Bacteroides salyersiae is a potent chondroitin sulfate-degrading species in the human gut microbiota.

Chondroitin sulfate (CS) has widely been used as a symptomatic slow-acting drug or a dietary supplement for the treatment and prevention of osteoarthritis. However, CS could not be absorbed after oral intake due to its polyanionic nature and large molecular weight. Gut microbiota has recently been proposed to play a pivotal role in the metabolism of drugs and nutrients. Nonetheless, how CS is degraded by the human gut microbiota has not been fully characterized. In the present study, we demonstrated that each human gut microbiota was characterized with a unique capability for CS degradation. Degradation and fermentation of CS by the human gut microbiota produced significant amounts of unsaturated CS oligosaccharides (CSOSs) and short-chain fatty acids. To uncover which microbes were responsible for CS degradation, we isolated a total of 586 bacterial strains with a potential CS-degrading capability from 23 human fecal samples. Bacteroides salyersiae was a potent species for CS degradation in the human gut microbiota and produced the highest amount of CSOSs as compared to other well-recognized CS-degraders, including Bacteroides finegoldii, Bacteroides thetaiotaomicron, Bacteroides xylanisolvens, and Bacteroides ovatus. Genomic analysis suggested that B. salyersiae was armed with multiple carbohydrate-active enzymes that could potentially degrade CS into CSOSs. By using a spent medium assay, we further demonstrated that the unsaturated tetrasaccharide (udp4) produced by the primary degrader B. salyersiae could serve as a "public goods" molecule for the growth of Bacteroides stercoris, a secondary CS-degrader that was proficient at fermenting CSOSs but not CS. Taken together, our study provides insights into the metabolism of CS by the human gut microbiota, which has promising implications for the development of medical and nutritional therapies for osteoarthritis. Video Abstract.

Hyaluronan and chondroitin sulfate in chicken-vegetable bone broth delay osteoporosis progression.

Bone broth has recently gained worldwide recognition as a superfood that supplements several nutrients lacking in modern human diets; however, little is known of its efficacy on osteoporosis. Therefore, we aimed to identify the components of chicken-vegetable bone broth (CVBB) that are associated with osteoporosis prevention and verified the efficacy of these components using in vivo studies. In biochemical and cell biological experiments, CVBB was fractionated using ion exchange chromatography (IEC), and the effect of each IEC fraction on osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and quantitative polymerase chain reaction analysis using mouse macrophage-like cells (RAW264 cell). In animal experiments, an ovariectomized (OVX) rat model was generated, followed by whole bone broth (OVX/CVBB) or IEC fraction (OVX/CVBB-Ext) administration and bone structural parameter characterization of OVX rat tibia based on micro-CT. Four CVBB fractions were obtained using IEC, and the fraction containing both hyaluronan and chondroitin sulfate (CVBB-Ext) led to the maximum inhibition of RAW264 cell differentiation. CVBB-Ext downregulated the expression of osteoclast differentiation marker genes. In animal experiments, the OVX group showed a clear decrease in bone density compared to that in the Sham operation group. The OVX/CVBB and OVX/CVBB-Ext groups showed increased bone mineral density and bone volume/tissue volume values compared to those in the OVX/control group. These results suggested that CVBB and CVBB-Ext slowed osteoporosis progression. Therefore, we conclude that hyaluronan and chondroitin sulfate in CVBB are key substances that impede osteoporosis progression. PRACTICAL APPLICATION: This study provides practical information on the effects of bone broth ingredients on osteoporosis to expand the current knowledge on the efficacy of bone broth, which is a widely consumed food. These results may help in the future development of bone broth as a dietary supplement for managing osteoporosis.

Open, Observational, Single-Arm, Multicenter Study Assessing the Effectiveness of a Dietary Supplement Containing Hydrolyzed Collagen, Chondroitin Sulfate, and Glucosamine for Osteoarthritis Pain Reduction.

Osteoarthritis (OA) is an age-related degenerative joint disease with a great impact on patients' well-being and quality of life. This is an observational, open, single-arm multicenter study aimed to evaluate the effectiveness of a nutritional supplement in patients with knee and/or hip OA. A total of 186 patients were recruited from Spanish centers and received a supplement containing hydrolyzed collagen (3000 mg), chondroitin sulfate (800 mg), glucosamine sulfate (700 mg), turmeric extract (250 mg) and devil's claw (150 mg), once daily during 6 months. The primary outcome was the patients' self-perceived pain in the affected joints measured with a visual analogue scale (VAS). Secondary outcome was the patient's functioning, measured with the Lequesne Functional Index and the Western Ontario and McMaster Universities Arthritis Index (WOMAC). Participants showed a significant reduction in self-perceived pain after 3 (mean reduction ± standard deviation, 1.99 ± 1.05) and 6 months (3.57 ± 1.39) of treatment (p < 0.0001 in both comparisons). Lequesne Functional Index score was significantly reduced at 3 months (3.86 ± 2.94) and at 6 months (6.73 ± 4.30) of treatment (p < 0.0001 in both comparisons). The WOMAC index was also significantly reduced after 3 (14.24 ± 10.04) and 6 months (26.43 ± 17.35) of treatment (p < 0.0001 in both comparisons). Significant reductions in WOMAC subdomains (p < 0.0001 in all comparisons) were observed. No severe adverse events were reported during the study. The main results arising from this study show that this nutritional supplementation can improve OA-related symptoms and physical function with a good safety profile in patients with hip and/or knee OA.

Effect of Oral Chondroitin Sulfate Supplementation on Acute Brain Injury in a Murine Necrotizing Enterocolitis Model.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating condition where inflammatory changes and necrosis in the gut results in activation of brain microglia and subsequent neurodevelopmental impairment. Chondroitin sulfate (CS) is a glycosaminoglycan in human breast milk that is absent in conventional formulas. We hypothesized that oral formula supplementation with CS during a murine model of experimental NEC would not only attenuate intestinal injury, but also brain injury. STUDY DESIGN: NEC was induced in mouse pups on postnatal days (PNDs) 5 to 8. Three conditions were studied: (1) breastfed controls, (2) NEC, and (3) NEC+enteral CS (formula+200 mg/kg/d of CS). Pups were euthanized on PND 9 or reunited with dams by the evening of PND 8. Intestinal segments were H&E stained, and immunohistochemistry was performed on brain tissue for Iba-1 to assess for microglial morphology and cortical changes. Neurodevelopmental assays were performed on mice reunited with foster dams on PND 9. Single-cell RNA-sequencing analysis was performed on human intestinal epithelial cells exposed to (1) nothing, (2) hydrogen peroxide (H 2 O 2 ) alone, or (3) H 2 O 2 + CS to look at the differential gene expression between groups. Groups were compared with ANOVA or Kruskal-Wallis tests as appropriate with p < 0.05 considered significant. RESULTS: Compared with NEC, mice treated with oral CS showed improved clinical outcomes, decreased intestinal injury, and attenuated microglial activation and deleterious cortical change. Mice with CS performed better on early neurodevelopmental assays when compared with NEC alone. Single-cell analysis of HIEC-6 cells demonstrated that CS treatment down regulated several inflammatory pathways including nuclear factor κB-suggesting an explanation for the improved Th17 intestinal cytokine profile. CONCLUSIONS: Oral CS supplementation improved both physiological, clinical, and developmental outcomes. These data suggest that CS is a safe compound for formula supplementation for the prevention of NEC.

Multicomponent analysis of dietary supplements containing glucosamine and chondroitin: comparative low- and high-field NMR spectroscopic study.

With the prevalence of glucosamine- and chondroitin-containing dietary supplements for people with osteoarthritis in the marketplace, it is important to have an accurate and reproducible analytical method for the quantitation of these compounds in finished products. NMR spectroscopic method based both on low- (80 MHz) and high- (500-600 MHz) field NMR instrumentation was established, compared and validated for the determination of chondroitin sulfate and glucosamine in dietary supplements. The proposed method was applied for analysis of 20 different dietary supplements. In the majority of cases, quantification results obtained on the low-field NMR spectrometer are similar to those obtained with high-field 500-600 MHz NMR devices. Validation results in terms of accuracy, precision, reproducibility, limit of detection and recovery demonstrated that the developed method is fit for purpose for the marketed products. The NMR method was extended to the analysis of methylsulfonylmethane, adulterant maltodextrin, acetate and inorganic ions. Low-field NMR can be a quicker and cheaper alternative to more expensive high-field NMR measurements for quality control of the investigated dietary supplements. High-field NMR instrumentation can be more favorable for samples with complex composition due to better resolution, simultaneously giving the possibility of analysis of inorganic species such as potassium and chloride.

High gastrointestinal digestive stability endows chondroitin sulfate-soluble undenatured type II collagen complex with high activity: Improvement of osteoarthritis in rats.

Previously, we prepared a chondroitin sulfate-soluble undenatured type II collagen complex (CS-SC II) with low salt content. This paper further explored the differences between CS-SC II and SC II in terms of gastrointestinal digestive characteristics and osteoarthritis (OA) improvement. In vitro and in vivo experiments showed that the gastric digestive stability of CS-SC II was high under both pH 2.0 and pH 3.0, the α1 chain and triple helix structure of type II collagen retained >60 %. However, SC II had high gastric digestive stability only under pH 3.0. Furthermore, intestinal digestion had little effect on α1 chains of CS-SC II and SC II, and distribution experiments showed that they might exert their biological activities in the intestine. CS-SC II had obvious improvement in OA rats at 1.0 mg/kg/d, that is, the joint swelling was significantly reduced and the weight-bearing ratio of the right hind limb was increased to 49 %, which was close to that of 4.0 mg/kg/d SC II. The wear of articular cartilage, Mankin and OARSI scores of rats in CS-SC II group were significantly reduced. The effects of low-dose CS-SC II on the proportion of regulatory T cells (Treg), mRNA expression of OA key biomarkers (Il6, Ccl7, MMP-3 and MMP13) and signaling pathway genes (NF-κB, AKT or AMPKα) were comparable to those of high-dose SC II. These results showed that CS-SC II might have greater potential to improve OA at a lower dose than SC II due to its high gastrointestinal digestive stability at a wide range of pH conditions.

Investigation of Drug-Interaction Potential for Arthritis Dietary Supplements: Chondroitin Sulfate, Glucosamine, and Methylsulfonylmethane.

Osteoarthritis is one of the leading conditions that promote the consumption of these dietary supplements. Chondroitin sulfate, glucosamine, and methylsulfonylmethane are among the prominent alternative treatments for osteoarthritis. In this study, these dietary supplements were incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes, and the formation of marker metabolites was measured to investigate their inhibitory potential on cytochrome P450 enzyme activities. The results revealed no significant inhibitory effects on seven CYPs, consistent with established related research data. Therefore, these substances are anticipated to have a low potential for cytochrome P450-mediated drug interactions with osteoarthritis medications that are likely to be co-administered. However, given the previous reports of interaction cases involving glucosamine, caution is advised regarding dietary supplement-drug interactions.

Minimally Invasive Intradiscal Delivery of BM-MSCs via Fibrous Microscaffold Carriers.

Current treatments of degenerated intervertebral discs often provide only temporary relief or address specific causes, necessitating the exploration of alternative therapies. Cell-based regenerative approaches showed promise in many clinical trials, but limitations such as cell death during injection and a harsh disk environment hinder their effectiveness. Injectable microscaffolds offer a solution by providing a supportive microenvironment for cell delivery and enhancing bioactivity. This study evaluated the safety and feasibility of electrospun nanofibrous microscaffolds modified with chitosan (CH) and chondroitin sulfate (CS) for treating degenerated NP tissue in a large animal model. The microscaffolds facilitated cell attachment and acted as an effective delivery system, preventing cell leakage under a high disc pressure. Combining microscaffolds with bone marrow-derived mesenchymal stromal cells demonstrated no cytotoxic effects and proliferation over the entire microscaffolds. The administration of cells attached to microscaffolds into the NP positively influenced the regeneration process of the intervertebral disc. Injectable poly(l-lactide-co-glycolide) and poly(l-lactide) microscaffolds enriched with CH or CS, having a fibrous structure, showed the potential to promote intervertebral disc regeneration. These features collectively address critical challenges in the fields of tissue engineering and regenerative medicine, particularly in the context of intervertebral disc degeneration.

A Review of Chondroitin Sulfate's Preparation, Properties, Functions, and Applications.

Chondroitin sulfate (CS) is a natural macromolecule polysaccharide that is extensively distributed in a wide variety of organisms. CS is of great interest to researchers due to its many in vitro and in vivo functions. CS production derives from a diverse number of sources, including but not limited to extraction from various animals or fish, bio-synthesis, and fermentation, and its purity and homogeneity can vary greatly. The structural diversity of CS with respect to sulfation and saccharide content endows this molecule with distinct complexity, allowing for functional modification. These multiple functions contribute to the application of CS in medicines, biomaterials, and functional foods. In this article, we discuss the preparation of CS from different sources, the structure of various forms of CS, and its binding to other relevant molecules. Moreover, for the creation of this article, the functions and applications of CS were reviewed, with an emphasis on drug discovery, hydrogel formation, delivery systems, and food supplements. We conclude that analyzing some perspectives on structural modifications and preparation methods could potentially influence future applications of CS in medical and biomaterial research.

Chondroitin sulfate supplementation improves clinical outcomes in a murine model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5-day-old pups. Six groups were studied (n = 9-15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well-established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p < 0.05 considered significant. CS supplementation in formula improved experimental NEC outcomes when compared to NEC alone. CS supplementation resulted in similar improvement in NEC in both the WT and eNOS KO mice. CS supplementation did not result in microbial changes when compared to NEC alone. Our data suggest that although CS supplementation improved outcomes in NEC, this protection is not conferred via the eNOS pathway or alteration of microbial diversity. CS therapy in NEC does improve the intestinal cytokine profile and further experiments will explore the mechanistic role of CS in altering immune pathways in this disease.

Novel Key Ingredients in Urinary Tract Health-The Role of D-mannose, Chondroitin Sulphate, Hyaluronic Acid, and N-acetylcysteine in Urinary Tract Infections (Uroial PLUS®).

Urinary tract infections represent a common and significant health concern worldwide. The high rate of recurrence and the increasing antibiotic resistance of uropathogens are further worsening the current scenario. Nevertheless, novel key ingredients such as D-mannose, chondroitin sulphate, hyaluronic acid, and N-acetylcysteine could represent an important alternative or adjuvant to the prevention and treatment strategies of urinary tract infections. Several studies have indeed evaluated the efficacy and the potential use of these compounds in urinary tract health. In this review, we aimed to summarize the characteristics, the role, and the application of the previously reported compounds, alone and in combination, in urinary tract health, focusing on their potential role in urinary tract infections.

Effect of glycosaminoglycans on the structure and composition of articular cartilage and bone of broilers.

This study aimed to assess the influence of glycosaminoglycan (chondroitin and glucosamine sulfates) supplementation in the diet of broilers on the expression of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 2 (TIMP-2) genes, the synthesis of proteoglycans, collagen type II and chondrocytes, bone and cartilage macroscopy, bone mineral densitometry, bone breaking strength and mineral profile. A completely randomized design was carried out in a 3 × 3 factorial scheme (3 levels of chondroitin sulfate: 0.00, 0.05, and 0.10%; and 3 levels of glucosamine sulfate: 0.00, 0.15, and 0.30%), totaling 9 treatments. At 21 and 42 d of age, broilers were slaughtered, and tibias and femurs were collected for evaluation. There was an interaction (P < 0.05) of sulfates for the expression of MMP-9 and its inhibitor TIMP-2 in femur articular cartilage, as well as for the number of chondrocytes, collagen type II and proteoglycans in tibia articular cartilage, bone and cartilage macroscopy and mineral profile (P < 0.05), with better results obtained with the inclusion of chondroitin and/or glucosamine sulfates in the feed. In conclusion, chondroitin and glucosamine sulfates can be used in broiler diets in order to favor the development of the structure of the locomotor system (bones and joints), thus preventing locomotion problems.

Sulfated and non-sulfated chondroitin affect the composition and metabolism of human colonic microbiota simulated in an in vitro fermentation system.

Chondroitin sulfate (CS) is a family of glycosaminoglycans and have a wide range of applications in dietary supplements and pharmaceutical drugs. In this study, we evaluated the effects of several types of CS, differing in their sulfated positions, on the human colonic microbiota and their metabolites. CS (CSA, CSC, and CSE) and non-sulfated chondroitin (CH) were added into an in vitro human colonic microbiota model with fecal samples from 10 healthy individuals. CS addition showed a tendency to increase the relative abundance of Bacteroides, Eubacterium, and Faecalibacterium, and CSC and CSE addition significantly increased the total number of eubacteria in the culture of the Kobe University Human Intestinal Microbiota Model. CSE addition also resulted in a significant increase in short-chain fatty acid (SCFA) levels. Furthermore, addition with CSC and CSE increased the levels of a wide range of metabolites including lysine, ornithine, and Ile-Pro-Pro, which could have beneficial effects on the host. However, significant increases in the total number of eubacteria, relative abundance of Bacteroides, and SCFA levels were also observed after addition with CH, and the trends in the effects of CH addition on metabolite concentrations were identical to those of CSC and CSE addition. These results provide novel insight into the contribution of the colonic microbiota to the beneficial effects of dietary CS.

[Influence of chronic pain in osteoarthritis on the risk of cardiovascular diseases and modern methods of drug prevention]. / Vliyanie khronicheskoi boli pri osteoartrite na risk razvitiya serdechno-sosudistykh zabolevanii i sovremennye sposoby ikh lekarstvennoi profilaktiki.

The purpose of the review of scientific medical literature was to evaluate the data of the epidemiology of osteoarthritis (OA) and cardiovascular diseases (CVD) with the analysis of risk factors, pathophysiological and pathobiochemical mechanisms of the relationship between OA and the risk of developing CVD in the presence of chronic pain, modern strategies for screening and management of this cohort of patients, the mechanism of action and pharmacological effects of chondroitin sulfate (CS). Conclusions were drawn about the need for additional clinical and observational studies of the efficacy and safety of the parenteral form of CS (Chondroguard) in patients with chronic pain in OA and CVD, improvement of clinical recommendations for the treatment of chronic pain in patients with OA and cardiovascular risk, with special attention to interventions that eliminate mobility restrictions in patients and the inclusion of basic and adjuvant therapy with DMOADs to achieve the goals of multipurpose monotherapy in patients with contraindications to standard therapy drugs.

Nano-elemental selenium particle developed via supramolecular self-assembly of chondroitin sulfate A and Na2SeO3 to repair cartilage lesions.

Cartilage repair is a significant clinical issue due to its restricted ability to regenerate and self-heal after cartilage lesions or degenerative disease. Herein, a nano-elemental selenium particle (chondroitin sulfate A­selenium nanoparticle, CSA-SeNP) is developed by the supramolecular self-assembly of Na2SeO3 and negatively charged chondroitin sulfate A (CSA) via electrostatic interactions or hydrogen bonds followed by in-situ reducing of l-ascorbic acid for cartilage lesions repair. The constructed micelle exhibits a hydrodynamic particle size of 171.50 ± 2.40 nm and an exceptionally high selenium loading capacity (9.05 ± 0.03 %) and can promote chondrocyte proliferation, increase cartilage thickness, and improve the ultrastructure of chondrocytes and organelles. It mainly enhances the sulfation modification of chondroitin sulfate by up-regulating the expression of chondroitin sulfate 4-O sulfotransferase-1, -2, -3, which in turn promotes the expression of aggrecan to repair articular and epiphyseal-plate cartilage lesions. The micelles combine the bio-activity of CSA with selenium nanoparticles (SeNPs), which are less toxic than Na2SeO3, and low doses of CSA-SeNP are even superior to inorganic selenium in repairing cartilage lesions in rats. Thus, the developed CSA-SeNP is anticipated to be a promising selenium supplementation preparation in clinical application to address the difficulty of healing cartilage lesions with outstanding repair effects.

Mixed Conductive, Injectable, and Fluorescent Supramolecular Eutectogel Composites.

Eutectogels are an emerging family of soft ionic materials alternative to ionic liquid gels and organogels, offering fresh perspectives for designing functional dynamic platforms in water-free environments. Herein, the first example of mixed ionic and electronic conducting supramolecular eutectogel composites is reported. A fluorescent glutamic acid-derived low-molecular-weight gelator (LMWG) was found to self-assemble into nanofibrillar networks in deep eutectic solvents (DES)/poly(3,4-ethylenedioxythiophene) (PEDOT): chondroitin sulfate dispersions. These dynamic materials displayed excellent injectability and self-healing properties, high ionic conductivity (up to 10-2  S cm-1 ), good biocompatibility, and fluorescence imaging ability. This set of features turns the mixed conducting supramolecular eutectogels into promising adaptive materials for bioimaging and electrostimulation applications.

Enzymatic preparation of chondroitin sulfate oligosaccharides and its alleviating effect on ovariectomy-induced osteoporosis in rats.

Postmenopausal osteoporosis is the most common type of osteoporosis. Chondroitin sulfate (CS) has been successfully employed as food supplement against osteoarthritis, while the therapeutic potential on postmenopausal osteoporosis is little explored. In this study, CS oligosaccharides (CSOs) were enzymatically prepared through the lysis of CS by a chondroitinase from Microbacterium sp. Strain. The alleviating effects of CS, CSOs and Caltrate D (a clinically used supplement) on ovariectomy (OVX) - induced rat's osteoporosis were comparatively investigated. Our data showed that the prepared CSOs was basically unsaturated CS disaccharide mixture of ∆Di4S (53.1%), ∆Di6S (27.7%) and ∆Di0S (17.7%). 12 weeks' intragastric administration of Caltrate D (250 mg/kg/d), CS or CSOs (500 mg/kg/d, 250 mg/kg/d, 125 mg/kg/d) could obviously regulate the disorder of serum indices, recover the mechanical strength and mineral content of bone, improve the cortical bones' density and the number and length of trabecular bones in OVX rats. Both CS and CSOs in 500 mg/kg/d and 250 mg/kg/d could restore more efficiently the serum indices, bone fracture deflection and femur Ca than Caltrate D. As compared with CS at the same dosage, CSOs exhibited a more significant alleviating effect. These findings suggested that there was great potential of CSOs as daily interventions for delaying the progression of postmenopausal osteoporosis.

Effects of chondroitin sulfate oligosaccharides on osteoclast differentiation of RAW264 cells, and myotube differentiation of C2C12 cells.

Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.

A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates.

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

Stabilization of anthocyanins by simultaneous encapsulation-copigmentation via protein-polysaccharide polyelectrolyte complexes.

This study prepared a series of polyelectrolyte complexes (PECs) composed of heated whey protein isolate (HWPI) and different polysaccharides for simultaneous encapsulation and copigmentation of anthocyanins (ATC) and their ultimate stabilization. Four polysaccharides including chondroitin sulfate, dextran sulfate, gum arabic, and pectin were chosen due to their abilities to simultaneously complex with HWPI and copigment ATC. At pH 4.0, these PECs were formed with an average particle size of 120-360 nm, the ATC encapsulation efficiency of 62-80%, and the production yield of 47-68%, depending on the type of polysaccharides. The PECs effectively inhibited the degradation of ATC during storage and when exposed to neutral pH, ascorbic acid, and heat. Pectin had the best protection, followed by gum arabic, chondroitin sulfate, and dextran sulfate. The stabilizing effects were associated with the hydrogen bonding, hydrophobic and electrostatic interactions between HWPI and polysaccharides, conferring dense internal network and hydrophobic microenvironment in the complexes.