Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts.

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

Expression of 42 kDa chitinase of Trichoderma asperellum (Ta-CHI42) from a synthetic gene in Escherichia coli.

Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42 kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6-8 and has an optimum temperature of 45°C with thermal stability in a range of 25-35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5 mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na+, Fe2+ or Mg2+ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi.

An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L ) cultivar L14

The present work aims to establish an efficient protocol for in vitro regeneration of peanut (Arachis hypogaea) cultivar L14. The study showed that de-embryonated cotyledon was a suitable explant for shoot multiplication on MS medium containing 4 mg/L BAP. The highest number of shoots per explant obtained after 4 weeks of culture was up to 6.8 shoots. Shoots in vitro were able to produce a large number of approximately 11 roots on MS medium supplemented with 0.5 mg/L NAA. These results will be very useful in establishing an in vitro regeneration protocol for peanut cultivar L14 during gene transfer in the next studies to improve their disease resistance.

Analysis of the Symptomatology, Disease Course, and Treatment of Postorgasmic Illness Syndrome in a Large Sample.

BACKGROUND: Postorgasmic illness syndrome (POIS) is a rare syndrome of unknown etiology where patients experience distressing physical and psychological after ejaculatory sequelae. AIM: To better characterize the epidemiology, symptomatology, disease course, and treatment of POIS, with specific interest placed on examining relationships between disease presentation and measures of disease burden. METHODS: A 30-item questionnaire was distributed to an online community of patients with POIS from June 2019 to January 2020. We assessed diagnostic criteria and clusters of symptomatology described in prior studies. Outcome measures include self-reported measures of symptom severity, disease burden, and behavioral changes. Statistical correlations were assessed with Pearson's chi-squared (χ2) and ordinal regression analyses. OUTCOMES: The main outcome measures of this study are self-reported measures of symptom severity, disease burden, and behavioral changes. RESULTS: The sample consisted of 302 men (mean age: 32.6 ± 11.4 years, mean age of onset: 19.1 ± 7.8 years) with 89% satisfying ≥3 diagnostic criteria. Common symptoms were difficulty concentrating (254, 84%), extreme fatigue (250, 83%), irritability (225, 74%), and muscle weakness (212, 70%). Common symptom clusters were general (219, 72%), muscle (137, 45%), and head (93, 31%). Common behavioral modifications were avoiding masturbation (215, 71%), schedule changes (213, 71%), and abstinence (186, 62%). Head and throat symptom clusters demonstrated worse disease burden outcomes. Professional medical advice was sought by 51% of participants. Attempted treatments included pharmacotherapy, vitamins, supplements, and herbs with variable efficacy. CLINICAL IMPLICATIONS: This study further characterizes POIS, including how patients respond to the condition, how it is treated in the community, and presentations that may be associated with more severe disease. STRENGTHS & LIMITATIONS: This is the largest study to date that concerns patients suffering from POIS and includes a diverse, global population. Limitations include that the survey was only administered in English and within 1 online community, that results were self-reported, and that the response rate was low (32%). CONCLUSIONS: Characterizing symptom cluster, but not number of diagnostic criteria, may offer prognostic value, and investigation to elucidate pathophysiology and potential treatments for POIS is necessary. Natale C, Gabrielson A, Tue Nguyen HM, et al. Analysis of the Symptomatology, Disease Course, and Treatment of Postorgasmic Illness Syndrome in a Large Sample. J Sex Med 2020;17:2229-2235.

Cloning, expression and characterization of catechol 1,2-dioxygenase from Burkholderia cepacia.

The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.

Intralesional Collagenase Clostridium histolyticum Causes Meaningful Improvement in Men with Peyronie's Disease: Results of a Multi-Institutional Analysis.

PURPOSE: In a multi-institutional setting we studied the efficacy and safety outcomes at multiple high volume centers where collagenase Clostridium histolyticum is used to treat Peyronie's disease. MATERIALS AND METHODS: We collected retrospective data on consecutive patients with Peyronie's disease who underwent treatment with collagenase C. histolyticum between April 2014 and March 2018 at a total of 5 institutions. Included in the study were 918 patients. Main outcomes of interest included the change in curvature after receiving collagenase C. histolyticum therapy and the frequency of serious treatment related adverse events. The 2-tailed paired Student t-test was used to compare continuous variables. Univariate and multivariate regression analyses were performed to assess predictors of the success of collagenase C. histolyticum therapy to improve curvature. RESULTS: In the cohort of 918 patients curvature improved from a mean of 48.2 degrees before treatment to 32.9 degrees after treatment, a 30.1% improvement from baseline (p <0.0001). Of the men 68.7% had a 20% or greater improvement in curvature. In the 502 patients who completed 4 or more cycles curvature improved from a mean of 49.7 degrees before to 32.7 degrees after treatment, a 33% improvement from baseline (p <0.0001). Of these men 74.4% experienced a 20% or greater improvement in curvature. A complication of treatment developed in 9% of patients. The number of cycles of collagenase C. histolyticum received was predictive of curvature improvement (p <0.0001). CONCLUSIONS: This large multi-institutional analysis confirms the safety and efficacy of collagenase C. histolyticum therapy in men with Peyronie's disease. Intralesional collagenase C. histolyticum for Peyronie's disease according to the IMPRESS (Investigation of Maximal Peyronie's Reduction Efficacy and Safety Studies) trial protocol produced an improvement in penile curvature in men with Peyronie's disease with a low rate of complications.

Review of Management Options for Active-Phase Peyronie's Disease.

INTRODUCTION: Peyronie's disease (PD) is a fibrotic disorder that leads to plaque formation in ∼3-9% of adult men. Connective tissue buildup results in plaque formation, penile curvature and shortening, and in some cases, pain. In the active phase of PD, penile pain is a common symptom, often accompanied by progression in curvature. Treatment options for PD include oral drugs, topical cream, intralesional injections, traction, and shock wave therapy. This article reviews the different treatment options for active-phase PD. AIM: To provide a comprehensive review of the current evidence-based treatments, focusing on active-phase PD. METHODS: A PubMed database search was performed for studies on conservative therapy. We reviewed current clinical practices and trials for the treatment of active-phase PD and assessed their applicability as treatment options. MAIN OUTCOME MEASURE: Define the most recent and effective therapies for the improvement in penile curvature in men presenting with acute PD. RESULTS: A number of trials have resulted in significant benefits in the treatment of PD. Many of the oral therapies studied improved patient outcomes, but the data are insufficient. Similarly, there is no approved topical therapy by guidelines. The best options are intralesional injections of collagenase Clostridium histolyticum (CCh) and interferon-α2b. Shock wave therapy is not recommended for treatment of curvature, but has been shown to improve pain. Traction therapy may have a role in improving PD curve and penile length. CONCLUSION: PD remains a challenge for urologists. Evidence suggests that, for oral therapy, a combination of drugs produces better results. Shock wave therapy is still unproven. Intralesional therapy delivers the best evidence for improvement in the active phase. CCh is the only FDA-approved treatment option available. Penile traction therapy may improve patient outcomes. Brimley SC, Yafi FA, Greenberg J, et al. Review of Management Options for Active-Phase Peyronie's Disease. Sex Med Rev 2019;7:329-337.

Racial Variation in the Outcome of Subsequent Prostate Biopsies in Men With an Initial Diagnosis of Atypical Small Acinar Proliferation.

BACKGROUND: African American (AA) men are known to have more aggressive prostate cancer (PCa) compared with Caucasian American men. We sought to determine predictors of subsequent detection and risk stratification of PCa in a racially diverse group of men with atypical small acinar proliferation (ASAP) on initial prostate biopsy. MATERIALS AND METHODS: A retrospective analysis was conducted on data from men with ASAP on initial prostate biopsy who subsequently received confirmatory biopsies between September 2000 and July 2015. Biopsies with more than 3 years between initial and confirmatory biopsies were excluded. Race, age, body mass index, transrectal ultrasound volume, serum prostate-specific antigen (PSA), PSA velocity, PSA density, and elapsed time between biopsies were assessed for predictive value in subsequent PCa diagnosis after an initial finding of ASAP. RESULTS: Of 106 men analyzed, 75 (71%) were AA and 31 (29%) were non-AA. Baseline variables revealed AA men had higher PSA levels, PSA velocity, and PSA density (all P < .05). PCa was diagnosed in subsequent biopsy in 42 (40%) patients without significant racial variation; 30 (40%) AA versus 12 (39%) non-AA. Of the 42 PCa patients, 25 (24%) met Epstein criteria for significant disease without racial variation; 18 (24%) AA versus 7 (23%) non-AA. Only 10 (9%) patients had any component of Gleason 4; 7 (9%) AA versus 3 (10%) non-AA. In multivariate analysis, increasing age, PSA level, and PSA density were significant predictors of PCa. CONCLUSION: AA men diagnosed with ASAP on initial prostate biopsy do not have increased risk of PCa on confirmatory biopsy compared with non-AA men.