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Introduction: Majority of available protocols for isolation of chondrocytes from articular cartilage tissue rely on the enzymatic digestion of the tissue by collagenase type 2. The yield of chondrocytes in such protocols is low. Herein, we designed a novel indigenous sequential digestion by dual enzyme Pronase and Collagenase Type 1 for isolating human Chondrocytes from articular cartilage. Methods: Articular cartilage of Osteoarthritis (OA) patients undergoing total knee replacement were collected for the isolation of chondrocyte cells and subjected to sequential digestion by Pronase for three hours followed by Collagenase 1 overnight. Pellet of cells collected after digestion was plated on culture flask in 5% CO2 incubator. Results: From day three onwards, round to elongated cells adhered to the flask were visible which developed into elongated cell population of homogenous morphology, expressed Aggrecan (Agg), Collagen 2a (Col2a) and SRY-box transcription factor (Sox9) and had chondrogenic differentiation similar to a commercially available healthy chondrocyte. These cells were negative for Alizarin red stain, thus confirming the purity of chondrocytes. Conclusion: We have successfully established a sequential dual enzyme digestion-based culture technique for isolating the human chondrocytes from the articular cartilage biopsy derived from OA knee joints. [ABSTRACT FROM AUTHOR]

Case summaryAn indoor-only 6-year-old spayed female domestic cat was evaluated for a history of stertorous respiration. Skull radiographs revealed increased soft tissue density within the caudal aspect of the left nasal cavity. CT and rhinoscopy revealed a mass lesion in the choana, plus a smaller lesion, nearly completely occluding flow through the nasal passages. Rhinoscopy was used to collect a biopsy specimen from a fleshy, tan-yellow mass visualized in the caudal nasopharynx. Histopathology was diagnostic for Cryptococcus species infection and systemic antifungal therapy with fluconazole was initiated. Following a series of discordant results, serum samples were submitted to a veterinary diagnostic laboratory that utilized a cryptococcal antigen latex agglutination system with pretreatment of serum with pronase. Twenty-three months after the initial diagnosis, the cat's serum cryptococcal antigen titer declined to 1:5 and the cat has responded well to continuing treatment.Relevance and novel informationThis case illustrates challenges associated with discordant test results for cryptococcal antigen among laboratories. Discordancies may be due to differences in assay design, or the underlying disease state itself, or whether serum is pre-treated with pronase; with some tests relying on the training and experience of the operator if the cryptococcal antigen detection test requires a subjective interpretation. It also resolves some confusion in the literature related to the assay types available and terminology used to describe them, and emphasizes the importance of considering cryptococcosis as an important differential for cats with upper respiratory signs, without nasal discharge, even if the cat is kept exclusively indoors.

Zebrafish are a powerful animal model for small molecule screening. Small molecule treatments of zebrafish embryos usually require that the chorion, an acellular envelope enclosing the embryo, is removed in order for chemical compounds to access the embryo from the bath medium. For large-scale studies requiring hundreds of embryos, manual dechorionation, using forceps, can be a time-consuming and limiting process. Pronase is a non-specific protease that is widely used as an enzymatic alternative for dechorionating zebrafish embryos. However, whether pronase treatments alter the effects of subsequent small molecule treatments has not been addressed. Here, we provide a detailed protocol for large-scale pronase dechorionation of zebrafish embryos. We tested whether pronase treatment can influence the efficacy of drug treatments in zebrafish embryos. We used a zebrafish model for Duchenne muscular dystrophy (DMD) to investigate whether the efficacies of trichostatin-A (TSA) or salermide + oxamflatin, small molecule inhibitors known to ameliorate the zebrafish dmd muscle degeneration phenotype, are significantly altered when embryos are treated with pronase versus manual dechorionation. We also tested the effects of pronase on the ability of the anthracycline cancer drug doxorubicin to induce cardiotoxicity in zebrafish embryos. When comparing pronase- versus forceps-dechorionated embryos used in these small molecule treatments, we found no appreciable effects of pronase on animal survival or on the effects of the small molecules. The significant difference that was detected was a small improvement in the ability of salermide + oxamflatin to ameliorate the dmd phenotype in pronase-treated embryos when compared with manual dechorionation. Our study supports the use of pronase treatment as a dechorionation method for zebrafish drug screening experiments. [ABSTRACT FROM AUTHOR]

Using electron microscopy, the effect of pronase on nerve ganglia of a mollusk, leech, and frog was studied for the first time. It was revealed that the effect of pronase causes the retraction and removal of glial membranes, as well as denudation of nerve fibers and neuronal bodies with a simultaneous convergence of neuromembranes of these structures, and leads to the formation of gap junctions. This effect of pronase on the membranes belongs to a number of unusual, unexpected functions, and we obtained the observed effect for the first time. Since we previously registered reverberation of a nerve impulse in the ganglia of a frog and a leech under the same conditions, we believe that the obtained morphological data represent an evidence of the formation of electrical synapses under the action of pronase on the nerve ganglia. [ABSTRACT FROM AUTHOR]

Abstract Background The embryo release from the zona pellucida is of prerequisites of successful implantation. Objectives Regarding the negative impact of embryo cryopreservation on the blastocysts hatchability, the aim of the present study was to investigate the effects of treating embryonic zona pellucida with pronase or acidic Tyrode's solution (ATS) before morula formation on the viability, freezability, and hatchability of vitrified‐warmed resulted blastocysts. Methods In the first experiment, the zona pellucida of 3‐ and 4‐day‐old embryos were treated with the above compounds for 30 or 45 s. Then, the competency of the treated embryos to reach to blastocyst stage and the hatchability of resulting blastocysts were investigated. In the second experiment, the cryo‐survivability and hatching rate of blastocysts resulting from 3‐day‐old embryos treated with pronase and ATS for 30 s were tested. Results In the first experiment and in contrast to the 45 s exposure, 30‐s exposure of embryos to pronase or ATS did not have negative effect on the viability and development of embryos to blastocyst stage. In the second experiment, the freezability of blastocysts derived from 3‐day‐old embryos treated with pronase and ATS for 30 s was not different from that of the control group. However, the hatching rate of the pronase group was significantly higher than that of the control group. Conclusion The results of the present study showed that reducing the thickness of zona pellucida of sheep embryos with pronase had no negative effect on the developmental competency and freezability of the treated embryos and improved the hatchability of vitrified‐warmed blastocysts.

Objective/hypothesisTo develop an injection-based enzymatic technique that selectively softens cartilage tissue for reshaping cartilaginous structures in the head and neck.Materials and methodsTwo groups were formed using fresh rabbit ears: (1) whole rabbit ear group; (2) composite graft group (2.5mm×3.0cm specimens sectioned from the central region of the pinna). Subperichondrial injections using three enzymes (hyaluronidase, pronase, and collagenase II) in sequence were performed for the experimental specimens from both groups. In the control specimens, phosphate buffered saline was injected in a similar fashion. The whole ear specimens were then photographed while held upright in the anatomical vertical position to evaluate for buckling, which corresponds to the integrity of the cartilage. In addition, backlight photography was performed for all specimens to further evaluate the effect of the enzymes, such that increased light intensity represents increased cartilage digestion.ResultsThe application of the digestive enzymes resulted in marked reduction of cartilage tissue matrix resiliency, while preserving overlying skin layers. Enzymatically treated whole pinnae buckled at the site where enzymes were delivered. Backlit images revealed increased local light intensity at the regions of digestion. There was no obvious destruction of the overlying skin upon visual inspection.ConclusionsThis study demonstrates the feasibility of injectable chondroplasty as a potential alternative method to conventional surgery for auricular cartilage reshaping. Sequential injection of hyaluronidase, pronase, and collagenase II into the subperichondrial space can be performed to digest and soften cartilage structure with minimal involvement of surrounding tissue. Future studies will need to include chondrocyte viability testing and optimization of delivery techniques.

Zebrafish are a powerful animal model for small molecule screening. Small molecule treatments of zebrafish embryos usually require that the chorion, an acellular envelope enclosing the embryo, is removed in order for chemical compounds to access the embryo from the bath medium. For large-scale studies requiring hundreds of embryos, manual dechorionation, using forceps, can be a time-consuming and limiting process. Pronase is a non-specific protease that is widely used as an enzymatic alternative for dechorionating zebrafish embryos. However, whether pronase treatments alter the effects of subsequent small molecule treatments has not been addressed. Here, we provide a detailed protocol for large-scale pronase dechorionation of zebrafish embryos. We tested whether pronase treatment can influence the efficacy of drug treatments in zebrafish embryos. We used a zebrafish model for Duchenne muscular dystrophy (DMD) to investigate whether the efficacies of trichostatin-A (TSA) or salermide + oxamflatin, small molecule inhibitors known to ameliorate the zebrafish dmd muscle degeneration phenotype, are significantly altered when embryos are treated with pronase versus manual dechorionation. We also tested the effects of pronase on the ability of the anthracycline cancer drug doxorubicin to induce cardiotoxicity in zebrafish embryos. When comparing pronase- versus forceps-dechorionated embryos used in these small molecule treatments, we found no appreciable effects of pronase on animal survival or on the effects of the small molecules. The significant difference that was detected was a small improvement in the ability of salermide + oxamflatin to ameliorate the dmd phenotype in pronase-treated embryos when compared with manual dechorionation. Our study supports the use of pronase treatment as a dechorionation method for zebrafish drug screening experiments.

Background: The embryo release from the zona pellucida is of prerequisites of successful implantation. Objectives: Regarding the negative impact of embryo cryopreservation on the blastocysts hatchability, the aim of the present study was to investigate the effects of treating embryonic zona pellucida with pronase or acidic Tyrode's solution (ATS) before morula formation on the viability, freezability, and hatchability of vitrified‐warmed resulted blastocysts. Methods: In the first experiment, the zona pellucida of 3‐ and 4‐day‐old embryos were treated with the above compounds for 30 or 45 s. Then, the competency of the treated embryos to reach to blastocyst stage and the hatchability of resulting blastocysts were investigated. In the second experiment, the cryo‐survivability and hatching rate of blastocysts resulting from 3‐day‐old embryos treated with pronase and ATS for 30 s were tested. Results: In the first experiment and in contrast to the 45 s exposure, 30‐s exposure of embryos to pronase or ATS did not have negative effect on the viability and development of embryos to blastocyst stage. In the second experiment, the freezability of blastocysts derived from 3‐day‐old embryos treated with pronase and ATS for 30 s was not different from that of the control group. However, the hatching rate of the pronase group was significantly higher than that of the control group. Conclusion: The results of the present study showed that reducing the thickness of zona pellucida of sheep embryos with pronase had no negative effect on the developmental competency and freezability of the treated embryos and improved the hatchability of vitrified‐warmed blastocysts. In this study, we show that treating the zona pellucida of ovine embryos with pronase at cleavage stage improves the hatchability of resulting blastocysts after vitrification and warming. This is significant because may have practical application and improve the implantation rate of IVP vitrified‐warmed embryos. [ABSTRACT FROM AUTHOR]

Background: Pronase pretreatment can reduce rituximab (RTX) interference by degrading CD20 in B-cell flow cytometry crossmatch (FCXM) testing. However, it may also reduce the assay sensitivity by degrading HLA molecules. We investigated the effects of various pronase concentrations on RTX interference and the analytical sensitivity of B-cell FCXM testing., Methods: Using 59 patient serum samples and 38 donor lymphocyte samples, we designed 97 recipient-donor pairs and divided them into three groups according to RTX use and the presence of weak-to-moderate donor HLA-specific antibody (DSA) reactions: RTX+/DSA-, RTX+/DSA+, and RTX-/DSA+. FCXM was performed after pretreating lymphocytes with six different pronase concentrations (0, 0.5, 1, 2, 3, and 4 mg/mL)., Results: With B-FCXM testing, false-positive results due to RTX in the RTX+/DSA- group markedly decreased with increasing pronase concentrations. The median channel shift values in the RTX+/DSA+ and RTX-/DSA+ groups did not significantly decrease when the pronase concentration was increased from 1 mg/mL to 2 or 3 mg/mL. All eight RTX+/DSA+ cases that were positive at 1 mg/mL pronase but negative at 2 or 3 mg/mL had mean fluorescence intensity (MFI) DSA values of less than 3,000 except for DQ5 (MFI: 5,226). With T-cell FCXM, false-positive results were observed in 2.9% of 315 FCXM tests with pronase pretreatment., Conclusions: Higher concentrations (2 or 3 mg/mL) of pronase effectively eliminated RTX interference but still carried a risk for false negativity for weak DSA reactions in B-cell FCXM. Higher pronase concentrations can be used as an auxiliary method to detect moderate-to-strong DSA reactions in RTX-treated patients.

Introduction: To investigate the efficacy and safety of preprocedural simethicone (S) and pronase (P) for optimal mucosal visualization during esophagogastroduodenoscopy with sedation. The effect of postural change combined with premedication on mucosal visibility was also examined., Methods: The study randomized 496 patients into 8 groups based on the type of premedication provided and whether a postural change occurred. The premedication in the control group was 100 mL of normal saline solution (NS). The remaining 3 intervention groups were administered 100 mL of simethicone alone (S), pronase solution alone (P), and simethicone plus pronase solution (S + P). Each group was classified into subgroups according to whether there was a postural change (PC). The mucosal visibility score (MVS), total mucosal visibility score (TVS), procedure time, water consumption for mucosal cleansing, and proportion of patients with diminutive lesions <5 mm were recorded., Results: The P and S groups had a significantly better TVS than the NS group (11.86 ± 3.36 in group P vs 14.52 ± 2.57 in group NS, P < 0.001; 12.36 ± 2.93 in group S vs 14.52 ± 2.57 in group NS, P = 0.006). The TVS was better in the P group than in the S group (11.86 ± 3.36 vs 12.36 ± 2.93, P = 0.037). The MVS was significantly better in the esophagus and duodenum and worse in the upper and lower gastric body in the S group than in the P group. The P + S group had a significantly better TVS than the P and S groups (9.81 ± 2.90 in group P + S vs 11.86 ± 3.36 in group P and 12.36 ± 2.93 in group S, respectively, P < 0.001),\ and had a reduced amount of flushing water during the procedure (0 [interquartile range [IQR]: 0-33] mL in group P + S vs 40 [IQR: 0-70] mL in group P, P < 0.01; 0 [IQR: 0-33] mL in group P + S vs 50 [IQR: 20-98] mL in group S, P < 0.001). The TVS was significantly better in the P + S + PC group than in the P + S group (8.44 ± 2.10 vs 9.81 ± 2.90, P = 0.003). The MVS was significantly better in the gastric antrum, fundus, and upper and lower gastric body in the P + S + PC group than in the P + S group. There was no significant difference in the detection rate of diminutive lesions among the different groups during an endoscopic examination ( P > 0.05)., Discussion: The combination of preprocedural administration with simethicone and pronase achieved superior mucosal visualization compared with saline, simethicone, or pronase alone in patients receiving upper endoscopy. Postural change maneuvers performed before endoscopy further improved the mucosal visibility in most parts of the stomach when used with preprocedural simethicone and pronase., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Crystal-storing histiocytosis (CSH), light chain proximal tubulopathy (LCPT), and light chain crystalline podocytopathy (LCCP) are rare complications of multiple myeloma (MM) or monoclonal gammopathy of renal significance, and their diagnoses are challenging., Case Presentation: In this case, a 69-year-old Chinese woman presented with suspicious Fanconi syndrome with renal insufficiency. Immunofixation electrophoresis of both serum and urine revealed elevated immunoglobulin G kappa (IgGkappa) and kappa light chain. Bone marrow aspirate revealed 15% plasma cells with considerable cytoplasmic granular inclusions and needle-shaped crystals. Renal biopsy confirmed the final pathologic diagnosis of kappa-restricted CSH, combined LCPT and LCCP by immunoelectron microscopy. A number of special casts were present which could easily be misdiagnosed as light chain cast nephropathy. Immunofluorescence on frozen tissue presented false negative for kappa light chain, as ultimately proven by paraffin-embedded tissue after pronase digestion. MM and CSH were diagnosed, and two cycles of chemotherapy were given. The patient subsequently refused further chemotherapy, and her renal function remained relatively stable during a 2.5-year follow-up period., Conclusions: In conclusion, we report a rare case of generalized kappa-restricted CSH involving bone marrow and kidney, combined with LCPT and LCCP, provide a comprehensive summary of renal CSH, and propose a new nomenclature of monoclonal immunoglobulin-induced crystalline nephrology. The presentation of monoclonal immunoglobulin and Fanconi syndrome should suggest the presence of monoclonal immunoglobulin-induced crystalline nephrology. Use of paraffin-embedded tissue after pronase digestion and immunoelectron microscopy is beneficial to improve the sensitivity of diagnosis.

Seeds often survive in the soil in a dormant state, but their persistence is endangered by micro-organisms that could feed on them. Seed–microbe interactions in the soil are, however, poorly understood. We used dormant caryopses of red rice to study the defence response induced by pronase, a mixture of proteases secreted by Streptomyces griseus , a non-pathogenic bacterium. Pronase was shown to activate the plant immune reaction, indicating that its activity was recognized as a potential microbial attack. The defence reaction included extracellular alkalinization and superoxide production, and the former was necessary to activate the latter, since buffering at pH 6 inhibited the oxidative burst. Alkalinization was sufficient to trigger the oxidative burst, as superoxide production increased when caryopses were incubated in buffered solutions of increasing pH without pronase. Release of proanthocyanidins was observed, with or without pronase. These diverse mechanisms are hypothesized to cooperate in reinforcing seed protection. Finally, time profiles of superoxide production by dormant and non-dormant red rice caryopses during imbibition did not support a relationship between extracellular superoxide and dormancy breaking or germination. Thus, the role of this reactive oxygen species in red rice imbibed caryopses appears to be essentially aimed at defence against attacks by challenging micro-organisms. [ABSTRACT FROM AUTHOR]

Background/AimsHelicobacter pylori colonizes on the apical surface of gastric surface mucosal cells and the surface mucous gel layer. Pronase is a premedication enzyme for endoscopy that can disrupt the gastric mucus layer. We evaluated the additive effects of pronase combined with standard triple therapy for H. pylori eradication.Methods : This prospective, single-blinded, randomized, controlled study was conducted between June and October 2012. A total of 116 patients with H. pylori infection were enrolled in the study (n=112 patients, excluding four patients who failed to meet the inclusion criteria) and were assigned to receive either the standard triple therapy, which consists of a proton pump inhibitor with amoxicillin and clarithromycin twice a day for 7 days (PAC), or pronase (20,000 tyrosine units) combined with the standard triple therapy twice a day for 7 days (PACE).Results : In the intention-to-treat analysis, the eradication rates of PAC versus PACE were 76.4% versus 56.1% (p=0.029). In the per-protocol analysis, the eradication rates were 87.5% versus 68.1% (p=0.027). There were no significant differences concerning adverse reactions between the two groups.Conclusion : sAccording to the interim analysis of the trial, pronase does not have an additive effect on the eradication of H. pylori infection (: NCT01645761).

Background: Premedication in upper gastrointestinal endoscopy for higher lesions detection rate has not been well studied so far. This study aimed to confirm whether premedication could improve the detection rate of early cancer or precancerous lesions and mucosal visibility.Method: From July 2015 to December 2015, 7200 participants from 6 centers were screened by endoscopy with one of the 4 following premedications randomly: (1) water (group D); (2) pronase (group A); (3) simethicone (group B); (4) pronase and simethicone (group C). Early cancer and precancerous lesions detection rates were taken as the primary endpoints, and mucosal visibility was taken as the secondary endpoint. They were compared among four groups to determine different premedication effects in terms of different anatomical sites. Trial was registered at Chinese Clinical Trial Registry; the registration number is ChiCTR-IOR-17010985.Results: The upper gastrointestinal overall precancerous lesion detection rates among four groups were 8.7, 8.4, 10.0, and 10.3%, the overall early cancer detection rates were 1.3, 1.4%, 1.5, and 1.6%, both without significant difference (p = 0.138 and 0.878). However, the visibility score distributions between control group (D) and premedication groups (A, B, and C) were all statistically significant, with all anatomical sites p values < 0.001. Subgroup analyses, from 2 centers without screening before, also showed significant difference in esophageal (3.9, 3.3, 4.5, and 8.4% with p = 0.004) and overall (7.0, 5.5, 7.3, and 12.0% with p = 0.004) precancerous lesion detection rate.Conclusions: Premedication with pronase and simethicone may not increase lesion detection rates but could significantly increase the upper gastrointestinal mucosal visibility. [ABSTRACT FROM AUTHOR]

An extremely halophilic archeon SWO25, affiliated to Haloarcula sp., isolated from sebkha of Ouargla (South Algeria) was screened for secretion of antagonistic substance. To enhance the growth and antagonistic activity, physicochemical conditions were optimized by using a full factorial design. The selected factors were NaCl, MgSO4 and temperature. At first, the antagonistic activity was partially characterized and suitability of Gompertz and Baranyi equations were evaluated for describing archaeal growth curves. The results show that the secreted antagonistic activity has broad spectrum, highest activity was obtained against Halobacterium salinarum and was completely abolished by proteinase K and pronase, suggesting that it is a halocin. The modified Gompertz equation was statistically sufficient to describe the growth data of strain SWO25. Secondary models for Growth rate (µ), lag-time (λ), LnODmax and antagonistic activity (Ac) followed a linear trend with lowest R² greater than 80% and lack of fit not significant (>0.05). A clear interaction effect between NaCl concentration and temperature on growth rate and anti-archaeal activity was shown; this interaction was affected by MgSO4 concentration in the case of antagonistic activity. Optimal conditions for growth parameters and anti-archaeal activity were different; they were simultaneously optimized at 20% NaCl, 7.5% MgSO4 and a temperature of 45°C. Under these conditions 1.6 and 1.4 fold increase in the specific growth rate and the maximal biomass respectively were reached but there was no further increase on anti-archaeal production. This study contributes to a large-scale biomass and anti-archaeal production for fundamental studies or potential applications. [ABSTRACT FROM AUTHOR]

ABO-incompatible (ABOi) kidney transplantation has become an established strategy to increase the number of available living donors. At our center, the conditioning protocol for ABOi patients is based on anti-A/B antibody removal and depletion of B cells with the anti-CD20 mAb rituximab (Mabthera®). It is known that even low amounts of remaining rituximab in serum of patients results in false positive B cell cross match results, masking detection of potentially harmful donor human leukocyte antigen (HLA) specific antibodies. Treatment of donor cells with high concentrations (>1 mg/mL) of pronase is currently standard procedure for elimination of rituximab (RIT) interference. It is, however, troublesome that recent reports indicate that pronase treatment per se can induce incorrect flow cytometry cross match (FCXM) results. The aim of this study was to evaluate an alternative pronase-free FCXM for crossmatching of patients treated with rituximab. FCXM with an anti-RIT monoclonal antibody (mAb) pre-blocking step were evaluated on normal human sera (NHS) and patient sera supplemented with RIT. NHS supplemented with RIT or patient sera, without donor specific antibodies (DSA), resulted in high B cell median channel shift (>200 IgG) above background. This shift was eliminated by a serum pre-blocking step with 2-fold excess of anti-RIT (clone MB2A4). Blocking with anti-RIT did not influence the T cells crossmatch results. We present data supporting proof-of-concept that blocking with anti-RIT antibody prior to XM can enable reliable detection of anti-HLA class I and II donor specific antibodies without use of pronase treated donor cells. [ABSTRACT FROM AUTHOR]

Background and Aim To investigate the efficacy and safety of premedication with simethicone/Pronase during esophagogastroduodenoscopy ( EGD) with sedation. Methods Six hundred and ten patients were randomly allocated to two groups based on type of premedication given. Premedication used in the control group was 10 mL lidocaine hydrochloride mucilage ( LHM, N = 314) and premedication used in the intervention group was 80 mL simethicone/Pronase solution plus 10 mL lidocaine hydrochloride mucilage ( SP/ LHM, N = 296). EGD was done under sedation. Visibility scores, number of mucosal areas that needed cleansing, water consumption for cleansing, time taken for examination, diminutive lesions, pathological diagnosis, patients' gag reflex and oxygenation (pulse oximetry) were recorded. Results SP/ LHM has significantly lower total visibility score than LHM (7.978 ± 1.526 vs 6.348 ± 1.097, P < 0.01). During the procedure, number of intragastric areas that needed cleansing and amount of water consumed were significantly less in the SP/ LHM than in the LHM group ( P < 0.01). In SP/ LHM ( P = 0.01), endoscopy procedure duration was significantly longer. Although there was no significant difference in rate of detection of diminutive lesions between LHM and SP/ LHM, the endoscopist carried out more biopsies in SP/ LHM. This led to a higher rate of diagnosis of atrophic gastritis ( P = 0.014) and intestinal metaplasia ( P = 0.024). There was no significant difference in gag reflex ( P = 0.604) and oxygenation during the endoscopy procedure for either group of patients. Conclusion Routine use of premedication with simethicone/Pronase should be recommended during EGD with sedation. [ABSTRACT FROM AUTHOR]

Background and aims Magnetically controlled capsule endoscopy (MCE) is a novel technique for which there is no agreed gastric preparation. We aimed to determine an optimal standardized gastric preparation regimen. Methods 120 patients referred for MCE were randomly assigned to gastric preparation with either water alone (A), water with simethicone (B) or water, simethicone and pronase (C). Image quality was assessed using cleanliness and visualization scores, higher scores equating to better image quality. Results The total cleanliness scores were (mean ± SD) 15.83 ± 2.41 (A), 21.35 ± 1.23 (B), and 20.82 ± 1.90 (C). The total visualization scores (mean ± SD) were 10.75 ± 2.02 (A), 15.20 ± 1.32 (B), and 15.08 ± 1.86 (C). While the image quality of the whole stomach in groups B and C were significantly better than group A (P < 0.0001), there was no statistical difference between group B and C (P > 0.05). MCE detected positive findings in 21 (52.5%), 27 (67.5%) and 21 (53.8%) patients in group A, B and C respectively, with no significant difference between groups (P > 0.5). Conclusions Simethicone swallowed with water prior to MCE produced the optimal gastric mucosal image quality. The addition of pronase had no demonstrable additional benefit. [ABSTRACT FROM AUTHOR]

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role. [ABSTRACT FROM AUTHOR]

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 μg/ml PRN followed by 50 μg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli . Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design. [ABSTRACT FROM AUTHOR]

Flow cytometry crossmatching ( FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors ( CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex ( MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and - II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay. [ABSTRACT FROM AUTHOR]

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removingListeria monocytogenes–Escherichia colidual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2= 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivableE. colicells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment. [ABSTRACT FROM PUBLISHER]

Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80–90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes. [ABSTRACT FROM AUTHOR]

A new lectin was purified to electrophoretic homogeneity from pronase treated human serum by a single-step of affinity chromatography on concanavalin A-Sepharose 4B. The isolated lectin agglutinated five types of vertebrate RBC, with highest titer against hen RBC. This activity was independent of divalent cations, insensitive to EDTA and specific to mannosamine, glucosamine as well as galactosamine. Purified lectin gave a single symmetrical peak in its native form with a molecular mass estimate of 6 kDa in FPLC analysis and 6.5 kDa by MALDI-TOF MS. SDS-PAGE analysis of the lectin revealed that it is a homo-oligomer of a 3 kDa subunit protein. Isolated lectin did possess both, hemagglutinating and phenoloxidase activities, but did not exhibit any antibacterial or antifungal activities. In addition, this lectin could oxidize all nine different phenolic substrates tested, with hydroquinone proving to be the best among them. Phenoloxidase inhibitors namely, phenylthiourea and tropolone inhibited this oxidation activity. [ABSTRACT FROM AUTHOR]

The effect of high hydrostatic pressure (HHP) on flaxseed protein structure and peptide profiles, obtained after protein hydrolysis, was investigated. Isolated flaxseed protein (1%, m/v) was subjected to HHP (600 MPa, 5 min or 20 min at 20 °C) prior to hydrolysis with trypsin only and trypsin-pronase. The results demonstrated that HHP treatment induced dissociation of flaxseed proteins and generated higher molecular weight aggregates as a function of processing duration. Fluorescence spectroscopy showed that HHP treatment, as well as processing duration, had an impact on flaxseed protein structure since exposition of hydrophobic amino acid tyrosine was modified. Except for some specific peptides, the concentrations of which were modified, similar peptide profiles were obtained after hydrolysis of pressure-treated proteins using trypsin. Finally, hydrolysates obtained using trypsin-pronase had a greater antioxidant capacity (ORAC) than control samples; these results confirmed that HHP enhanced the generation of antioxidant peptides. [ABSTRACT FROM AUTHOR]

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here, we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals, but varied >10 fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans. [ABSTRACT FROM AUTHOR]

The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endotoxins from Bacillus thuringiensis kurstaki BNS3 was studied. Both toxins bound specifically to the BBMV of E. kuehniella . The result of the ligand blot showed that Cry1Ac bound to three putative receptors of about 100, 65 and 80 kDa and Cry1Aa interacted only with a 100 kDa protein. Pronase digestion of the BBMV-bound toxins was used to analyze the toxin insertion. Both toxins inserted into the BBMV as monomers however, a 14 kDa peptide of α4-α5 which correspond to the oligomeric form of this peptide was detected in case of Cry1Ac only. Analysis of the in vitro oligomerisation of these toxins in the presence of the BBMV of E. kuehniella showed reduced oligomer formation in case of Cry1Aa in comparison with Cry1Ac. Taken together, these results strongly suggest that the difference of toxicity between Cry1Aa and Cry1Ac to E. kuehniella is due to a deficient oligomerisation of Cry1Aa. [ABSTRACT FROM AUTHOR]

The effects of pronase (PRN), cellulase (CEL) or DNaseI alone or combined with benzalkonium chloride (BAC) againstListeria monocytogenes-carrying biofilms were assayed. The best removal activity againstL. monocytogenes–Escherichia colibiofilms was obtained using DNaseI followed by PRN and CEL. Subsequently, a modified logistic model was used to quantify the combined effects of PRN or DNaseI with BAC. A better BAC performance after PRN compared to DNaseI eradicatingL. monocytogeneswas observed. InE. colithe effects were the opposite. Finally, effects of DNaseI and DNaseI–BAC treatments were compared against two differentL. monocytogenes-carrying biofilms. DNaseI–BAC was more effective againstL. monocytogeneswhen co-cultured withE. coli. Nonetheless, comparing the removal effects after BAC addition, these were higher in mixed-biofilms withPseudomonas fluorescens.However, a high number of released viable cells was observed after combined treatments. These results open new perspectives of enzymes as an anti-biofilm strategy for environmental pathogen control. [ABSTRACT FROM PUBLISHER]

Background and Aims: Chromoendoscopy with Lugol's staining is used to screen for early esophageal squamous cell carcinoma (ESCC). Its efficacy is greatly limited by unstandardized defoaming preparation. This study aimed to confirm whether pre-procedure oral administration of pronase could improve the diagnostic performance of Lugol chromoendoscopy in high-risk patients being screened for early ESCC., Methods: A total of 955 patients at-risk were prospectively recruited for screening for ESCC. Patients were randomly assigned (1:1) to groups with or without (control group) pronase administration. Endoscopic diagnosis of early ESCC was based on the presence of pink-color sign in Lugol's unstained area, and a biopsy was routinely conducted if the Lugol's unstained lesion was larger than 0.5 cm. The early cancer detection rate was used as the primary endpoint., Results: Pre-procedure oral administration of pronase improved mucosal visibility during Lugol chromoendoscopy (P = 0.008). There were no differences in the number of Lugol's unstained lesions between the 2 groups (23.27% [111/477] vs. 25.11% [120/478], P = 0.508). Meaningfully, the detection rate of ESCC (confirmed by histopathology) was significantly higher in the pronase group than in the control group (27.03% [30/111] vs. 17.50% [21/120], P = 0.041), as well as the detection rate of lesions with pink-color sign during chromoendoscopy (35.14% [39/111] vs. 13.33% [16/120], P < 0.001). The diagnostic performance of Lugol chromoendoscopy had improved with the use of pronase (area under the curve = 0.85 vs. 0.69, P = 0.019), accompanied by an increased sensitivity (86.67% vs. 47.62%, P = 0.004). There was no difference in the adverse events between the 2 groups (P = 0.793)., Conclusions: Pre-procedure oral administration of pronase significantly increased the detection rate of early ESCC and optimized the diagnostic performance of Lugol chromoendoscopy, which should be recommended during routine endoscopic screening for early ESCC in high-risk patients., Trial Registration: Pronase improves efficacy of Lugol chromoendoscopy screening on esophageal cancerous lesions (NCT02030769)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus–oocyte complexes were exposed to 10 7 ​tissue culture-infective doses (TCID 50 )/mL of an ncp-BVDV strain during IVM ( in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription–polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection. [ABSTRACT FROM AUTHOR]

Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N -glycans have been reported recently. However, there are no previous reports of the O -glycosylation of rhGM-CSF from plant cells, and so this study investigated O -glycans, O -glycosylation sites, and their structural role in rrhGM-CSF. Monosaccharide analysis revealed the presence of O -glycans comprising arabinose and galactose. Eight O -glycans comprising four arabinose residues with zero to seven galactose residues along with their relative quantities were analyzed. Analysis of pronase-digested glycopeptides indicated that the O -glycans are partially attached to Ser 5, Ser 7, Ser 9, or Thr 10 residues, and glycan heterogeneity was confirmed at each site. Pro-to-hydroxyproline conversions occurred at Pro 2, Pro 6, and Pro 8 residues. The preparation of deglycosylated rrhGM-CSFs revealed that deglycosylation greatly affects their α-helix structures. These findings indicate that O -glycans of rrhGM-CSF are essential for maintaining its structural stability and result in an extended in vivo half-life, but without affecting its biological function. This is the first report on the O -glycosylation of rhGM-CSF derived from plant cells. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate the effectiveness of pronase combined with simethicone emulsion for premedication of gastroscopy. METHOD: 180 patients undergoing painless electronic gastroscopy admitted into People’s Hospital of Liaoning Province from Jul. 2017 to Jul. 2018 were selected and divided into observation group and control group via random number table, with 90 cases in each group. The control group was treated with simethicone emulsion alone, while the observation group was given pronase combined with simethicone emulsion. Mucosa visibility of gastric antrum, upper stomach, lower stomach, and gastric fundus of the two groups was scored, differences in detection of microscopic lesions and incidences of adverse drug reactions between two groups were compared. RESULTS: The mucosa visibility of gastric antrum, upper stomach, lower stomach, and gastric fundus of observation group were significantly higher than those of the control group, with statistically significant differences(P<0.05); the detectable rate of polyps, sunken, hemorrhagic spot and erosion of observation group were significantly higher than those of the control group, with statistically significant differences(P<0.05); the incidences of adverse drug reactions of observation group and control group were respectively 2.2%(2/90)and 3.3%(3/90), the difference had no statistical significance(P>0.05). CONCLUSIONS: The combination of pronase and simethicone emulsion for premedication of gastroscopy is beneficial for clearing bubbles and mucus, obtaining clearer vision, increasing the visibility of gastric mucosa and improving detectable rate of lesions. [ABSTRACT FROM AUTHOR]

Some of the products generated by the Maillard reaction are desired and very important for defining consumer acceptance of breakfast cereals. However, in recent years there has been an increased concern about compounds that are potentially harmful such as furfurals. The aim of this work was to analyze the effectiveness of protein hydrolysis with pronase as a pretreament for the evaluation of furfurals generated by the Maillard reaction during toasting of cornflakes and the parallel development of brown and fluorescent compounds. Furfurals were more accurately quantified with the pronase hydrolysis pretreatment because the protein matrix binds furfurals and fluorescent compounds. For control of the early reaction steps in the toasting process, the most sensitive parameter was fluorescence.