Enterococcus faecalis is a common cause of healthcare-acquired bloodstream infections and catheter-associated urinary tract infections (CAUTIs) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but the function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, ΔhylA exhibited defects in bladder colonization and dissemination to the bloodstream, and ΔhylB exhibited a defect in kidney colonization. Furthermore, a ΔhylAΔhylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both hyaluronic acid (HA) and chondroitin sulfate in vitro, while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during the stationary phase and also contributed to dampening of lipopolysaccharide-mediated NF-κB activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

In non-muscle invasive bladder cancer, Bacillus Calmette-Guérin (BCG) responders benefit from strong Th1-type inflammatory and T cell responses mediating tumor rejection. However, the corresponding lack of anti-inflammatory Th2-type immunity impairs tissue repair in the bladder wall and facilitates the development of cystitis, causing urinary pain, urgency, incontinence, and frequency. Mechanistically, the leakage of the glycosaminoglycan (GAG) layer enables an influx of potassium ions, bacteria, and urine solutes towards the underlying bladder tissue, promoting chronic inflammation. Treatments directed towards re-establishing this mucopolysaccharide-based protective barrier are urgently needed. We discuss the pathomechanisms, as well as the therapeutic rationale of how chondroitin and hyaluronic acid instillations can reduce or prevent BCG-induced irritative bladder symptoms. Moreover, we present a case series of five patients with refractory BCG-induced cystitis successfully treated with combined chondroitin and hyaluronic acid instillations.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

The nervous system is primarily composed of neurons and glia, and the communication between them plays profound roles in regulating the development and function of the brain. Neuron-glia signal transduction is known to be mediated by secreted or juxtacrine signals through ligand-receptor interactions on the cell membrane. Here, we report a novel mechanism for neuron-glia signal transduction, wherein neurons transmit proteins to glia through extracellular vesicles, activating glial signaling pathways. We find that in the amphid sensory organ of Caenorhabditis elegans, different sensory neurons exhibit varying aging rates. This discrepancy in aging is governed by the crosstalk between neurons and glia. We demonstrate that early-aged neurons can transmit heat shock proteins (HSP) to glia via extracellular vesicles. These neuronal HSPs activate the IRE1-XBP1 pathway, further increasing their expression in glia, forming a positive feedback loop. Ultimately, the activation of the IRE1-XBP-1 pathway leads to the transcriptional regulation of chondroitin synthases to protect glia-embedded neurons from aging-associated functional decline. Therefore, our studies unveil a novel mechanism for neuron-glia communication in the nervous system and provide new insights into our understanding of brain aging.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-β-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-β-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-β-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the β-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

The formulation of microparticles composed of a mixture of carriers represents an innovative approach for lung drug delivery of dry powder. The carriers used can significantly influence the properties of the microparticles, such as size, shape, surface area, hygroscopicity, or aggregation, thus improving the aerosolization of the drugs after inhalation. The properties mentioned above are crucial for effective  pulmonary  therapy. The  combination of carriers of a carbohydrate nature and gelling agents is advantageous for controlled drug release. The experimental work aimed to prepare by spray drying and subsequently evaluate ten batches of microparticles composed of sugar-based carriers (mannitol, maltodextrin, dextran) and gelling polymers (chitosan, chondroitin sulfate) and to select a suitable combination for follow-up experimental work aimed at drug incorporation into the microparticle matrix. The most suitable parameters were exhibited by batches whose aerodynamic diameter was close to 5 µm, particles prepared from a combination of mannitol and dextran, chitosan and chondroitin, or maltodextrin and chondroitin. These batches also showed the highest fine particle fraction value (> 43%). From a processability point of view, the batch with maltodextrin and chondroitin is preferable due to the lower viscosity of the dispersion and the more regular shape of the final microparticles.

Colon cancer is a common cancer with high mortality globally. The role of chondroitin polymerizing factor (CHPF) has been elucidated in various cancers. However, its role and mechanism remain unknown in colon cancer. CHPF expression was examined by GEPIA database, reverse transcription-quantitative polymerase chain reaction and western blot. The relationship between CHPF expression and the clinicopathologic characteristics as well as miR-214-3p level was determined in colon cancer patients. The role and mechanism of CHPF in the growth, ferroptosis, and glycolysis of colon cancer cells were evaluated by cell counting kit-8, biochemical detections, luciferase, and western blot experiments. Additionally, the role of CHPF was explored in xenografted mice. CHPF expression was increased and was related to advanced TNM stage, poor differentiation and shorter overall survival in patients with colon cancer. Knockdown of CHPF inhibited colon cancer cell growth, and downregulated the expression of proteins involving in ferroptosis and glycolysis both in vitro and in vivo. Besides, CHPF silencing increased the levels of ferrous iron and ROS, but decreased glucose uptake, lactate product, and ATP level in vitro. Mechanically, miR-214-3p directly targeted CHPF and negatively regulated its expression. Upregulation of miR-214-3p reduced cell viability, glucose uptake, lactate product, and ATP level, but increased the levels of ferrous iron and ROS, which were reversed by the overexpression of CHPF. Upregulation of CHPF predicted poor prognosis, and miR-214-3p/CHPF axis inhibited growth, downregulated the levels of glycolysis-related indexes, and promoted ferroptosis in colon cancer cells.

The formulation of microparticles composed of a mixture of carriers represents an innovative approach for lung drug delivery of dry powder. The carriers used can significantly influence the properties of the microparticles, such as size, shape, surface area, hygroscopicity, or aggregation, thus improving the aerosolization of the drugs after inhalation. The properties mentioned above are crucial for effective  pulmonary  therapy. The  combination of carriers of a carbohydrate nature and gelling agents is advantageous for controlled drug release. The experimental work aimed to prepare by spray drying and subsequently evaluate ten batches of microparticles composed of sugar-based carriers (mannitol, maltodextrin, dextran) and gelling polymers (chitosan, chondroitin sulfate) and to select a suitable combination for follow-up experimental work aimed at drug incorporation into the microparticle matrix. The most suitable parameters were exhibited by batches whose aerodynamic diameter was close to 5 µm, particles prepared from a combination of mannitol and dextran, chitosan and chondroitin, or maltodextrin and chondroitin. These batches also showed the highest fine particle fraction value (> 43%). From a processability point of view, the batch with maltodextrin and chondroitin is preferable due to the lower viscosity of the dispersion and the more regular shape of the final microparticles.

Reprogramming metabolic flux is a promising approach for constructing efficient microbial cell factories (MCFs) to produce chemicals. However, how to boost the transmission efficiency of metabolic flux is still challenging in complex metabolic pathways. In this study, metabolic flux is systematically reprogrammed by regulating flux size, flux direction, and flux rate to build an efficient MCF for chondroitin production. The ammoniation pool for UDP-GalNAc synthesis and the carbonization pool for UDP-GlcA synthesis are first enlarged to increase flux size for providing enough precursors for chondroitin biosynthesis. Then, the ammoniation pool and the carbonization pool are rematched using molecular valves to shift flux direction from cell growth to chondroitin biosynthesis. Next, the adaptability of polymerization pool with the ammoniation and carbonization pools is fine-tuned by dynamic and static valve-based adapters to accelerate flux rate for polymerizing UDP-GalNAc and UDP-GlcA to produce chondroitin. Finally, the engineered strain E. coli F51 is able to produce 9.2 g L-1 chondroitin in a 5-L bioreactor. This strategy shown here provides a systematical approach for regulating metabolic flux in complex metabolic pathways for efficient biosynthesis of chemicals.

The gastrointestinal (GI) mucosal barrier is often exposed to inflammatory and erosive insults, resulting in gastric lesions. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), chondroitin sulfate (CS), and N-acetylglucosamine (NAG) have shown potential beneficial effects as GI protectants. This study aimed to evaluate the gastroprotective effects of oral GAGs in rats with indomethacin-induced GI lesions. Forty-five Sprague-Dawley rats (8-9 weeks-old, 228 ± 7 g) were included in the study, divided into five study groups, and given, administered orally, either sucralfate (positive control group; PC), NAG (G group), sodium alginate plus HA and CS (AHC group), sodium alginate plus HA, CS, and NAG (AHCG group), or no treatment (negative control group; NC). Animals were administered 12.5 mg/kg indomethacin orally 15 min after receiving the assigned treatment. After 4 h, stomach samples were obtained and used to perform a macroscopic evaluation of gastric lesions and to allow histological assessment of the gastric wall (via H/E staining) and mucous (via PAS staining). The AHCG group showed significant gastroprotective improvements compared to the NC group, and a similar efficacy to the PC group. This combination of sodium alginate with GAGs might, therefore, become a safe and effective alternative to prescription drugs for gastric lesions, such as sucralfate, and have potential usefulness in companion animals.