Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling.

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFß signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFß and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

Effect of subcritical water flash release processing on buckwheat flour properties.

BACKGROUND: Buckwheat (Fagopyrum esculentum) is rich in bioactive components. However, many of these components are trapped within cellular structures, making them inaccessible. Buckwheat flour was hydrothermally modified using subcritical water coupled with a flash pressure release (SCWF). The effects of the SCWF parameters (120, 140, and 160 °C and hold times of 0, 15, and 30 min) on the flour's structure, physicochemical, and functional properties were studied relative to the raw flour. RESULTS: Treatment deepened the flour color with increasing processing temperatures and hold times. Starch content remained unchanged though its granular structure was disrupted. SCWF treatments lowered total phenolic content compared with the raw flour, except for 160 °C-30 min, where total phenolic content increased by 12.7%. The corresponding antioxidant activities were found consistent with phenolic content. Soluble and insoluble dietary fiber amounts were not substantially influenced at 120 and 140 °C, whereas treatments at 160 °C (15 and 30 min hold) decreased soluble dietary fiber while increasing insoluble dietary fiber. Protein content increased 70-109% in some treatments, suggesting greater protein accessibility. Water-holding capacity significantly increased for flour treated at 120 °C, whereas only slight improvements occurred at 140 and 160 °C. CONCLUSIONS: Subcritical water flash processing can modify the compositional and functional properties of buckwheat flour depending on the choice of reaction conditions. Observed changes were consistent with alteration of the flour's cellular structure and allow some components to become more accessible. The resulting SCWF-modified buckwheat flours provide new food ingredients for potential use in ready-to-eat foods and spreads with improved health benefits. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

ADAMTS6 cleaves the large latent TGFß complex and increases the mechanotension of cells to activate TGFß.

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFß. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFß homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFß-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFß homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFß activation. We found that their expression increases TGFß activation in a dose dependent manner, following stimulation with mature TGFß1. For ADAMTS6, the catalytically active protease is required for effective TGFß activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFß complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFß is perturbed ADAMTS6 can influence TGFß activation via two mechanisms. It directly cleaves the latent TGFß complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFß from large latent complexes which may contribute to disease pathogenesis.

Pulse processing affects gas production by gut bacteria during in vitro fecal fermentation.

Flatulence is one barrier to pulse consumption for many people. Therefore, we examined how processing affects gas production by the microbiome in three classes of pulses. Processing did not affect gas production from Navy beans. However, in Pardina lentils and green peas, (-1.9 ± 0.3 mL/24 h, p < 0.001; -2.3 ± 0.3 mL/24 h, p < 0.001, respectively). In Pardina lentils and green peas, germination diminished carbohydrate utilization by the microbiome compared with unprocessed samples. In Pardina lentils germination reduced abundance germination resulted in the greatest reduction in gas production among six processing methods of amplicon sequence variants (ASVs) from Bacteroides and Lachnospiraceae and reduced propionate production compared with unprocessed samples. In green peas, germination reduced ASVs from Lachnospiraceae, including one from Roseburia, and reduced proportion of butyrate production during fermentation. Three ASVs from Clostridium sensu stricto (cluster 1), Megasphaera elsdenii, and unclassified Veillonellaceae, were strongly associated with increased gas production across all samples (ρ = 0.67-0.69, p < 0.001). This study showed that processing can reduce gas production by the microbiome in some pulses, but also reduces saccharolytic fermentation and production of beneficial microbial metabolites.

Structural studies of elastic fibre and microfibrillar proteins.

Elastic tissues owe their functional properties to the composition of their extracellular matrices, particularly the range of extracellular, multidomain extensible elastic fibre and microfibrillar proteins. These proteins include elastin, fibrillin, latent TGFß binding proteins (LTBPs) and collagens, where their biophysical and biochemical properties not only give the matrix structural integrity, but also play a vital role in the mechanisms that underlie tissue homeostasis. Thus far structural information regarding the structure and hierarchical assembly of these molecules has been challenging and the resolution has been limited due to post-translational modification and their multidomain nature leading to flexibility, which together result in conformational and structural heterogeneity. In this review, we describe some of the matrix proteins found in elastic fibres and the new emerging techniques that can shed light on their structure and dynamic properties.

Quantitative NIR determination of isoflavone and saponin content of ground soybeans.

Over 3200 discrete soybean samples were obtained from production locations around the United States during the years 2012-2016. Ground samples were scanned on near infrared spectrometers (NIRS) and analyzed by HPLC for total isoflavone and total saponin composition, as well as total carbohydrate composition. Multiple Linear Regression (MLR) analysis of preprocessed spectral data was used to develop optimized models to predict isoflavone content. The selection of a suitable calibration model was based on a high regression coefficient (R2), and lower standard error of calibration (SEC) values. Robust validated predictions were obtained for isoflavones, however less than robust calibrations were obtained for the total saponins. The correlations were not as robust for predicting the carbohydrate composition. NIRS is a suitable, rapid, nondestructive method to determine isoflavone composition in ground soybeans. Useful isoflavone composition predictions for large numbers of soybean samples can be obtained from quickly obtained NIRS scans.

Molecular Cloning, Lentiviral Transduction, and Expression of Recombinant ADAMTSL2 and ADAMTSL4.

Lentiviral systems have proven advantageous in the delivery and long-term integration of gene sequences into the genome of several cell types in vitro, in vivo, as well as in clinical trials. Here we detail the protocols involved in the molecular cloning of ADAMTSL2 and ADAMTSL4 into the human immunodeficiency virus (HIV)-derived pCDH lentiviral system. We also describe the lentiviral transduction of ADAMTSL2 and ADAMTSL4 into mammalian HEK293-EBNA cells to create stable cell lines, as well as their recombinant expression.

Purification of Recombinant ADAMTSL2.

Recombinantly produced proteins are used in many biological disciplines. However, their purity and quality are vital for downstream applications used to determine their structure and functions. Several purification and detection strategies can be used in combination to obtain protein samples with homogeneity and structural conformity. Here we detail the protocols involved in the purification of ADAMTSL2 from mammalian cells. We also describe the protocols used to validate the purity of the protein samples.

The role of fibrillin and microfibril binding proteins in elastin and elastic fibre assembly.

Fibrillin is a large evolutionarily ancient extracellular glycoprotein that assembles to form beaded microfibrils which are essential components of most extracellular matrices. Fibrillin microfibrils have specific biomechanical properties to endow animal tissues with limited elasticity, a fundamental feature of the durable function of large blood vessels, skin and lungs. They also form a template for elastin deposition and provide a platform for microfibril-elastin binding proteins to interact in elastic fibre assembly. In addition to their structural role, fibrillin microfibrils mediate cell signalling via integrin and syndecan receptors, and microfibrils sequester transforming growth factor (TGF)ß family growth factors within the matrix to provide a tissue store which is critical for homeostasis and remodelling.

Fibrillin microfibrils and elastic fibre proteins: Functional interactions and extracellular regulation of growth factors.

Fibrillin microfibrils are extensible polymers that endow connective tissues with long-range elasticity and have widespread distributions in both elastic and non-elastic tissues. They act as a template for elastin deposition during elastic fibre formation and are essential for maintaining the integrity of tissues such as blood vessels, lung, skin and ocular ligaments. A reduction in fibrillin is seen in tissues in vascular ageing, chronic obstructive pulmonary disease, skin ageing and UV induced skin damage, and age-related vision deterioration. Most mutations in fibrillin cause Marfan syndrome, a genetic disease characterised by overgrowth of the long bones and other skeletal abnormalities with cardiovascular and eye defects. However, mutations in fibrillin and fibrillin-binding proteins can also cause short-stature pathologies. All of these diseases have been linked to dysregulated growth factor signalling which forms a major functional role for fibrillin.

ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-ß superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill-Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions.

ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10.

Properties of Cookies Made with Natural Wax-Vegetable Oil Organogels.

The aim of this study was to examine the feasibility of cookies in which the conventional margarine is replaced with an organogel of vegetable oil (VO) and natural wax. New cookies from VO organogels contain no trans fats and much less saturated fats than cookies made with a conventional margarine. To understand the effects of different kinds of waxes, organogels were prepared from 4 different waxes including sunflower wax (SW), rice bran wax (RBW), beeswax, and candelilla wax and properties of cookie dough and cookie were evaluated. To investigate the effects of different VOs on the properties of cookies, 3 VOs including olive oil, soybean oil and flaxseed oil representing oils rich in oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3), respectively, were used. Both the wax and VO significantly affected properties of organogel such as firmness and melting behavior shown in differential scanning calorimetry. The highest firmness of organogel was observed with SW and flaxseed oil. Properties of dough such as hardness and melting behavior were also significantly affected by wax and VO while trends were somewhat different from those for organogels. SW and RBW provided greatest hardnesses to cookie dough. However, hardness, spread factor, and fracturability of cookie containing the wax-VO organogel were not significantly affected by different waxes and VOs. Several cookies made with wax-VO organogels showed similar properties to cookies made with a commercial margarine. Therefore, this study shows the high feasibility of utilization of the organogel technology in real foods such as cookies rich in unsaturated fats.

Physical and chemical changes in whey protein concentrate stored at elevated temperature and humidity.

In a case study, we monitored the physical properties of 2 batches of whey protein concentrate (WPC) under adverse storage conditions to provide information on shelf life in hot, humid areas. Whey protein concentrates with 34.9 g of protein/100g (WPC34) and 76.8 g of protein/100g (WPC80) were stored for up to 18 mo under ambient conditions and at elevated temperature and relative humidity. The samples became yellower with storage; those stored at 35 °C were removed from the study by 12 mo because of their unsatisfactory appearance. Decreases in lysine and increases in water activity, volatile compound formation, and powder caking values were observed in many specimens. Levels of aerobic mesophilic bacteria, coliforms, yeast, and mold were <3.85 log10 cfu/g in all samples. Relative humidity was not a factor in most samples. When stored in sealed bags, these samples of WPC34 and WPC80 had a shelf life of 9 mo at 35 °C but at least 18 mo at lower temperatures, which should extend the market for these products.

Detection of Corn Adulteration in Brazilian Coffee (Coffea arabica) by Tocopherol Profiling and Near-Infrared (NIR) Spectroscopy.

Coffee is a high-value commodity that is a target for adulteration, leading to loss of quality and causing significant loss to consumers. Therefore, there is significant interest in developing methods for detecting coffee adulteration and improving the sensitivity and accuracy of these methods. Corn and other lower value crops are potential adulterants, along with sticks and coffee husks. Fourteen pure Brazilian roasted, ground coffee bean samples were adulterated with 1-20% of roasted, ground corn and were analyzed for their tocopherol content and profile by HPLC. They were also analyzed by near-infrared (NIR) spectroscopy. Both proposed methods of detection of corn adulteration displayed a sensitivity of around 5%, thus representing simple and fast analytical methods for detecting adulteration at likely levels of contamination. Further studies should be conducted to verify the results with a much larger sample size and additional types of adulterants.

Navy Bean Flour Particle Size and Protein Content Affect Cake Baking and Batter Quality(1).

Whole navy bean flour and its fine and coarse particle size fractions were used to completely replace wheat flour in cakes. Replacement of wheat flour with whole bean flour significantly increased the protein content. The protein content was adjusted to 3 levels with navy bean starch. The effect of navy bean flour and its fractions at 3 levels of protein on cake batter rheology and cake quality was studied and compared with wheat flour samples. Batters prepared from navy bean flour and its fractions had higher viscosity than the cake flour. Reducing the protein content by addition of starch significantly lowered the viscosity of cake batters. The whole navy bean flour and coarse bean fraction cakes were softer than cakes made with wheat flour but had reduced springiness. Principal component analysis showed a clear discrimination of cakes according to protein. It also showed that low protein navy bean flour cakes were similar to wheat flour cakes. Navy bean flour with protein content adjusted to the level of cake (wheat) flour has potential as a healthy alternative in gluten-free cakes.

Preparation of margarines from organogels of sunflower wax and vegetable oils.

UNLABELLED: It was previously reported that sunflower wax (SW) had high potential as an organogelator for soybean oil-based margarine and spread products. In this study, 12 other vegetable oils were evaluated in a margarine formulation to test feasibility of utilization of SW as an alternative to solid fats in margarine and spread products containing these oils. The minimum quantity of SW required to form a gel with these oils ranged from 0.3% to 1.0% (wt.). Organogels were prepared from the vegetable oils with 3%, 5% and 7% SW and were tested for firmness as well as melting behaviors using differential scanning calorimetry. These organogels were also incorporated into a margarine formulation. All of the vegetable oil organogels produced relatively firm margarines. The margarines prepared from organogels containing 3% (wt.) SW had greater firmness than commercial spreads, whereas margarines made from 7% SW were softer than commercial stick margarines. However, dropping points of the margarine samples were higher than those of commercial spread and margarine products. Margarine firmness was modestly inversely correlated with the amount of polar compounds in the oils and did not correlate with fatty acid compositions. This study demonstrates the feasibility of using a number of healthy vegetable oils rich in polyunsaturated fatty acids to make healthy margarine and spread products by utilizing SW as an organogelator. PRACTICAL APPLICATION: This study showed that sunflower wax could be used as an alternative to traditional solid fats for the development of new margarine and spread products from a variety of healthy vegetable oils.

Amylose-potassium oleate inclusion complex in plain set-style yogurt.

Health and wellness aspirations of U.S. consumers continue to drive the demand for lower fat from inherently beneficial foods such as yogurt. Removing fat from yogurt negatively affects the gel strength, texture, syneresis, and storage of yogurt. Amylose-potassium oleate inclusion complexes (AIC) were used to replace skim milk solids to improve the quality of nonfat yogurt. The effect of AIC on fermentation of yogurt mix and strength of yogurt gel was studied and compared to full-fat samples. Texture, storage modulus, and syneresis of yogurt were observed over 4 weeks of storage at 4 °C. Yogurt mixes having the skim milk solids partially replaced by AIC fermented at a similar rate as yogurt samples with no milk solids replaced and full-fat milk. Initial viscosity was higher for yogurt mixes with AIC. The presence of 3% AIC strengthened the yogurt gel as indicated by texture and rheology measurements. Yogurt samples with 3% AIC maintained the gel strength during storage and resulted in low syneresis after storage for 4 wk.

A simple method to fluorescently label pericytes in the CNS and skeletal muscle.

Pericytes play important roles in vascular control and may form an important part of the blood brain barrier. Here we introduce a simple method for fluorescently labelling pericytes to enable further studies in live or fixed tissue of rats and mice. Following intraperitoneal injection, the fluorescent tracer Fluorogold was rapidly taken up into vascular endothelial cells, and within 3h in the central nervous system appeared within small perivascular cells with a morphology consistent with pericytes. These Fluorogold labelled cells were pericytes since they displayed immunoreactivity for platelet derived growth factor receptor ß and were closely associated with isolectin B4 binding to endothelial cells. Pericytes in skeletal muscle were also labelled with this method, but not those within the heart, lungs or kidney. This simple method could therefore be applied for labelling pericytes in a wide variety of studies, including live cell imaging or immunohistochemistry.

Effect of purified oat ß-glucan on fermentation of set-style yogurt mix.

UNLABELLED: Effect of oat ß-glucan on the fermentation of set-style yogurt was investigated by incorporating 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% of purified oat ß-glucan into the yogurt mix. It was found that levels up to 0.3% resulted in yogurts with quality characteristics similar to the control yogurt. Higher levels of ß-glucan however retarded the fermentation process with noticeable difference in the characteristics of the yogurt. Examination of the morphologies of yogurt with and without ß-glucan revealed that ß-glucan formed aggregates with casein micelle and did not form phase-separated domains. This research demonstrated that ß-glucan could be added to yogurt up to 0.3%, which meets the nutrient guidelines, to have added nutritional benefits. PRACTICAL APPLICATION: Yogurt is known for its beneficial effects on human health and nutrition. Yogurt production and consumption is increasing in the United States every year. However, it is lacking in ß-glucans, which are recognized for their nutritional importance as functional bioactive ingredients. The main objective was to develop and characterize low-fat yogurts with added ß-glucan. This research demonstrated that ß-glucan could be added to yogurt up to 0.3%, which meets the nutrient guidelines for added nutritional benefits, without affecting the characteristics of yogurt significantly. This study will benefit the dairy industry by generating new products offering healthy alternatives.