Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

Lignin biodegradation by white-rot fungi is pivotal to the earth's carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems.

Mn²âº-deficiency reveals a key role for the Pleurotus ostreatus versatile peroxidase (VP4) in oxidation of aromatic compounds.

The manganese peroxidase gene family (mnps) is a part of the ligninolytic system of Pleurotus ostreatus. This gene family is comprised of nine members, mnp1-9, encoding short manganese peroxidases (short-MnPs) or versatile peroxidases (VPs). We show that unlike in Mn(2+)-amended glucose-peptone (GP) medium, where redundancy among mnps was reported, in Mn(2+)-deficient GP medium mnp4 [encoding versatile peroxidase isoenzyme 4 (VP4)] has a key and nonredundant function. The abundance of mnps transcripts at time points corresponding to the tropophase (active growth), early idiophase, and idiophase indicates that mnp4 is the predominantly expressed mnp gene and that its relative predominance is dependent on the age of the culture. In this medium, azo dye, Orange II (OII) decolorization occurs only during the idiophase and a Δmnp4 strain showed a drastic reduction in this decolorization. Three degradation metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS), indicating both asymmetric and symmetric enzymatic cleavage of the azo-bond. In addition, the culture filtrate of Δmnp4 showed negligible values of oxidation capability of four typical VP substrates: Mn(2+), 2,6-dimethoxyphenol, phenol red, and Reactive Black 5 (RB5), compared to the wild-type strain PC9. We concluded that under Mn(2+)-deficient GP culture, VP4 (encoded by mnp4) is the main active ligninolytic enzyme able to oxidize Mn(2+) as well as high and low redox potential aromatic substrate, including dyes. Furthermore, other VPs/MnPs do not compensate for the lack of VP4 activity.

Redundancy among manganese peroxidases in Pleurotus ostreatus.

Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn(2+) amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn(2+)-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn(2+)-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn(2+)-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn(2+)-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members.

From pseudo to real-time dynamics of T cell thymic differentiation.

Numerous methods have recently emerged for ordering single cells along developmental trajectories. However, accurate depiction of developmental dynamics can only be achieved after rescaling the trajectory according to the relative time spent at each developmental point. We formulate a model which estimates local cell densities and fluxes, and incorporates cell division and apoptosis rates, to infer the real-time dimension of the developmental trajectory. We validate the model using mathematical simulations and apply it to experimental high dimensional cytometry data obtained from the mouse thymus to construct the true time profile of the thymocyte developmental process. Our method can easily be implemented in any of the existing tools for trajectory inference.

N-acetylneuraminic acid links immune exhaustion and accelerated memory deficit in diet-induced obese Alzheimer's disease mouse model.

Systemic immunity supports lifelong brain function. Obesity posits a chronic burden on systemic immunity. Independently, obesity was shown as a risk factor for Alzheimer's disease (AD). Here we show that high-fat obesogenic diet accelerated recognition-memory impairment in an AD mouse model (5xFAD). In obese 5xFAD mice, hippocampal cells displayed only minor diet-related transcriptional changes, whereas the splenic immune landscape exhibited aging-like CD4+ T-cell deregulation. Following plasma metabolite profiling, we identified free N-acetylneuraminic acid (NANA), the predominant sialic acid, as the metabolite linking recognition-memory impairment to increased splenic immune-suppressive cells in mice. Single-nucleus RNA-sequencing revealed mouse visceral adipose macrophages as a potential source of NANA. In vitro, NANA reduced CD4+ T-cell proliferation, tested in both mouse and human. In vivo, NANA administration to standard diet-fed mice recapitulated high-fat diet effects on CD4+ T cells and accelerated recognition-memory impairment in 5xFAD mice. We suggest that obesity accelerates disease manifestation in a mouse model of AD via systemic immune exhaustion.

Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus.

Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn(2+)-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn(2+)-dependent and Mn(2+)-independent peroxidase activity under Mn(2+)-deficient culture conditions.

RNAi as a potential tool for biotechnological applications in fungi.

RNA interference (RNAi) is a post-transcriptional gene-silencing (PTGS) phenomenon in which double-stranded RNA (dsRNA) triggers the degradation of homologous mRNA species, thereby reducing gene expression. In fungi, the use of RNAi as a tool for reverse genetics, aimed at modification of gene expression, is constantly growing, with more than 40 species already proven to be responsive. This technology has the ability to co-down-regulate the expression of several genes; however, this trait also makes it susceptible to non-target effects, which can be addressed using both available and developing bioinformatic tools. Moreover, the functionality of absorbed exogenous RNAi molecules, and the various classes of small RNAs found in fungi, offer great versatility and flexibility in acquiring the desired effects on gene expression, even without the necessity to genetically modify the targeted strain. There is an emerging role for RNAi as a potential tool for biotechnological applications. This is evident from current investigations in fungi, demonstrating the contribution of RNAi to progress research and applications in biomaterials production, bioconversion, plant fungal interactions and virulence factors of human pathogens. Possible problems and prospects for the use of RNAi in fungal biotechnology are discussed.

Correction: Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

[This corrects the article DOI: 10.18632/oncotarget.27438.].

Inhibition of fibroblast secreted QSOX1 perturbs extracellular matrix in the tumor microenvironment and decreases tumor growth and metastasis in murine cancer models.

Extracellular matrix (ECM) plays an important role in tumor development and dissemination, but few points of therapeutic intervention targeting ECM of the tumor microenvironment have been exploited to date. Recent observations suggest that the enzymatic introduction of disulfide bond cross-links into the ECM may be modulated to affect cancer progression. Specifically, the disulfide bond-forming activity of the enzyme Quiescin sulfhydryl oxidase 1 (QSOX1) is required by fibroblasts to assemble ECM components for adhesion and migration of cancer cells. Based on this finding and the increased QSOX1 expression in the stroma of aggressive breast carcinomas, we developed monoclonal antibody inhibitors with the aim of preventing QSOX1 from participating in pro-metastatic ECM remodeling. Here we show that QSOX1 inhibitory antibodies decreased tumor growth and metastasis in murine cancer models and had added benefits when provided together with chemotherapy. Mechanistically, the inhibitors dampened stromal participation in tumor development, as the tumors of treated animals showed fewer myofibroblasts and poorer ECM organization. Thus, our findings demonstrate that specifically targeting excess stromal QSOX1 secreted in response to tumor-cell signaling provides a means to modulate the tumor microenvironment and may complement other therapeutic approaches in cancer.

Effects of cre1 modification in the white-rot fungus Pleurotus ostreatus PC9: altering substrate preference during biological pretreatment.

BACKGROUND: During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation. RESULTS: Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1-overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1. CONCLUSIONS: The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected "mirror image" (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity.

A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells.

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly).We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein.rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models.Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn(2+) supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn(2+) on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn(2+)-deficient media, whereas strongly repressed (to approximately 1%) in Mn(2+)-supplemented media. Accordingly, in-vitro Mn(2+)-independent activity was found to be negligible. We tested whether release of mnp4 from Mn(2+) repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the ß-tubulin promoter was produced. Now, despite the presence of Mn(2+) in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to ß-tubulin) and the activity was comparable to the typical activity of PC9 in Mn(2+)-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [(14)C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn(2+) suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

Pleurotus ostreatus manganese-dependent peroxidase silencing impairs decolourization of Orange II.

Decolourization of azo dyes by Pleurotus ostreatus, a white-rot fungus capable of lignin depolymerization and mineralization, is related to the ligninolytic activity of enzymes produced by this fungus. The capacity of P. ostreatus to decolourize the azo dye Orange II (OII) was dependent and positively co-linear to Mn(2+) concentration in the medium, and thus attributed to Mn(2+)-dependent peroxidase (MnP) activity. Based on the ongoing P. ostreatus genome deciphering project we identified at least nine genes encoding for MnP gene family members (mnp 1-9), of which only four (mnp 1-4) were previously known. Relative real-time PCR quantification analysis confirmed that all the nine genes are transcribed, and that Mn(2+) amendment results in a drastic increase in the transcript levels of the predominantly expressed MnP genes (mnp 3 and mnp 9), while decreasing versatile peroxidase gene transcription (mnp 4). A reverse genetics strategy based on silencing the P. ostreatus mnp 3 gene by RNAi was implemented. Knock-down of mnp 3 resulted in the reduction of fungal OII decolourization capacity, which was co-linear with marked silencing of the Mn(2+)-dependent peroxidase genes mnp 3 and mnp 9. This is the first direct genetic proof of an association between MnP gene expression levels and azo dye decolourization capacity in P. ostreatus, which may have significant implication on understanding the mechanisms governing lignin biodegradation. Moreover, this study has proven the applicability of RNAi as a tool for gene function studies in Pleurotus research.