COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction.

Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.

Platelet hyaluronidase 2 enrichment in acute coronary syndromes: a conceivable role in monocyte-platelet aggregate formation.

Acute Coronary Syndromes (ACS) with plaque erosion display dysregulated hyaluronan metabolism, with increased hyaluronidase-2 (HYAL2) expression. However, the expression and the role of this enzyme on platelets has never been explored. We evaluated the platelet's HYAL2 (pltHYAL2) levels on I) stable angina (SA) and II) ACS patients, furtherly sub-grouped in Intact-Fibrous-Cap (IFC) and Ruptured-Fibrous-Cap (RFC), according to Optical Coherence Tomography. We assessed the HYAL2 role through an in vitro model setting of co-cultured monocytes and platelets, before and after treatment with low-molecular-weight hyaluronic acid (HA) as pro-inflammatory stimulus and with or without HYAL2-antibody to inhibit HYAL2 activity. ACS patients exhibit higher pltHYAL2 levels comparing to SA, with the higher expression for IFC group. The addition of HYAL2-antibody significantly reduced the percentage of monocyte-platelet binding, suggesting that pltHYAL2 enrichment at the site of the culprit lesion is a key mediator in the systemic thrombo-inflammatory status of ACS presenting with plaque erosion.

Serum KIAA1199 is an advanced-stage prognostic biomarker and metastatic oncogene in cholangiocarcinoma.

BACKGROUND: Cell proliferation and migration are the determinants of malignant tumor progression, and a better understanding of related genes will lead to the identification of new targets aimed at preventing the spread of cancer. Some studies have shown that KIAA1199 (CEMIP) is a transmembrane protein expressed in many types of noncancerous cells and cancer cells. However, the potential role of KIAA1199 in the progression of cholangiocarcinoma (CCA) remains unclear. RESULTS: Analysis of cancer-related databases showed that KIAA1199 is overexpressed in CCA. ELISA, immunohistochemistry, Western blotting and qPCR indicated high expression levels of KIAA1199 in serum, CCA tissues and CCA cell lines. In the serum (n = 41) and large sample validation (n = 177) cohorts, higher KIAA1199 expression was associated with shorter overall survival and disease-free survival times. At the cellular level, KIAA1199 overexpression (OE) promoted CCA growth and metastasis. Subcutaneous tumor xenograft experiments showed that KIAA1199 enhances CCA cell proliferation. Additionally, the expression levels of components in the EMT-related TGF-ß pathway changed significantly after KIAA1199 upregulation and silencing. CONCLUSION: KIAA1199 is a promising new diagnostic molecule and therapeutic target in CCA. The serum KIAA1199 level can be used as a promising clinical tool for predicting the overall postoperative outcomes of patients with CCA. METHODS: CCA-related KIAA1199 data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. To assess the prognostic impact of KIAA1199, an enzyme-linked immunosorbent assay (ELISA) was used to measure the serum level of KIAA1199 in 41 patients who underwent surgical resection. Immunohistochemical staining, Western blotting and qPCR were used to verify and retrospectively review the expression levels of KIAA1199 in cancer tissue specimens from 177 CCA patients. The effect of KIAA1199 on CCA was evaluated by cell-based functional assays and subcutaneous tumor xenograft experiments. The expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and activation of relevant signaling pathways were measured via Western blotting.

The role of hyaluronic acid and hyaluronidase-1 in obstructive sleep apnoea.

Biological functions of hyaluronic acid (HA) depend on its molecular size. High-molecular weight HA (HMW-HA) is an important component of the endothelial wall and has anti-inflammatory and antioxidant properties. Under inflammation or hypoxia, HMW-HA is degraded by hyaluronidases, such as HYAL-1 resulting in pro-inflammatory low-molecular weight fragments. Obstructive sleep apnoea (OSA) is characterised by intermittent hypoxia and systemic inflammation. Our aim was to evaluate circulating HMW-HA and HYAL-1 in OSA. We recruited 68 patients with OSA and 40 control volunteers. After full-night sleep study blood samples were taken for HMW-HA and HYAL-1 measurements. HYAL-1 levels were significantly higher in patients with OSA compared to controls (0.59/0.31-0.88/ng/mL vs. 0.31/0.31-0.58/ng/mL; p = 0.005) after adjustment for gender, age, BMI and smoking. There was a trend for reduced HMW-HA concentrations in OSA (31.63/18.11-59.25/ng/mL vs. 46.83/25.41-89.95/ng/mL; p = 0.068). Significant correlation was detected between circulating HMW-HA and apnoea-hypopnoea-index (r = - 0.195, p = 0.043), HYAL-1 and apnoea-hypopnoea-index (r = 0.30, p < 0.01) as well as oxygen desaturation index (r = 0.26, p < 0.01). Our results suggest that chronic hypoxia is associated with increased plasma HYAL-1 concentration and accelerated HMW-HA degradation. Altered hyaluronan metabolism may be involved in the inflammatory cascade potentially leading to endothelial dysfunction in OSA.

HYAL-1-induced autophagy facilitates pancreatic fistula for patients who underwent pancreaticoduodenectomy.

The main mechanism of hyaluronidase 1(HYAL-1) in the development of postoperative pancreatic fistula (POPF) after pancreatoduodenectomy (PD) was unknown. In this study, a comprehensive inventory of pre-, intra-, and postoperative clinical and biological data of two cohorts (62 pancreatic cancer [PCa] and 111 pancreatic ductal adenocarcinoma [PDAC]) which could induce POPF were retrospectively analyzed. Then, a total of 7644 genes correlated with HYAL-1 was predicted in PDAC tissues and the enriched pathway, kinase targets and biological process of those correlated genes were evaluated. Finally, a mouse pancreatic fistula (PF) model was first built and in vitro studies were performed to investigate the effects of HYAL-1 on PF progression. Our data indicated that preoperative serum HYAL-1 level, pancreatic fibrosis score, and pancreatic duct size were valuable factors for detecting POPF of Grade B and C. The serum HYAL-1 level of 2.07 mg/ml and pancreatic fibrosis score of 2.5 were proposed as the cutoff values for indicating POPF. The bioinformatic analysis and in vitro and in vivo studies demonstrated that HYAL-1 facilitates pancreatic acinar cell autophagy via the dephosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and signal transducers and activators of transcription 3 (STAT3) signaling pathways, which exacerbate pancreatic secretion and inflammation. In summary, the preoperative serum HYAL-1 was a significant predictor for POPF in patients who underwent PD. Tumor-induced HYAL-1 is one of core risk in accelerating PF and then promoting pancreatic secretion and acute inflammation response through the AMPK and STAT3-induced autophagy.

Effect of Liposomal Delivery of Oxidized Dextran with Isonicotinic Acid Hydrazide on Component Profile of Pulmonary Extracellular Matrix in Mice with BCG-Induced Granulomatosis.

Intraperitoneal injections of isonicotinic acid hydrazide (INH), dextrazide (oxidized dextran+INH), or liposomes loaded with dextrazide (INH dose of 14 mg/kg) over 2 months to mice with BCG-induced granulomatosis started from postinfection day 90 induced qualitative and quantitative changes in composition of pulmonary extracellular matrix. Both dextrazide and its liposomal form decreased the levels of sulfated glycosaminoglycans and uronic acids. In contrast to INH, both preparations did not decrease the levels of total glycosaminoglycans, proteins, and galactose. This difference is explained by the fact both free and liposomal dextrazide activated MMP, but did not increase the content of TIMP-1 and TIMP-2, whereas injection of INH was followed by an increase in TIMP-2 content and a decrease in the level of free hydroxyproline, which attested to down-regulation of collagen degradation and maintenance of the conditions for pulmonary fibrosis in mice of this group.

Knockdown of KIAA1199 suppresses IL-1ß-induced cartilage degradation and inflammatory responses in human chondrocytes through the Wnt/ß-catenin signalling pathway.

The overproduction of proteolytic enzymes and dysregulation of extracellular matrix (ECM) metabolism have been shown to accelerate the degradation process of articular cartilage. The purpose of this study was to investigate the role of KIAA1199 and its association with the pathophysiology of osteoarthritis (OA). We found that the expression of KIAA1199 was significantly upregulated in OA cartilage compared with normal tissues. Serum levels of KIAA1199 were higher in OA patients than in non-OA patients. Furthermore, knockdown of KIAA1199 inhibited interleukin-1 beta (IL-1ß)-induced ECM metabolic imbalance by regulating the expression of A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5; matrix metallopeptidase-13; aggrecan; and COL2A1. In addition, silencing of KIAA1199 significantly decreased the expression of inflammatory mediators such as prostaglandin E2, IL-6, and TNF-α. Mechanistic analyses further revealed that IL-1ß-induced activation of the Wnt/ß-catenin pathway was suppressed during KIAA1199 knockdown. Moreover, KIAA1199 expression was also upregulated in an in vivo rat OA model. Together, these results increase our understanding of the emerging role of KIAA1199 in the process of OA degeneration, and may lead to a novel molecular target to prevent cartilage degradation.

Analyzing Hyaluronidases in Biological Fluids.

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.

Serum levels of hyaluronic acid are associated with COPD severity and predict survival.

Hyaluronic acid (HA) and its degradation products play an important role in lung pathophysiology and airway remodelling in chronic obstructive pulmonary disease (COPD).We investigated if HA and its degrading enzyme hyaluronidase (HYAL)-1 are associated with COPD severity and outcome.Serum HA was assessed in a discovery cohort of 80 COPD patients at stable state and exacerbations. HA, HYAL-1 and HYAL-1 enzymatic activity were evaluated at stable state, exacerbations and 4 weeks after exacerbations in 638 COPD patients from the PROMISE validation cohort.In the discovery cohort, serum HA was higher at exacerbations compared with the stable state (p=0.015). In the validation cohort, HA was higher at moderate and severe exacerbations than at baseline (p<0.001), and remained higher after 4 weeks (p<0.001). HA was strongly predictive for overall survival since it was associated with time to death (p<0.001) independently of adjusted Charlson score, annual exacerbation rate and BODE (body mass, airflow obstruction, dyspnoea, exercise capacity) index. Serum HYAL-1 was increased at moderate (p=0.004) and severe (p=0.003) exacerbations, but decreased after 4 weeks (p<0.001). HYAL-1 enzymatic activity at stable state was inversely correlated with FEV1 % pred (p=0.034) and survival time (p=0.017).Serum HA is associated with COPD severity and predicts overall survival. Degradation of HA is associated with airflow limitation and impairment of lung function.

Increased OGA Expression and Activity in Leukocytes from Patients with Diabetes: Correlation with Inflammation Markers.

O-linked-ß-N-Acetylglucosaminylation (O-GlcNAcylation), a reversible post-translational modification involved in diabetic complications, is regulated by only two enzymes, O-linked N-acetylglucosamine transferase (OGT) and ß-N-Acetylglucosaminidase (OGA). Increased OGA expression has been described previously in blood cells from patients with diabetes and was interpreted as an adaptative response to hyperglycemia-induced O-GlcNAcylation. OGA expression was thus proposed to have potential utility as a diagnostic marker. The present work was undertaken to determine whether determination of OGA enzymatic activity in blood cells could constitute a more rapidly accessible marker than OGA expression level measurements.Blood samples were obtained from patients with type 2 diabetes from the Department of Diabetology of the Cochin Hospital and healthy volunteers from the French blood Agency. OGA enzymatic activity and OGA mRNA expression levels were evaluated in leucocytes from patients with type 2 diabetes and from healthy donors.OGA activity was higher in leucocytes from patients with diabetes compared to control individuals. Surprisingly, OGA activity was not correlated hyperglycaemia markers (blood glucose, fructosamine, HbA1c) but was positively correlated with the inflammatory marker C-reactive protein. OGA mRNA levels were also increased in leucocytes from patients with diabetes and were correlated with mRNA coding for two pro-inflammatory proteins, TNFα and TxNIP.Therefore, OGA activity in leucocytes might be a more easily accessible biomarker than OGA expression levels. However, changes in OGA activity observed in patients with type 2 diabetes may reflect the inflammatory rather than the glycaemic status of these patients.

Effect of diabetes and hyaluronidase on the retinal endothelial glycocalyx in mice.

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW = 155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW = 376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice.

BACKGROUND: Increased vascular permeability is a pathophysiological hallmark of sepsis and results in increased transcapillary leakage of plasma fluid, hypovolemia, and interstitial edema formation. 6% hydroxyethyl starch (HES 130/0.4) is commonly used to treat hypovolemia to maintain adequate organ perfusion and oxygen delivery. The present study was designed to investigate the effects of 6% HES 130/0.4 on glycocalyx integrity and vascular permeability in lipopolysaccharide (LPS)-induced pulmonary inflammation and systemic inflammation in mice. METHODS: 6% HES 130/0.4 or a balanced electrolyte solution (20 ml/kg) was administered intravenously 1 h after cecal ligation and puncture (CLP) or LPS inhalation. Sham-treated animals receiving 6% HES 130/0.4 or the electrolyte solution served as controls. The thickness of the endovascular glycocalyx was visualized by intravital microscopy in lung (LPS inhalation model) or cremaster muscle (CLP model). Syndecan-1, hyaluronic acid, and heparanase levels were measured in blood samples. Vascular permeability in the lungs, liver, kidney, and brain was measured by Evans blue extravasation. RESULTS: Both CLP induction and LPS inhalation resulted in increased vascular permeability in the lung, liver, kidney, and brain. 6% HES 130/0.4 infusion led to significantly reduced plasma levels of syndecan-1, heparanase, and hyaluronic acid, which was accompanied by a preservation of the glycocalyx thickness in postcapillary venules of the cremaster (0.78 ± 0.09 µm vs. 1.39 ± 0.10 µm) and lung capillaries (0.81 ± 0.09 µm vs. 1.49 ± 0.12 µm). CONCLUSIONS: These data suggest that 6% HES 130/0.4 exerts protective effects on glycocalyx integrity and attenuates the increase of vascular permeability during systemic inflammation.

A Polysaccharide from Amusium Pleuronectes Combined with Praziquantel Treatment Ameliorates Hepatic Fibrosis in Schistosoma Japonicum-Infected Mice.

BACKGROUND Polysaccharides from bivalves have multiple bioactivities in various aspects of biology. However, the role of a polysaccharide derived from Amusium pleuronectes on potential hepatoprotective effects remains unclear. MATERIAL AND METHODS A water-soluble polysaccharide was isolated from Amusium pleuronectes (APS-1) using ultrasound-assisted hot-water extraction. The molecular weight of APS-1 was approximately 11.7 kDa and was determined by calibration with dextran. APS-1 was analyzed by high-performance liquid chromatography (HPLC), and mainly consisted of a uniform glucose polymer. The protective effect of APS-1 on Schistosoma japonicum-induced liver fibrosis was investigated in a mouse model. RESULTS Treatment with APS-1 increased serum levels of interleukin (IL)-12 and interferon (IFN)-γ, increased superoxide dismutase (SOD) activity, and decreased levels of IL-13 and IL-5, and hyaluronidase activity. Moreover, immunohistochemical analysis revealed that the collagen content of hepatic tissue of APS-1-treated mice, including that of collagen I, II, and IV, was dramatically decreased. Furthermore, our data showed that combined treatment of APS-1 with praziquantel had more pronounced effects than treatment with either APS-1 or praziquantel alone. CONCLUSIONS Our findings suggest that the treatment using APS-1 in combination with praziquantel attenuated S. japonicum egg-induced hepatic fibrosis, and possessed potent hepatoprotective activity.

Phase 1 trials of PEGylated recombinant human hyaluronidase PH20 in patients with advanced solid tumours.

BACKGROUND: Hyaluronan accumulation in tumour stroma is associated with reduced survival in preclinical cancer models. PEGPH20 degrades hyaluronan to facilitate tumour access for cancer therapies. Our objective was to assess safety and antitumour activity of PEGPH20 in patients with advanced solid tumours. METHODS: In HALO-109-101 (N=14), PEGPH20 was administered intravenously once or twice weekly (0.5 or 50 µg kg-1) or once every 3 weeks (0.5-1.5 µg kg-1). In HALO-109-102 (N=27), PEGPH20 was administered once or twice weekly (0.5-5.0 µg kg-1), with dexamethasone predose and postdose. RESULTS: Dose-limiting toxicities included grade ⩾3 myalgia, arthralgia, and muscle spasms; the maximum tolerated dose was 3.0 µg kg-1 twice weekly. Plasma hyaluronan increased in a dose-dependent manner, achieving steady state by Day 8 in multidose studies. A decrease in tumour hyaluronan level was observed in 5 of the 6 patients with pretreatment and posttreatment tumour biopsies. Exploratory imaging showed changes in tumour perfusion and decreased tumour metabolic activity, consistent with observations in animal models. CONCLUSIONS: The tumour stroma has emerging importance in the development of cancer therapeutics. PEGPH20 3.0 µg kg-1 administered twice weekly is feasible in patients with advanced cancers; exploratory analyses indicate antitumour activity supporting further evaluation of PEGPH20 in solid tumours.

[COMPARATIVE CHARACTERIZATION OF HYALURONIDASE ISOFORMS OF THE 1ST TYPE IN SOMATIC TISSUES AND SERUM OF WHITE RATS].

There are identified 3 proteins with molecular mass of 63, 73 and 132 kDa, exhibiting the properties of the hyaluronidase of the 1st type in the liver, spleen, renal papilla, skin and serum by the method of zymography. A protein with a molecular mass of 73 kDa was detected in all studied tissues and serum, while protein with a molecular mass of 63 kDa was present only in the tissues, and the protein with molecular mass of 132 kDa was present only in the serum. It is discussed the possibility of aggregation of 63 kDa protein molecules in the dimers with the formation of a 132 kDa protein in the serum.

Selective V2-Agonist of Vasopressin Desmopressin Stimulates Activity of Serum Hyaluronidase.

Effects of desmopressin (arginine vasopressin V2 receptor agonist) were studied for elucidation of the possible role of the kidneys in the regulation of blood hyaluronidase activity. Similar to vasopressin, intraperitoneal administration of desmopressin to Wistar rats increased blood hyaluronidase activity by 30%. Against the background of water load, nether desmopressin nor vasopressin increased blood hyaluronidase activity. The possibility of renal secretion of hyaluronidase into the blood as a mechanism of antidiuretic activity of vasopressin and its role in the regulation of water metabolism are discussed.

An upconversion luminescence nanoprobe for the ultrasensitive detection of hyaluronidase.

A new upconversion luminescence nanoprobe for the detection of hyaluronidase has been developed by coupling the hyaluronic acid-bearing upconversion fluorescence nanoparticles (HA-UCNPs) with poly(m-phenylenediamine) (PMPD) nanospheres via covalent linkage. The nanoprobe alone exhibits an extremely low background signal owing to the effective fluorescence quenching by electron-rich PMPD and the near-infrared excitation characteristic (λex = 980 nm) of HA-UCNPs; upon reaction with hyaluronidase, however, a more than 31-fold fluorescence enhancement is produced. Compared with the corresponding nanosystem assembled via physical adsorption, the prepared nanoprobe shows a largely increased stability and a much higher signal-to-background ratio, which offers an ultrasensitive assay for hyaluronidase, with a detection limit of 0.6 ng/mL. The nanoprobe has been successfully used to determine hyaluronidase in human serum samples from both colorectal cancer patients and healthy people, disclosing that the serum hyaluronidase level in colorectal cancer patients is roughly 3 times higher than that in healthy people. Furthermore, the nanoprobe has also been employed to study the activity change of hyaluronidase affected by different concentrations of arsenate (a potential carcinogen), and the results show that even a low dosage of arsenate (50 µg/L) can raise the activity of hyaluronidase by about one-third, revealing the relationship between arsenate and the enzyme. The proposed method is not only simple but also highly sensitive, making it useful to assay hyaluronidase in relevant clinical samples.

The relationship between aortic stiffness and serum hyaluronidase levels in patients with diabetes mellitus and hypertension.

The aim of this study was to investigate the association of serum hyaluronidase and nitric oxide (NO) levels with arterial stiffness in patients with hypertension (HT) and diabetes mellitus (DM). A total of 101 patients with diagnosis of DM and HT were enrolled in this study. The patients were divided into three groups as follows: only hypertensive (I), only diabetic (II) and both diabetic and hypertensive (III). Serum hyaluronidase levels were negatively correlated with aortic strain (AS) and aortic distensibility (AOD) in all groups, whereas a significant positive correlation was noted between serum hyaluronidase levels and aortic strain index (ASI) (all p-values < 0.05). There was a significant negative correlation between serum hyaluronidase and NO levels in all patients (p < 0.001). When the correlation between serum hyaluronidase and serum NO levels was investigated in the individual patient groups, a negative correlation was found in groups I, II and III (p = 0.017, p < 0.001 and p < 0.001, respectively). A significant relationship between plasma hyaluronidase level and parameters of aortic stiffness was found in patients with HT and/or DM. We suggest that the pathophysiological mechanisms responsible for the development of arterial stiffness in subjects with impaired endothelial function may involve pathological changes in the HA metabolism.

DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood.

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC.

Ratiometric fluorescent biosensor for hyaluronidase with hyaluronan as both nanoparticle scaffold and substrate for enzymatic reaction.

Hyaluronidases (HAase) are involved in various physiological and pathological processes and have been reported as urinary marker for bladder cancer. In this study, a novel ratiometric fluorescent sensing system based on both aggregation-induced emission (AIE) and aggregation-induced quenching (ACQ) was developed to quantitatively assess hyaluronidase level. First, a tetraphenylethylene derivative with positive charges (TPE-2N(+), typical AIE molecule) at both ends and an anthracene derivative with positive charge at one end (AN-N(+), typical ACQ molecule) was synthesized. These two positively charged compounds were then mixed with a negatively charged hyaluronan (HA), which induced the aggregation of the compounds as well as the nanoparticles formation as a result of electrostatic complexation, with TPE-2N(+) acting as cross-linking agent. The aggregation also caused the efficient quenching of the emission of AN-N(+) due to ACQ effect, as well as the fluorescence enhancement of TPE-2N(+) due to AIE effect. In the presence of HAase, the enzymatic reaction led to the degradation of HA and triggered disassembly of the nanoparticles; as a result, the emission of AN-N(+) was restored and that of TPE-2N(+) was suppressed. This fluorescence variation affords the system a robust ratiometric biosensor for HAase, and the ratio of fluorescence intensity for AN-N(+) (I414) to that for TPE-2N(+) (I474) can be used as the sensing signal for detecting HAase activity. In this system, hyaluronan serves not only as the scaffold for nanoparticle formation but also as the substrate for enzymatic reaction. This assay system is operable in aqueous media with very low detection limit of 0.0017 U/mL and is capable of detecting HAase in biological fluids such as serum and urine. This strategy may provide a new and effective approach for developing other enzyme assays.