An Artificial Enzyme for Asymmetric Nitrocyclopropanation of α,ß-Unsaturated Aldehydes - Design and Evolution.

The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,ß-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction in high yield with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.

Preoperative contrast-enhanced CT-based radiomics nomogram for differentiating benign and malignant primary retroperitoneal tumors.

OBJECTIVES: This study evaluated the ability of a preoperative contrast-enhanced CT (CECT)-based radiomics nomogram to differentiate benign and malignant primary retroperitoneal tumors (PRT). METHODS: Images and data from 340 patients with pathologically confirmed PRT were randomly placed into training (n = 239) and validation sets (n = 101). Two radiologists independently analyzed all CT images and made measurements. Key characteristics were identified through least absolute shrinkage selection combined with four machine-learning classifiers (support vector machine, generalized linear model, random forest, and artificial neural network back propagation) to create a radiomics signature. Demographic data and CECT characteristics were analyzed to formulate a clinico-radiological model. Independent clinical variables were merged with the best-performing radiomics signature to develop a radiomics nomogram. The discrimination capacity and clinical value of three models were quantified by the area under the receiver operating characteristics (AUC), accuracy, and decision curve analysis. RESULTS: The radiomics nomogram was able to consistently differentiate between benign and malignant PRT in the training and validation datasets, with AUCs of 0.923 and 0.907, respectively. Decision curve analysis manifested that the nomogram achieved higher clinical net benefits than did separate use of the radiomics signature and clinico-radiological model. CONCLUSIONS: The preoperative nomogram is valuable for differentiating between benign and malignant PRT; it can also aid in treatment planning. KEY POINTS: • A noninvasive and accurate preoperative determination of benign and malignant PRT is crucial to identifying suitable treatments and predicting disease prognosis. • Associating the radiomics signature with clinical factors facilitates differentiation of malignant from benign PRT with improved diagnostic efficacy (AUC) and accuracy from 0.772 to 0.907 and from 0.723 to 0.842, respectively, compared with the clinico-radiological model alone. • For some PRT with anatomically special locations and when biopsy is extremely difficult and risky, a radiomics nomogram may provide a promising preoperative alternative for distinguishing benignity and malignancy.

Deep Learning Radiomics Nomogram to Predict Lung Metastasis in Soft-Tissue Sarcoma: A Multi-Center Study.

Objectives: To build and evaluate a deep learning radiomics nomogram (DLRN) for preoperative prediction of lung metastasis (LM) status in patients with soft tissue sarcoma (STS). Methods: In total, 242 patients with STS (training set, n=116; external validation set, n=126) who underwent magnetic resonance imaging were retrospectively enrolled in this study. We identified independent predictors for LM-status and evaluated their performance. The minimum redundancy maximum relevance (mRMR) method and least absolute shrinkage and selection operator (LASSO) algorithm were adopted to screen radiomics features. Logistic regression, decision tree, random forest, support vector machine (SVM), and adaptive boosting classifiers were compared for their ability to predict LM. To overcome the imbalanced distribution of the LM data, we retrained each machine-learning classifier using the synthetic minority over-sampling technique (SMOTE). A DLRN combining the independent clinical predictors with the best performing radiomics prediction signature (mRMR+LASSO+SVM+SMOTE) was established. Area under the receiver operating characteristics curve (AUC), calibration curves, and decision curve analysis (DCA) were used to assess the performance and clinical applicability of the models. Result: Comparisons of the AUC values applied to the external validation set revealed that the DLRN model (AUC=0.833) showed better prediction performance than the clinical model (AUC=0.664) and radiomics model (AUC=0.799). The calibration curves indicated good calibration efficiency and the DCA showed the DLRN model to have greater clinical applicability than the other two models. Conclusion: The DLRN was shown to be an accurate and efficient tool for LM-status prediction in STS.

Proton density fat fraction measurements of rotator cuff muscles: Accuracy, repeatability, and reproducibility across readers and scanners.

PURPOSE: To determine the accuracy, repeatability, and reproducibility of magnetic resonance imaging-based proton density fat fraction (MRI-PDFF) measurements of rotator cuff muscles between two readers and three different scanners. METHODS: Twenty-seven volunteers underwent serial shoulder MRI examinations of both left and right sides on one 1.5-T MRI scanner and two 3.0-T MRI scanners. Two independent readers measured muscular PDFF of the supraspinatus, infraspinatus/teres minor muscle, and subscapularis. MR spectroscopy-based proton density fat fraction (MRS-PDFF) was regarded as the reference standard for assessing accuracy. A "coffee break" examination method was used to test the repeatability of each scanner. Bland-Altman plots, Pearson correlation, and linear regression analysis were used to assess bias and linearity. The Wilcoxon signed-rank test and Friedman test were applied to evaluate repeatability and reproducibility. RESULTS: MRI-PDFF measurements indicated strong linearity (R2 = 0.749) and small bias (-0.18%) in comparison with the MRS-PDFF measurements. A very strong positive Pearson correlation (r = 0.955-0.986) between the PDFF estimates of the two repeat scans indicated excellent repeatability. The PDFF measurements showed high reproducibility, with a strong positive Pearson correlation (r = 0.668-0.698) and a small mean bias (-0.04 to -0.10%) across different scanners. CONCLUSION: MRI-PDFF measurements of rotator cuff muscles were highly accurate, repeatable, and reproducible across different readers and scanners, leading us to the conclusion that PDFF can be a reliable and robust quantitative imaging biomarker for longitudinal or multi-center studies.

Imaging Characteristics of Invasive Pulmonary Fungal Infection Secondary to Hematological Diseases and Comparison before and after Treatment.

Fungal infections have become crucial factors that threaten the prognosis and survival of blood disease patients. Here, we aim to analyze the epidemiological characteristics and early and advanced CT (computed tomography) manifestations of patients with invasive pulmonary fungal infections secondary to blood system diseases. 65 hospitalized patients from October 2018 to October 2020 with invasive pulmonary fungal infections secondary to blood diseases were enrolled. Blood diseases were recorded according to clinical and imaging data, and the serum galactomannan test (GM test) was conducted. Two senior radiologists analyzed the CT data and recorded the distribution of the lesions and CT signs. We analyzed and counted the first chest CT scan images of patients with nodule/mass type secondary to hematological diseases and invasive pulmonary fungal infection. The first CT nodules or mass-type lesions were statistically significant in nodule size, the number of lesions, distribution, and accompanying signs. Pulmonary fungal infection was common in both lungs during 7-day, 14-day, and 30-day follow-up CT. We also found that the nodular mass type was the main manifestation in the positive group of the GM test. Both the positive group and the negative group had the highest incidence of nodules. The incidence of air crescent signs in nodules or mass lesions in the positive group was higher than in the negative group, and the difference was statistically significant. To conclude, follow-up CT signs after antifungal treatment were highly sensitive to the early diagnosis of hematological diseases and secondary invasive pulmonary Eumycetes infection, which could be used for clinical treatment to provide help. GM test results were also related to CT manifestations such as air crescent sign, cavity, and halo sign.

Radiomics Nomograms Based on Multi-Parametric MRI for Preoperative Differential Diagnosis of Malignant and Benign Sinonasal Tumors: A Two-Centre Study.

OBJECTIVES: To investigate the efficacy of multi-parametric MRI-based radiomics nomograms for preoperative distinction between benign and malignant sinonasal tumors. METHODS: Data of 244 patients with sinonasal tumor (training set, n=192; test set, n=52) who had undergone pre-contrast MRI, and 101 patients who underwent post-contrast MRI (training set, n=74; test set, n=27) were retrospectively analyzed. Independent predictors of malignancy were identified and their performance were evaluated. Seven radiomics signatures (RSs) using maximum relevance minimum redundancy (mRMR), and the least absolute shrinkage selection operator (LASSO) algorithm were established. The radiomics nomograms, comprising the clinical model and the RS algorithms were built: one based on pre-contrast MRI (RNWOC); the other based on pre-contrast and post-contrast MRI (RNWC). The performances of the models were evaluated with area under the curve (AUC), calibration, and decision curve analysis (DCA) respectively. RESULTS: The efficacy of the clinical model (AUC=0.81) of RNWC was higher than that of the model (AUC=0.76) of RNWOC in the test set. There was no significant difference in the AUC of radiomic algorithms in the test set. The RS-T1T2 (AUC=0.74) and RS-T1T2T1C (RSWC, AUC=0.81) achieved a good distinction efficacy in the test set. The RNWC and the RNWOC showed excellent distinction (AUC=0.89 and 0.82 respectively) in the test set. The DCA of the nomograms showed better clinical usefulness than the clinical models and radiomics signatures. CONCLUSIONS: The radiomics nomograms combining the clinical model and RS can be accurately, safely and efficiently used to distinguish between benign and malignant sinonasal tumors.

A CT-based radiomics nomogram for differentiation of lympho-associated benign and malignant lesions of the parotid gland.

OBJECTIVES: Preoperative differentiation between benign lymphoepithelial lesion (BLEL) and mucosa-associated lymphoid tissue lymphoma (MALToma) in the parotid gland is important for treatment decisions. The purpose of this study was to develop and validate a CT-based radiomics nomogram combining radiomics signature and clinical factors for the preoperative differentiation of BLEL from MALToma in the parotid gland. METHODS: A total of 101 patients with BLEL (n = 46) or MALToma (n = 55) were divided into a training set (n = 70) and validation set (n = 31). Radiomics features were extracted from non-contrast CT images, a radiomics signature was constructed, and a radiomics score (Rad-score) was calculated. Demographics and CT findings were assessed to build a clinical factor model. A radiomics nomogram combining the Rad-score and independent clinical factors was constructed using multivariate logistic regression analysis. The performance levels of the nomogram, radiomics signature, and clinical model were evaluated and validated on the training and validation datasets, and then compared among the three models. RESULTS: Seven features were used to build the radiomics signature. The radiomics nomogram incorporating the clinical factors and radiomics signature showed favorable predictive value for differentiating parotid BLEL from MALToma, with AUCs of 0.983 and 0.950 for the training set and validation set, respectively. Decision curve analysis showed that the nomogram outperformed the clinical factor model in terms of clinical usefulness. CONCLUSIONS: The CT-based radiomics nomogram incorporating the Rad-score and clinical factors showed favorable predictive efficacy for differentiating BLEL from MALToma in the parotid gland, and may help in the clinical decision-making process. KEY POINTS: • Differential diagnosis between BLEL and MALToma in parotid gland is rather difficult by conventional imaging modalities. • A radiomics nomogram integrated with the radiomics signature, demographics, and CT findings facilitates differentiation of BLEL from MALToma with improved diagnostic efficacy.

An Inulin-Specific Lectin with Anti-HIV-1 Reverse Transcriptase, Antiproliferative, and Mitogenic Activities from the Edible Mushroom Agaricus bitorquis.

A novel lectin (ABL) was purified from the dried fruiting bodies of Agaricus bitorquis. An efficient 3-step purification protocol involved two consecutive steps of ion exchange chromatography on Q-Sepharose and SP-Sepharose and gel filtration by FPLC on Superdex 75. ABL is a monomeric protein with the molecular mass of 27.6 kDa, which is different from other lectins from genus Agaricus. Its N-terminal amino acid sequence is EYTISIRVYQTNPKGFNRPV which is unique and sharing considerably high similarity of other mushroom lectins. The hemagglutinating activity of the lectin was inhibited by inulin. Based on hemagglutination tests, ABL prefers rabbit, human type A, and AB erythrocytes to human type B and O erythrocytes. The lectin inhibits the activity of HIV-1 reverse transcriptase and the proliferation of leukemia cell (L1210) with an IC50 value of 4.69 and 4.97 µM, respectively. Furthermore, ABL demonstrates the highest mitogenic activity with a response of 24177.7 ± 940.6 [3H-methyl] thymidine counts per minute (CPM) at a concentration of 0.91 µM.

Chondromyxoid fibroma of the temporal bone: A case report and review of the literature.

BACKGROUND: Chondromyxoid fibroma (CMF) is a rare benign bone tumour of cartilaginous origin, which usually affects the metaphysis of the long bone. Involvement of the temporal bone is extremely rare. Patients with CMF in the temporal bone can present some neurological deficits due to involvement of surrounding neural structures. CASE SUMMARY: We present the first case of histopathologically proven CMF originating in the temporal bone and involving the hypoglossal canal in a 40-year-old woman. Hypoglossal nerve paralysis was identified on the cranial nerve examination. The patient underwent surgical excision and was neurologically normal except for mild left facial palsy on 5-mo follow-up examination after surgery. In the current report, the major characteristics and computed tomography/magnetic resonance imaging features of the lesion are discussed. Furthermore, previous literature regarding this pathology is reviewed. CONCLUSION: The current study presents the first case of temporal bone CMF involving the hypoglossal canal.

Morphometric MRI changes in intracranial hypertension due to cerebral venous thrombosis: a retrospective imaging study.

AIM: To evaluate whether some magnetic resonance imaging (MRI) signs suggesting idiopathic intracranial hypertension (IIH) could also be found in intracranial hypertension (IH) due to cerebral venous thrombosis (CVT) and to assess their possible contribution to diagnosing this disorder. MATERIALS AND METHODS: Thirty-one patients with IH due to CVT were evaluated prospectively using MRI. A group of 33 age- and sex-matched healthy volunteers served as controls. The optic nerve and sheath, pituitary gland, and ventricles were assessed. The prevalence of each imaging feature was compared between the two groups. RESULTS: Optic nerve sheath (ONS) dilatation and decreased pituitary gland height were the most valid signs suggesting IH in CVT patients: sensitivity 70.97% and 87.1%, respectively; specificity 96.97% and 72.73%, respectively; area under the curve 0.840 and 0.809, respectively. The MRI finding that showed the strongest association with IH in CVT patients was ONS dilatation (odds ratio 78.5). CONCLUSIONS: The combination of T1-weighted volumetric MRI and magnetic resonance venography could be helpful for diagnosing IH with CVT. Abnormalities of the ONS and the pituitary gland were reliable diagnostic signs for IH due to CVT.

A novel laccase with potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from mycelia of mushroom Coprinus comatus.

A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2'-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K(m) values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 µM, 4.95 µM, and 5.85 µM, respectively, signifying that it is an antipathogenic protein.

Antifungal and antiviral products of marine organisms.

Marine organisms including bacteria, fungi, algae, sponges, echinoderms, mollusks, and cephalochordates produce a variety of products with antifungal activity including bacterial chitinases, lipopeptides, and lactones; fungal (-)-sclerotiorin and peptaibols, purpurides B and C, berkedrimane B and purpuride; algal gambieric acids A and B, phlorotannins; 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy)phenol, spongistatin 1, eurysterols A and B, nortetillapyrone, bromotyrosine alkaloids, bis-indole alkaloid, ageloxime B and (-)-ageloxime D, haliscosamine, hamigeran G, hippolachnin A from sponges; echinoderm triterpene glycosides and alkene sulfates; molluscan kahalalide F and a 1485-Da peptide with a sequence SRSELIVHQR; and cepalochordate chitotriosidase and a 5026.9-Da antifungal peptide. The antiviral compounds from marine organisms include bacterial polysaccharide and furan-2-yl acetate; fungal macrolide, purpurester A, purpurquinone B, isoindolone derivatives, alterporriol Q, tetrahydroaltersolanol C and asperterrestide A, algal diterpenes, xylogalactofucan, alginic acid, glycolipid sulfoquinovosyldiacylglycerol, sulfated polysaccharide p-KG03, meroditerpenoids, methyl ester derivative of vatomaric acid, lectins, polysaccharides, tannins, cnidarian zoanthoxanthin alkaloids, norditerpenoid and capilloquinol; crustacean antilipopolysaccharide factors, molluscan hemocyanin; echinoderm triterpenoid glycosides; tunicate didemnin B, tamandarins A and B and; tilapia hepcidin 1-5 (TH 1-5), seabream SauMx1, SauMx2, and SauMx3, and orange-spotted grouper ß-defensin. Although the mechanisms of antifungal and antiviral activities of only some of the aforementioned compounds have been elucidated, the possibility to use those known to have distinctly different mechanisms, good bioavailability, and minimal toxicity in combination therapy remains to be investigated. It is also worthwhile to test the marine antimicrobials for possible synergism with existing drugs. The prospects of employing them in clinical practice are promising in view of the wealth of these compounds from marine organisms. The compounds may also be used in agriculture and the food industry.

Purification and characterization of a protein with antifungal, antiproliferative, and HIV-1 reverse transcriptase inhibitory activities from small brown-eyed cowpea seeds.

A 36-kDa protein, with an N-terminal sequence highly homologous to polygalacturonase (PG) inhibiting proteins, was isolated from small brown-eyed cowpea seeds. The protein was unadsorbed on diethylaminoethyl cellulose but adsorbed on both Affi-gel blue gel and SP-sepharose. It inhibited mycelial growth in the fungus Mycosphaerella arachidicola with an half-maximal (50%) inhibitory concentration (IC50 ) of 3.3 µM. It reduced [methyl-(3) H] thymidine incorporation into MBL2 lymphoma and L1210 leukemia cells with an IC50 of 7.4 and 5.4 µM, respectively. It inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 12.9 µM. However, it did not inhibit PG. The potent antifungal and antitumor activities of the protein suggest that it can be developed into an antifungal agent for combating M. arachidicola invasion in crops and an agent for cancer therapy in humans.

Purification and characterization of a ribonuclease from the wild edible mushroom Armillaria luteo-virens.

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70 degrees C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U) > poly(C) > poly (G) > poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.

A phytase characterized by relatively high pH tolerance and thermostability from the shiitake mushroom Lentinus edodes.

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37 °C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0-9.0, considerable thermostability with more than 60% residual activity at 70 °C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > ß -glycerophosphate. The phytase activity was moderately stimulated by Ca(2+), but inhibited by Al(3+), Mn(2+), Zn(2+), and Cu(2+) at a tested concentration of 5 mM.

Purification and characterization of a novel acid phosphatase from the split gill mushroom Schizophyllum commune.

A monomeric acid phosphatase (ACP) with a molecular mass of 72.5 kDa was purified from fresh fruiting bodies of cultured Schizophyllum commune mushroom. The isolation procedure entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. It demonstrated a unique N-terminal amino acid sequence of NAPWAQIDEV, which exhibited 60% amino acid identity to that of S. commune hypothetical histidine ACP based on its genome sequence, but less than 30% amino acid identity to that of other fungal ACPs previously reported. The ACP exhibited an optimum temperature at 50 °C, an optimum pH at pH 4.6, and was considerably stable at a pH range of 4.0 to 9.0, and a temperature range of 20-40 °C. The Km of the purified enzyme for ρ-nitrophenyl phosphate (ρNPP) was 0.248 mM and the Vmax was 9.093 × 10(-3) µM/min. ACP activity was strongly inhibited by Al(3+) and Fe(3+) , but enhanced by Co(2+) , Mg(2+) , and Ca(2+) at a concentration of 0.5 mM.

Purification and characterization of a novel lectin with antiphytovirus activities from the wild mushroom Paxillus involutus.

A novel lectin was isolated from the dried fruiting bodies of the wild mushroom Paxillus involutus. Isolation was conducted by anion exchange chromatography on DEAE-Cellulose, Q-Sepharose and gel filtration on Superdex 75 using a fast protein liquid chromatography (FPLC) system. This lectin had a molecular mass of 28 kDa and was composed of four identical subunits, each with a molecular mass of 7 kDa. N-terminal amino acid sequence of the P. involutus lectin was determined to be CTCAVFLNNTTVKS, which showed a low level of similarity to mushroom lectin sequences reported previously. The biochemical properties of this lectin were determined, and the hemagglutinating activity was inhibited by inulin and O-Nitrophenyl-ß-D-galacto-pyranoside. Additionally, Ca2+, Zn2+, Cd2+, Fe2+, and Al3+ inhibited its hemagglutinating activity, while Cu2+ promoted this activity. This lectin exhibited poor thermostability and was sensitive to HCl, but it had a high tolerance to NaOH exposure. In terms of biological properties, this lectin manifested antiphytovirus activity towards tobacco mosaic virus (TMV) with a 70.61% inhibition at a concentration of 200 µg/mL. This lectin was devoid of inhibitory activities toward pathogenic fungi and HIV-1 reverse transcriptase, and antiproliferative activities were observed in tumor cell lines including lung cancer A-549 and human colon cancer HCT-8 cells.

A laccase with antiproliferative and HIV-I reverse transcriptase inhibitory activities from the mycorrhizal fungus Agaricus placomyces.

A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7'-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, K(m) values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC(50) of 1.8 µM, 1.7 µM, and 1.25 µM, respectively, signifying that it is an antipathogenic protein.

Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis.

A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.

A novel aspartic protease with HIV-1 reverse transcriptase inhibitory activity from fresh fruiting bodies of the wild mushroom Xylaria hypoxylon.

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 µM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.