Genome-Wide Characterization of the Hyaluronidase Gene Family and Their Potential Roles in Viviparous Black Rockfish(Sebastes schlegeli)

Black rockfish(Sebastes schlegelii)has evolved viviparous reproduction mode.Different from the oviparous fishes,the spermatozoa of black rockfish are stored on the surface of ovarian follicle for over four months until the egg mature in ovary,while the adaptive evolution of fertilization-related genes remains to be studied.In the present study,based on the genome and transcriptome information of black rockfish,a total of 10 hyaluronidase genes were identified by phylogenetic and sequence analyses,including hyal1,hyal2,hyal3,hyal4,hyal6 and spam1a,spam1b,spam1c and spam1d.The spam1 subfamilies,including spam1a,spam1b,spam1c and spam1d,were significantly expanded compared to other species.The adaptive evolution of hyaluronidase was further investigated by selection pressure analysis of branch model and branch site model.The results showed that only spam1 subfamily was positively selected with a large number of positive selection sites,and the evolution rate was significantly higher thanthose of other teleosts.Two positively selected sites,LYS-171 and GLY-164,were located in the neutral hyaluronidase activity domain,which was pivotal for SPAM1 to participate in enzymatic hydrolysis of the extracellular hyaluronic acid matrix and mediate acrosome reaction in mammals.Furthermore,spam1 was mainly expressed in spermatocytes,secondary spermatocytes,and spermatids of testis,whereas it was not found in metamorphic sperm and mature sperm based on the spatiotemporal expression analysis.All results indicated that spam1 might originate from the testis and might be an adaptation of viviparous trait in black rockfish.

Hyaluronidase in sub-Tenon's anesthesia for phacoemulsification,a double-blind randomized clinical trial

AIM:To investigate the effect of hyaluronidase use on the quality of sub-Tenon's anaesthesia for phacoemulsification.· METHODS:This was a randomized,double-blind clinical trial which was conducted at Nikookari Eye Hospital for 5 months.Forty-two eyes of candidates for phacoemulsification under sub-Tenon's anaesthesia were randomly allocated to two equal groups and received either 2 mL of lidocaine 2% solution with(LH),or without(L) addition of hyaluronidase(150IU/mL).Akinesia was assessed 15 minutes after sub-Tenon's injection.Patients and surgeon's satisfaction,as well as the postoperative pain(the visual analogue scale,VAS) were investigated after operation.The contingency tables(including the Chi-square or Fisher's exact tests,when appropriate) and parametric analysis(the independent samples t test) were used for statistical analysis.· RESULTS:Complete akinesia(33.3% vs 4.8%,P =0.04),as well as the patients(85.7% vs 57.1%,P =0.04)and surgeon's satisfaction(87.5% vs 52.4%,P =0.02) were significantly more frequent in LH than in L group.The mean VAS was significantly lower in the same group(1.90±1.45 vs 3.00±1.55,P =0.04).· CONCLUSION:Addition of hyaluronidase to lidocaine solution for sub-Tenon's anesthesia significantly improves the ocular akinesia,enhances the intra-operative patients and surgeons' satisfaction,and attenuates the postoperative pain.

Analysis of Hyaluronidase Expression by qPCR in Egyptian Clinical Isolates of <i>Staphylococcus aureus</i>and Its Correlation with Phenotypic Plate Assay

Hyaluronidase enzyme (HysA) is an extracellular enzyme that is considered to be an important virulence factor for Staphylococcus aureus. We screened the production of HysA enzyme in the spent media of Egyptian clinical isolates (32 isolates) via phenotypic plate assay. We found that 75% of the isolates (24 isolates) were able to produce HysA enzyme. We designed primers for qPCR analysis of hysA mRNA expression that was derived from the alignment of hysA gene sequences of 41 strains of S. aureus. The designed primers could be used for the amplification of hysA in 79.2% of the isolates (19 isolates) that were positive for HysA production as demonstrated by phenotypic plate assay. A significant positive correlation, as indicated by Pearson correlation analysis (r = 0.84 at P < 0.001), was found between phenotypic plate assay and qPCR of mRNA expression of hysA in the investigated isolates of S. aureus. In conclusion, we analyzed for the first time hysA mRNA expression via qPCR in S. aureus. Additionally, our work showed a good agreement between the phenotypic assay of HysA production via plate assay and hysA expression in S. aureus. The qPCR analysis of this study could be used as a more reliable quantitative method for hysA expression analysis particularly in infected animal models of S. aureus.

Therapeutic Use of Hyaluronidase in Obstetrics

Introduction: Hyaluronidases are a group of enzymes that permit greater diffusion of fluid through the tissues. These enzymes have the ability to reduce the viscosity of hyaluronic acid and increase cellular membrane and blood vessel permeability. This review discusses the indication, usage, effects, and safety of hyaluronidases in obstetrics. Materials and Methods: MEDLINE/ PubMed and the Cochrane Library were searched using the following terms: (“hyaluronidase” [Title/Abstract]) OR (“hyaluronidases” [Title/Abstract]) AND (“therapeutic use” [Title/Abstract]) OR (“therapeutic uses” [Title/Abstract]) OR (“perineal trauma” [Title/Abstract]) OR (“perineal tear” [Title/Abstract]) OR (“cervical ripening” [Title/Abstract]). Results: This review included four randomized controlled trials (RCT) that randomized a total of 642 pregnant women and two interventional non-RCTs that included a total of 2824 pregnant women. The data from two RCTs suggested that the incidence of perineal trauma was significantly lower in the intervention (perineal hyaluronidase injection in stage two of labor) than the control group (no intervention). However, both groups did not differ significantly in the incidence of first- and second-degree tears. Similarly, no significant difference in the incidence of episiotomy was found between both groups. In one interventional non-RCT, the administration of intracervical hyaluronidase was associated with a statistically significant acceleration and shortening of labor by approximately 1.95 hours after the injection of intracervical hyaluronidase. However, it had no effect on uterine contractions or the duration of stages two and three labor. Conclusions: While it is safe, clinicians should consider patient acceptance and the effectiveness of hyaluronidase compared to conventional less invasive methods.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Cationic conjugated polymers for homogeneous and sensitive fluorescence detection of hyaluronidase

The cationic charged water-soluble polyfluorenes containing 2,1,3-benzothiadiazole (BT) units (P1-3) have been synthesized and characterized. These polymers demonstrate intramolecular energy transfer from the fluorene units to the BT sites when oppositely charged hyaluronan is added due to the formation of electrostatic complexes, followed by a shift in emission color from blue to green or brown. Upon adding hyaluronidase, the hyaluronan is cleaved into fragments. The relatively weak electrostatic interactions of hyaluronan fragments with polyfluorenes keep their main chains separated and energy transfer from the fluorene units to the BT sites is inefficient, and the polyfluorenes recover their blue emissions. The complexes of conjugated polymers/hyaluronan can be utilized as probes for sensitive and facile fluorescence assays for hyaluronidase. The new assay method interfaces with the aggregation and light harvesting properties of conjugated polymers.

Sperm hyaluronidase is critical to mammals'fertilization for its ability to disperse cumulus-oocyte complex layer

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus-oocyte complex(COC);however,their role in mammalian fertilization remains unclear.Previously,we have shown that hyaluronidase 5(Hyal5)/Hyal7 double-knockout(dKO)mice produce significantly fewer offspring than their wild-type(WT)counterparts because of defective COC dispersal.Male infertility is mainly caused by a low sperm count.It can be further exacerbated by the deficiency of sperm hyaluronidase,which disperses the cumulus cells of the outer layer of the COC.In the current study,we evaluated the effects of a low count of Hyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model.Our results demonstrated that a low sperm count further decreases the in vitro fertilization(IVF)rate of Hyal-deficient dKO spermatozoa.In addition,the dKO spermatozoa resulted in a fertilization rate of 12.5%upon fertilizing COCs with a thick cumulus layer,whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized.Finally,we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal.Our results suggest that a deficiency of proteins involved in fertilization,such as sperm hyal,has a vital role in fertilization.

Two-dimensional LDH nanodisks modified with hyaluronidase enable enhanced tumor penetration and augmented chemotherapy

The condensed tumor extracellular matrix(ECM)consisting of cross-linked hyaluronic acid(HA)is one of the key factors that result in the aberrant tumor microenvironment and severely impair drug delivery and tumor penetration.Herein,we report a simple design of a hyaluronidase(HAase)-modified layered double hydroxide(LDH)nanoplatform loaded with anticancer drug doxorubicin(DOX)for enhanced tumor penetration and augmented chemotherapy.In our approach,LDH nanodisks were synthesized via a co-precipitation method,modified with HAase by electrostatic attraction,and finally physically loaded with DOX.The formulated DOX/LDH-HAase complexes show a high DOX loading percentage of 34.2%with good colloidal stability,retain 86.1%of the enzyme activity,and release DOX in a pH-responsive manner having a faster release rate under slightly acidic tumor microenvironment than that under a physiological condition.With the catalytic activity of HAase to digest the HA nearby the cancer cells,the developed DOX/LDH-HAase complexes enable more significant uptake by cancer cells and penetration in 3-dimensional tumor spheroids than enzyme-free DOX/LDH complexes,thus displaying much better antitumor efficacy in vitro than the latter.The more significant tumor penetration and inhibition of the DOX/LDH-HAase complexes than that of the DOX/LDH complexes was further demonstrated by in vivo tumor imaging and therapeutic activity assessments.Our study suggests a unique nanomedicine platform combined with both anticancer drug and enzyme for improved tumor penetration and chemotherapy,which is promising for effective chemotherapy of different types of stroma-rich tumors.

A Novel Enzyme-Responded Controlled Release Electrochemical Biosensor for Hyaluronidase Activity Detection

Hyaluronidase(HAase)has been regarded as one of the potential cancer biomarkers.In this study,a novel enzyme-responded controlled release electrochemical biosensor has been constructed for HAase detection.Methylene blue(MB)can be adsorbed on hyaluronic acid(HA)to form macromolecular HA–MB complex first through electrostatic interaction between anionic HA and positively charged MB.The prepared complex cannot be filtered out through centrifugal ultrafiltration,and small electrochemical signal can be recorded from the ultrafiltrate.With the help of HAase,HA–MB complex can be cleaved into small pieces through enzyme digestion.The small pieces of HA–MB complex can be easily released from the macromolecular HA–MB complex to the ultrafiltrate by centrifugal ultrafiltration.Then,the enhanced electrochemical signal from MB can be detected in the above-mentioned ultrafiltrate.The enhanced diff erential pulse voltammetric response of the system has a linear relationship with HAase concentration in the range of 4.0–60 U·mL^(-1),with the limit of detection of 0.28 U·mL^(-1).The developed strategy has been successfully applied to detect HAase activity in urine samples.

Overview, prevention and management of chemotherapy extravasation

Chemotherapy extravasation remains an accidental complication of chemotherapy administration and may result in serious damage to patients. We review in this article the clinical aspects of chemotherapy extravasation and latest advances in definitions, classification, pre-vention, management and guidelines. We review the grading of extravasation and tissue damage according to various chemotherapeutic drugs and present an update on treatment and new antidotes including dexrazoxane for anthracyclines extravasation. We highlight the importance of education and training of the oncology team for prevention and prompt pharmacological and non-pharmacological management and stress the availability of new antidotes like dexrazoxane wherever anthracyclines are being infused.

Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism

Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency(Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized.

Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 &mu;L, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 &plusmn;87) GC per antral follicle of which 82% &plusmn;5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 &plusmn;16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.

Abscess Formation as a Complication of Injectable Fillers

Importance: Dermal filler use in aesthetic clinics, are now widespread and although complications are rare, the formation of granulomas or abscesses and subsequent defects can be devastating to the patient. Design: Retrospective chart review of 4 cases over the period of 10 years, from 2002-2012 were examined from The Nasal and Facial Plastic Cosmetic Surgery Institute. Results: Four female patients experienced delayed onset reactions (>2 weeks) with sterile abscess formation and eventual resolution with serial drainage and macrolide antibiotics were observed over a prolonged period until resolution occurred. Only 1 case identified an organism (streptococci) on culture after 8 months, however, the initial culture still showed only sterile abscess. All 4 had a history of previous injectable fillers, 2 patients had evidence of pre existing autoimmune disorders. Conclusions and Relevance: Since the treatment of all of these patients, there is new evidence that infections may present as delayed onset sterile abscesses due to biofilm formation. Fluorescent in situ hybridization (FISH) test has shown to be as specific in identifying responsible organisms in biofilm infections as simple culture but is more sensitive;thus preventing misdiagnosis of sterile abscess. Counter intuitively steroid injection may promote abscesses, while hyaluronidase may be useful.

Hyaluronic acid association with bacterial,fungal and viral infections:Can hyaluronic acid be used as an antimicrobial polymer for biomedical and pharmaceutical applications?

The relationships between hyaluronic acid(HA)and pathological microorganisms incite new understandings on microbial infection,tissue penetration,disease progression and lastly,potential treatments.These understandings are important for the advancement of next generation antimicrobial therapeutical strategies for the control of healthcare-associated infections.Herein,this review will focus on the interplay between HA,bacteria,fungi,and viruses.This review will also comprehensively detail and discuss the antimicrobial activity displayed by various HA molecular weights for a variety of biomedical and pharmaceutical applications,including microbiology,pharmaceutics,and tissue engineering.

An oxygen reservoir-irrigated photoimmunotherapy of malignant melanoma

Tumor hypoxia is the pivotal factor limiting the therapeutic efficacy of photodynamic therapy(PDT),and can be partly improved by either the oxygen economizing or the oxygen supplementation strategies.Nevertheless,the current studies scarcely integrated the merits of both strategies and neglected the bottleneck of poor oxygen infiltration in deep tumors,resulting in PDT resistance.Herein,we developed an oxygen reservoir-irrigated PDT which integrates oxygen supply,oxygen economizing,and oxygen infiltration altogether.Specifically,mitochondria-targeted mesoporous prussian blue nanoparticles(Ce6@TPB)were fabricated to bridge the gap between oxygen economizing and oxygen supplementation by reducing oxygen output while increasing oxygen input.Hyaluronidase-loaded microneedles were further developed to pave the way for deep PDT with increased infusion of oxygen and photosensitizer by degrading dense extracellular matrix.The modulation of tumor oxygenation and permeability during PDT leads to the complete eradication of primary melanoma and strong immunogenic cell death.Its further combination with checkpoint-blockade inhibitor greatly suppressed the proliferation of distal tumors by reprogramming immune microenvironments,as evidenced by the depletion of M2 macrophage,increased infiltration of anti-tumor immune cells,and elevated excretion of immune cytokines.Therefore,such an oxygen reservoir-irrigated PDT potentiates powerful photoimmunotherapy and provides a favorable prospect for tumor treatment.

A HAase/NIR responsive surface on titanium implants for treating bacterial infection and improving osseointegration

Bacterial infection and insufficient osseointegration are critical factors affecting the long-term success of titanium-based implants.Unfortunately,the direct application of antibiotic on Ti implants easily leads to poor cytocompatibility,as well as the production of drug-resistant bacteria.So,in this work,we designed a prospective antibacterial strategy by combining photothermal and ciprofloxacin(CIP).The synergistic effect of photothermal and antibiotic may provide an effective bacteriostatic efficacy without sacrificing osteogenesis at a mild condition of moderate temperature and less antibiotic.Herein,CIP was loaded into mesoporous polydopamine(MPDA)nanoparticles(MPDA@CIP),which were anchored on the surface of titanium and finally covered with sodium hyaluronate-catechol(HAc)coating.The hydrophilic HAc layer could inhibit the early adhesion of bacteria,and some bacteria could secrete bacterial hyaluronidase to accelerate the degradation of HAc.This enabled smart enzyme-triggered release of antimicrobials at the site of infection on-demand and avoided unwanted side effects on normal tissues.In addition,NIR light irradiation had a positive influence on both CIP release and MPDA nanoparticle’s photothermal effect.Moreover,before anchoring MPDA@CIP,by the construction of hydroxyapatite microstructure on Ti sur-face with micro-arc oxidation and alkali heat treatment,the ability of bone formation of Ti could be promoted also.Both in vitro as well as in vivo assays demonstrated that functional Ti has an excellent antibacterial effect and osteogenic ability.

Folic acid-modified Exosome-PH20 enhances the efficiency of therapy via modulation of the tumor microenvironment and directly inhibits tumor cell metastasis

High accumulation of hyaluronan(HA)in the tumor microenvironment leads to an increase in the interstitial pressure and reduction perfusion of drugs.Furthermore,high molecular-weight(HMW)-HA suppresses M1 macrophage polarization,enhances M2 polarization,and induces immunosuppression.Hyaluronidase treatment have attempted to decrease the quantity of HA in tumors.However,hyaluronidase-driven HA degradation driven accelerates tumor cell metastasis,which is a major cause of mortality in cancer patients.Thus,we designed a novel exosome-based drug delivery system(DDS),named Exos-PH20-FA,using genetic engineering to express human hyaluronidase(PH20)and self-assembly techniques to modify the exosomes with folic acid(FA).Our results show that Exos-PH20-FA degraded HMW-HA to low-molecular-weight(LMW)-HA.Moreover,LMW-HA polarized macrophages to the M1 phenotype and reduced the number of relevant immunosuppressive immunocytes which changed the immune microenvironment from an immunosuppressive to immunosupportive phenotype.Furthermore,we demonstrated Exos-PH20-FA directly reduced hyaluronidase-induced metastasis of tumor cells.This tumor treatment also allowed an enhanced delivery of chemotherapy by tumor-targeting effect with FA modification.Our findings indicate that Exos-PH20-FA improves tumor treatment efficiency and reduces the side effects of hyaluronidase treatment,namely tumor cell metastasis.This all-in-one exosome-based HA targeting DDS maybe a promising treatment that yields more efficient and safer results.