A hyaluronic acid fluorescent hydrogel based on fluorescence resonance energy transfer for sensitive detection of hyaluronidase.

Due to its important role in tumor development and treatment, hyaluronidase (HAase) has been widely investigated in vitro and in vivo. However, such investigation was limited by the absence of sensitive and in situ detection methods. Herein, a hyaluronic acid (HA) hydrogel based on the fluorescence resonance energy transfer (FRET) effect was constructed for the detection of HAase. FITC and AuNPs were covalently coupled with two HA derivatives respectively to form a fluorescent donor-acceptor pair. In the presence of HAase, the hydrogel established by cross-linking of HA derivatives was hydrolyzed specifically. The FRET effect in the hydrogel disappeared and the fluorescence intensity increased proportionally with the changes in the concentration of the HAase. Experiments proved that the HAase sensing system had a wide response range (0.5-100 U/mL), good anti-interference, and excellent biocompatibility. When the hydrogel was used for 3D culture of lung cancer cells, in situ fluorescent response could be achieved. Graphical abstract.

Sensitive Hyaluronidase Biosensor Based on Target-Responsive Hydrogel Using Electronic Balance as Readout.

The development of simple but sensitive methods for hyaluronidase (HAase) detection has been paid a great deal of attention because HAase is a potential cancer marker. In this work, a novel system coupled with a controlled release system has been designed for HAase determination without complex analytical instruments and skilled technicians. Pt@SiO2 nanoparticles (NPs), which can catalyze the breakdown of H2O2 into O2 and H2O, was embedded in the hydrogel constructed by polyethylenimine (PEI) and hyaluronic acid (HA). In the presence of HAase, the hydrogel was broken down as HAase can catalyze the degradation of HA and hence the Pt@SiO2 NPs in the hydrogel was released. The released Pt@SiO2 NPs mixed with H2O2 solution in a drainage device, and then O2 was generated due to the decomposition of H2O2, resulting in an enhancement of pressure in the drainage device because of the low solubility of O2. A certain amount of H2O overflowed from the drainage device because the difference of the pressure between the inner and outer of the drainage device. The overflowed H2O was collected by a tube, and its amount was easily measured by an electronic balance. The weight of the H2O has a linear relationship with the HAase concentration in the range of 1-60 U/mL (120 min enzymatic hydrolysis time) and 0.2-10 U/mL (240 min enzymatic hydrolysis time). The developed system has been applied to detect the activity of HAase in urine samples with satisfied results.

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 µg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes.

Chondroitin sulfate (CS), a linear polysaccharide, is a major component of the cartilage matrix. Although CS plays various roles in several biological and pathological processes, most details regarding its metabolism are still poorly understood. Some CS-degrading enzymes have been identified in mammals, but their expression patterns and localizations remain unclear. Here we present a simple zymography procedure to detect CS-degrading enzymes using salmon nasal cartilage proteoglycans as substrates. This method should be useful to explore CS-degrading enzymes.

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.

Hyaluronic Acid-Modified Porous Silicon Films for the Electrochemical Sensing of Bacterial Hyaluronidase.

The development of enzyme-responsive hyaluronic acid methacrylate (HYAMA)-coated porous silicon (pSi) films and their application in electrochemical diagnostic devices for the in situ detection of the enzyme hyaluronidase (hyal), which is secreted by Staphylococcus aureus (S. aureus) bacteria, are reported. The approach relies on a HYAMA-pSi electrode made of thermally hydrocarbonized pSi (pSi-THC) that is impregnated with crosslinked HYAMA/polyethylene glycol diacrylate (PEGDA) hydrogels. The enzymatic degradation of HYAMA by bacterial hyal is monitored by differential pulse voltammetry (DPV) utilizing pSi-THC as a working electrode and ferro/ferricyanide (FF) as external redox probe. The degradation of HYAMA results in reduced diffusion of the redox probe through the partially charged film, thereby enabling the detection of hyal by DPV. In addition to the determination of the concentration-dependent response in NaOAc buffer (pH 5.2), the detection of hyal as indicator for the presence of S. aureus bacteria above a threshold level in bacterial supernatants and artificial wound fluid is highlighted.

Cationic Carbon Dots for Modification-Free Detection of Hyaluronidase via an Electrostatic-Controlled Ratiometric Fluorescence Assay.

Carbon dots (CDs) emerge as excellent fluorescent nanomaterials, but the full exploitation and application of their exceptional properties in the development of fluorescence assay are still rare. In this work, cationic carbon dots (C-CDs) covered with plenty of positive charges on the surface were synthesized through a facile ultrasonic method. Negatively charged hyaluronic acid (HA) caused the aggregation of positively charged C-CDs and neutral red (NR) along its linear chain via electrostatic adsorption, leading to a remarkable Förster resonance energy transfer (FRET) from C-CDs to NR. However, the presence of hyaluronidase (HAase) resulted in the enzymolysis of HA, as well as the liberation of C-CDs and NR. The corresponding change of fluorescence color from red to green-yellow afforded a reliable ratiometric assay for HAase. Also the ratio of fluorescence intensity for C-CDs (I525) to that for NR (I630) was used for quantitative detection of HAase. The proposed sensing system was easily operated in aqueous media with a detection limit of 0.05 U/mL. This strategy provides a new approach for the wider application of some special CDs in detecting biomolecules.

In Vitro Biochemical Assays for O-GlcNAc-Processing Enzymes.

O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are the only enzymes that regulate the dynamics of protein O-GlcNAcylation. Protein O-GlcNAcylation is an important post-translational modification (PTM) of nuclear and cytoplasmic proteins with O-linked ß-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc and its enzymes are involved in a wide variety of cellular processes and are linked to the pathological progression of chronic diseases. Considering their emerging biological significance, systematic and rapid methods to determine the activities of OGT and OGA have become essential, and several chemical/biochemical methods for measuring the activities of these enzymes have been developed. This minireview mainly focuses on the various biochemical assay methods developed to date, while also providing a description of the fundamental principles underlying the monitoring of O-GlcNAc enzyme activities.

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.

Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells.

Mouse liver lysosomes contain enzymatically active processed forms of Hyal-1.

It has long been known that liver lysosomes contain an endoglycosidase activity able to degrade the high molecular mass glycosaminoglycan hyaluronic acid (HA). The identification and cloning of a hyaluronidase with an acidic pH optimum, Hyal-1, suggested it might be responsible for this activity. However, we previously reported that this hydrolase could only be detected in pre-lysosomal compartments of the mouse liver using a zymography technique that allows the detection of Hyal-1 activity after SDS-PAGE ("renatured protein zymography"). Present work reveals that the activity highlighted by this technique belongs to a precursor form of Hyal-1 and that the lysosomal HA endoglycosidase activity of the mouse liver is accounted for by a proteolytically processed form of Hyal-1 that can only be detected using "native protein zymography". Indeed, the distribution of this form follows the distribution of ß-galactosidase, a well-established lysosomal marker, after fractionation of the mouse liver in a linear sucrose density gradient. In addition, both activities shift toward the lower density region of the gradient when a specific decrease of the lysosomal density is induced by Triton WR-1339 injection. The fact that only native protein zymography but not renatured protein zymography is able to detect Hyal-1 activity in lysosomes points to a non-covalent association of Hyal-1 proteolytic fragments or the existence of closely linked partners supporting Hyal-1 enzymatic activity. The knockdown of Hyal-1 results in an 80% decrease of total acid hyaluronidase activity in the mouse liver, confirming that Hyal-1 is a key actor of HA catabolism in this organ.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis.

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Enhancing hyaluronidase enzyme activity: Insights from advancement in bovine and ovine testicular hyaluronidase purification.

This essay investigates the use of an affinity resin named Capto lentil lectin for the purification of bovine and ovine testicular hyaluronidase. Hyaluronidase, an enzyme that degrades hyaluronic acid, is used widely in medical fields like dermatology, orthopedics, and ophthalmology. The research highlights the importance of optimizing the purification process to increase enzyme activity and purity. A new purification method is proposed, which begins with ammonium sulfate precipitation, followed by Blue Sepharose and Capto Lentil Lectin chromatography. This novel approach significantly increases the yield, purity, and activity of the enzyme. This study paves the way for further research into improving the purification process. The study further discusses challenges in identifying hyaluronidase bands using SDS-PAGE and highlights the necessity of using Western blotting for precise results.

Immunotherapy with commercial venoms is efficacious for anaphylactic reactions to Vespa orientalis stings.

BACKGROUND: Vespa orientalis (VO) stings occasionally induce anaphylaxis. In the absence of commercial VO venom, allergists use commercial venoms for immunotherapy, despite having no indication regarding efficacy. We attempted to examine the effectiveness of immunotherapy with commercial venoms in patients with VO allergy and to identify the venom accountable for this effect. METHODS: Patients who unequivocally identified VO as the culprit insect were administered venom immunotherapy with the commercial venoms available in Israel to which they had positive skin tests. Patients were also skin tested with VO venom sac extracts and, after reaching the maintenance dose, were sting challenged by a live insect. The allergenic components in the venom were determined by immunoblotting. RESULTS: Twelve patients were recruited and, based on their skin test results, all were treated with yellow jacket (YJ) venom, either alone or combined with other venoms. All 8 patients who were sting challenged by VO demonstrated positive skin test responses to VO venom. Six of the stung patients tolerated the sting challenge uneventfully. Two patients developed minimal transient symptoms that resolved spontaneously. SDS-PAGE with patient sera suggested cross-reactivity between VO and YJ venoms at molecular weights of 39-42 kDa. Using phospholipases, antigen 5 and hyaluronidase derived from several Vespa, Dolichovespula and Vespula species, hyaluronidase is possibly accountable for inducing the allergic reaction. CONCLUSION: In the absence of commercial VO venom the practice of treating patients allergic to this insect with available commercial venoms seems to be efficacious and YJ venom is probably responsible for this effect.

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.

Presence of hyaluronidase isoforms in nasal polyps.

BACKGROUND: Nasal polyps are benign lesions originating from the nasal mucosa or paranasal sinuses. The most important etiological factor seems to be increased hydration of epithelium and hyperplasia of the extracellular matrix, which may involve hyaluronan, a high molecular mass extracellular glycosaminoglycan. Degradation of hyaluronan proceeds through the action of specific hyaluronidases. OBJECTIVE: The aim of the present study was to investigate the hydrodynamic size of hyaluronan and the presence of the various hyaluronidase isoforms in nasal polyps. METHODS: Samples of polypoid mucosal tissue and normal nasal mucosa were obtained from twenty patients suffering from nasal polyposis. Zymographic analysis and western blotting were used to detect hyaluronidase activity. RESULTS: The results indicated the presence of hyaluronan of small molecular mass in all samples examined. About one third of it has a mean molecular mass of 240 kDa, exactly that required for the expression of inflammatory response. Laboratory analysis suggested that degradation of hyaluronan occurred through the action of three hyaluronidase isoforms: Hyal-1, Hyal-2 and PH-20. CONCLUSIONS: Since hyaluronan fragments of 200-250 kDa induce the expression of inflammatory cytokines, a specific role of hyaluronidases in the development or progression of nasal polyps may be concluded. Therefore, new treatment protocols may be proposed.

A new method for determination of hyaluronidase activity in biological samples using capillary zone electrophoresis.

The aim of the study was to develop a new capillary zone electrophoresis (CZE) method for determination of enzymatic activity of hyaluronidase. The method permits monitoring of the process of hyaluronic acid digestion by hyaluronidase. Studies were performed using CZE instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter 75 µm. Separation was performed in the phosphate buffer (pH 8.10) in the electric field of 20 kV, λ = 220 nm. The procedure was based on mixing a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate the activity of hyaluronidase originating from different sources with high precision and accuracy. t-Tests showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The developed method is characterized by a short duration of analysis, low volume of analyzed sample, small amount of buffers used and low cost of analysis.

Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1ß stimulated human chondrocytes cells.

BACKGROUND: Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities. METHODS: A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1ß (IL-1ß) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated. RESULTS: Bark extract of Payena dasyphylla (100 µg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1ß (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 µg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 µg/mL. CONCLUSION: These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property.

Hyaluronidase Availability in Emergency Rooms: A Statewide Analysis.

Objective: Authors sought to determine the immediate availability of hyaluronidase (HYAL) among emergency rooms (ERs) in California. Background: Hyaluronic acid (HA) fillers are regarded as a safe procedure; however, major ischemic complications do exist, notably blindness and tissue necrosis. The successful management of these vascular events relies on an injector's immediate HYAL, the enzymatic reversal agent for HA. Unfortunately, many barriers exist for injector sites to stock HYAL. As a result, ERs serve as unofficial safety nets in cases when providers encounter an ischemic complication and do not have HYAL in supply. Materials and Methods: Telephone survey inquiring about HYAL availability in all California ERs. Results: This study included 330 California ERs and achieved an 89.7% response rate (n = 296). 45.6% of the surveyed ERs did not have immediate access to HYAL. HYAL availability was positively associated with level I-III adult trauma center status, pediatric trauma center status, children's hospital status, higher bed counts, and regional geography (p < 0.05, all). Conclusions: HYAL availability is unreliable among Californian ERs, posing a potential risk to patient safety.

Haskap Berry Leaves (Lonicera caerulea L.)-The Favorable Potential of Medical Use.

The presented research evaluates the medical use potential of Lonicera caerulea leaves, which are waste plants in cultivating berries. The study's screening activity included the leaves of five varieties of Lonicera caerulea: Atut, Duet, Wojtek, Zojka, and Jugana. The microbiological analysis confirmed the safety of using Lonicera caerulea leaves without significant stabilization. Lonicera caerulea leaves standardization was carried out based on the results of the chromatographic analysis, and it showed differences in the contents of active compounds (loganic, chlorogenic and caffeic acids, and rutin), which are attributed to biological activity. For the Lonicera caerulea leaves varieties tested, the differences in the content of total polyphenol content, chlorophylls, and carotenoids were also confirmed. The screening of biological activity of five Lonicera caerulea leaf varieties was carried out concerning the possibility of inhibiting the activity of α-glucosidase, lipase, and hyaluronidase as well, and the antioxidant potential was determined. The defined profile of the biological activity of Lonicera caerulea leaves makes it possible to indicate this raw material as an essential material supporting the prevention and treatment of type II diabetes. However, this research showed that tested enzymes were strongly inhibited by the variety Jugana. The health-promoting potential of Lonicera caerulea leaves was correlated with the highest chlorogenic acid and rutin content in the variety Jugana.