreover, YCJP-2 demonstrated superior anti-gastric cancer effects compared to YCJP-1. This could potentially be attributed to YCJP-2's higher glucose content and narrower molecular weight distribution.

Structural Characterization of Polygonatum Cyrtonema Polysaccharide and Its Immunomodulatory Effects on Macrophages.

A neutral Polygonatum cyrtonema polysaccharide (NPCP) was isolated and purified from Polygonatum cyrtonema by various chromatographic techniques, including DEAE-52 and Sephadex-G100 chromatography. The structure of NPCP was characterized by HPLC, HPGPC, GC-MS, FT-IR, NMR, and SEM. Results showed that NPCP is composed of glucose (55.4%) and galactose (44.6%) with a molecular weight of 3.2 kDa, and the sugar chain of NPCP was →1)-α-D-Glc-(4→1)-ß-D-Gal-(3→. In vitro bioactivity experiments demonstrated that NPCP significantly enhanced macrophages proliferation and phagocytosis while inhibiting the M1 polarization induced by LPS as well as the M2 polarization induced by IL-4 and IL-13 in macrophages. Additionally, NPCP suppressed the secretion of IL-6 and TNF-α in both M1 and M2 cells but promoted the secretion of IL-10. These results suggest that NPCP could serve as an immunomodulatory agent with potential applications in anti-inflammatory therapy.

Optimization of Composite Enzymatic Extraction, Structural Characterization and Biological Activity of Soluble Dietary Fiber from Akebia trifoliata Peel.

In order to reduce the waste of Akebia trifoliata peel and maximize its utilization, in this study, on the basis of a single-factor experiment and the response surface method, the optimum technological conditions for the extraction of soluble dietary fiber from Akebia trifoliata peel with the compound enzyme method were obtained. The chemical composition, physical and chemical properties, structural characterization and biological activity of the purified soluble dietary fiber (AP-SDF) from the Akebia trifoliata peel were analyzed. We discovered that that the optimum yield was 20.87% under the conditions of cellulase addition 600 U/g, enzymolysis time 100 min, solid-liquid ratio 1:24 g/mL and enzymolysis temperature 51 °C. At the same time, AP-SDF was a porous network structure cellulose type I acidic polysaccharose mainly composed of arabinoxylan (36.03%), galacturonic acid (27.40%) and glucose (19.00%), which possessed the structural characteristic peaks of the infrared spectra of polysaccharides and the average molecular weight (Mw) was 95.52 kDa with good uniformity. In addition, the AP-SDF exhibited high oil-holding capacity (15.11 g/g), good water-holding capacity and swelling capacity, a certain antioxidant capacity in vitro, hypoglycemic activity in vitro for α-glucosidase inhibition and hypolipidemic activity in vitro for the binding ability of bile acids and cholesterol. These results will provide a theoretical basis for the development of functional products with antioxidant, hypoglycemic and hypolipidemic effects, which have certain application value in related industries.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

Integrated neuromuscular training intervention applied in schools induces a higher increase in salivary high molecular weight adiponectin and a more favorable body mass index, cardiorespiratory fitness and muscle strength in children as compared to the traditional physical education classes.

Background: High-molecular-weight adiponectin (HMW-adiponectin) is a cardio-metabolic health protector. Objectives: (1) to compare body mass index (BMI), cardiorespiratory fitness (CRF) and muscle strength (MS) in healthy school-children depending on their baseline salivary-HMW-adiponectin concentration; and (2) to apply a 3-month integrated neuromuscular training (INT) and evaluate its effects on salivary-HMW-adiponectin concentration, BMI, CRF and MS in the same children. Additional goal: to identify if any potential changes during the 3-month period may be related to a potential change in salivary-HMW-adiponectin concentration. Methods: Ninety children (7.4 ± 0.3 years) were recruited in primary schools and randomly allocated into control or intervention group. The intervention consisted of a 3-month INT applied during physical education (PE) classes, twice-weekly, while the control group had traditional PE classes. Body mass and height were measured, BMI was calculated and HMW-adiponectin was quantified in saliva. To assess CRF and MS, 800 m-run and hand-dynamometry were applied, respectively. All measurements were performed twice, at baseline and after 3 months. Results: Children with higher baseline salivary-HMW-adiponectin have more favorable BMI (p = 0.006) and slightly higher CRF (p = 0.017) in comparison to the children with lower baseline salivary-HMW-adiponectin. There were no big changes after the 3-month-period neither in the control, nor the INT group. However, it is worthy to note that the INT induced slightly higher increase in salivary-HMW-adiponectin (p = 0.007), and a slightly higher improvement in BMI (p = 0.028), CRF (p = 0.043) and MS (p = 0.003), as compared to the traditional PE classes. Finally, the INT-induced improvement in CRF was associated with the increased post-salivary-HMW-adiponectin concentration (p = 0.022). Conclusion: Main findings may suggest the potential utility of an INT as a cost-effective strategy that can be applied in schools to induce cardio-protective effects in school-children.

Isolation and identification of milk-clotting proteases from Prinsepia utilis Royle and its application in cheese processing.

The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %-50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4-6 and good thermal stability at 30-80 °C. The addition of CaCl2 (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.

Insights into modifiers effects in differential mobility spectrometry: A data science approach for metabolomics and peptidomics.

Utilizing a data-driven approach, this study investigates modifier effects on compensation voltage in differential mobility spectrometry-mass spectrometry (DMS-MS) for metabolites and peptides. Our analysis uncovers specific factors causing signal suppression in small molecules and pinpoints both signal suppression mechanisms and the analytes involved. In peptides, machine learning models discern a relationship between molecular weight, topological polar surface area, peptide charge, and proton transfer-induced signal suppression. The models exhibit robust performance, offering valuable insights for the application of DMS to metabolites and tryptic peptides analysis by DMS-MS.

Amyloid-like Aggregation of Wheat Gluten and Its Components during Cooking: Mechanisms and Structural Characterization.

Amyloid-like aggregation widely occurs during the processing and production of natural proteins, with evidence indicating its presence following the thermal processing of wheat gluten. However, significant gaps remain in understanding the underlying fibrillation mechanisms and structural polymorphisms. In this study, the amyloid-like aggregation behavior of wheat gluten and its components (glutenin and gliadin) during cooking was systematically analyzed through physicochemical assessment and structural characterization. The presence of amyloid-like fibrils (AFs) was confirmed using X-ray diffraction and Congo red staining, while Thioflavin T fluorescence revealed different patterns and rates of AFs growth among wheat gluten, glutenin, and gliadin. AFs in gliadin exhibited linear growth curves, while those in gluten and glutenin showed S-shaped curves, with the shortest lag phase and fastest growth rate (t1/2 = 2.11 min) observed in glutenin. Molecular weight analyses revealed AFs primarily in the 10-15 kDa range, shifting to higher weights over time. Glutenin-derived AFs had the smallest ζ-potential value (-19.5 mV) and the most significant size increase post cooking (approximately 400 nm). AFs in gluten involve interchain reorganization, hydrophobic interactions, and conformational transitions, leading to additional cross ß-sheets. Atomic force microscopy depicted varying fibril structures during cooking, notably longer, taller, and stiffer AFs from glutenin.

Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles.

BACKGROUND: In common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles. RESULTS: By integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7 OE, Glu-1Bx13, Glu-1Bx14 (-) , Glu-1Bx14 (+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14 (+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14 (+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14 (-), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14 (+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis. CONCLUSIONS: Seven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

A clinical study of the local injection of a freshly manufactured 35 kDa hyaluronan fragment for treating chronic wounds.

This study manufactured a 35 kDa hyaluronan fragment (HA35) by enzymatically degrading high-molecular-weight HA using hyaluronidase PH20 derived from bovine testis. The research then examined the therapeutic efficacy of locally administered, tissue-permeable HA35 in alleviating chronic wounds and their associated neuropathic pain. For 20 patients with nonhealing wounds and associated pain lasting over three months, 100 mg of HA35 was injected daily into the healthy skin surrounding the chronic wound for 10 days. Self-assessments before and after treatment indicated that HA35 significantly enhanced wound healing. This was evidenced by the formation of fresh granulation tissue on the wounds (p < 0.0001); reduced darkness, redness, dryness, and damage in the skin surrounding the wounds (p < 0.0001), and a decrease in wound size (p < 0.001). Remarkably, HA35 injections alleviated pain associated with chronic wounds within 24 hours (p < 0.0001). It can be concluded that the low-molecular-weight hyaluronan fragment HA35 potentially enhances the immune response and angiogenesis during wound healing.