miR-29c-3p acts as a tumor promoter by regulating ß-catenin signaling through suppressing DNMT3A, TET1 and HBP1 in ovarian carcinoma.

Ovarian Carcinoma (OvCa) is characterized by rapid and sustained growth, activated invasion and metastasis. Studies have shown that microRNAs recruit and alter the expression of key regulators to modulate carcinogenesis. Here, we find that miR-29c-3p is increased in benign OvCa and malignant OvCa compared to normal ovary. Univariate and multivariate analyses report that miR-29c-3p overexpression is associated with poor prognosis in OvCa. Furthermore, we investigate that expression of miR-29c-3p is inversely correlated to DNA methyltransferase (DNMT) 3 A and Ten-Eleven-Translocation enzyme TET1. The high-throughput mRNA sequencing, bioinformatics analysis and pharmacological studies confirm that aberrant miR-29c-3p modulates tumorigenesis in OvCa cells, including epithelial-mesenchymal transition (EMT), proliferation, migration, and invasion. This modulation occurs through the regulation of ß-catenin signaling by directly targeting 3'UTR of DNMT3A, TET1 and the HMG box transcription factor HBP1 and suppressing their expression. The further 3D spheres assay clearly shows the regulatory effects of miR-29c-3p on OvCa tumorigenesis. Additionally, the receiver operating characteristic (ROC) curve analysis of miR-29c-3p and the clinical detection/diagnostic biomarker CA125 suggests that miR-29c-3p may be conducive for clinical diagnosis or co-diagnosis of OvCa. These findings support miR-29c-3p functions as a tumor promoter by targeting its functional targets, providing new potential biomarker (s) for precision medicine strategies in OvCa.

Geniposide alleviated hydrogen peroxide-induced apoptosis of human hepatocytes via altering DNA methylation.

Geniposide (GP) is the homology of medicine and food with bioactive effects of antioxidation and resistance to apoptosis in the liver. It's of great significance to explore the biosafety exposure limits and action mechanisms of GP. This study detected the global DNA methylation microenvironment and the regulation of specific genes in GP against cellular apoptosis induced by hydrogen peroxide (H2O2) of human hepatocyte L-02 cells. The half inhibitory concentration (IC50) of GP on normal L-02 cells was 57.7 mg/mL. GP exerted new epigenetic activity, increased DNMT1, decreased TET1 and TET2 expression, and reversed the demethylation effect to some extent, thereby increasing the overall genomic DNA methylation level at the concentration of 900 µg/mL. GP pretreatment could also adjust the level of P53, Bcl-2 and AKT altered by H2O2, reducing their specific DNA methylation levels in the promoter regions of AKT and Bcl-2 to inhibit apoptosis. Taken together, GP regulates the global DNA methylation level and controls the expression changes of P53, Bcl-2 and AKT, jointly inhibiting the occurrence of apoptosis in human hepatocytes and providing the newly theoretical references for its safety evaluation.

Flavin containing monooxygenase 2 regulates renal tubular cell fibrosis and paracrine secretion via SMURF2 in AKI­CKD transformation.

In the follow­up of hospitalized patients with acute kidney injury (AKI), it has been observed that 15­30% of these patients progress to develop chronic kidney disease (CKD). Impaired adaptive repair of the kidneys following AKI is a fundamental pathophysiological mechanism underlying renal fibrosis and the progression to CKD. Deficient repair of proximal tubular epithelial cells is a key factor in the progression from AKI to CKD. However, the molecular mechanisms involved in the regulation of fibrotic factor paracrine secretion by injured tubular cells remain incompletely understood. Transcriptome analysis and an ischemia­reperfusion injury (IRI) model were used to identify the contribution of flavin­containing monooxygenase 2 (FMO2) in AKI­CKD. Lentivirus­mediated overexpression of FMO2 was performed in mice. Functional experiments were conducted using TGF­ß­induced tubular cell fibrogenesis and paracrine pro­fibrotic factor secretion. Expression of FMO2 attenuated kidney injury induced by renal IRI, renal fibrosis, and immune cell infiltration into the kidneys. Overexpression of FMO2 not only effectively blocked TGF secretion in tubular cell fibrogenesis but also inhibited aberrant paracrine activation of pro­fibrotic factors present in fibroblasts. FMO2 negatively regulated TGF­ß­mediated SMAD2/3 activation by promoting the expression of SMAD ubiquitination regulatory factor 2 (SMURF2) and its nuclear translocation. During the transition from AKI to CKD, FMO2 modulated tubular cell fibrogenesis and paracrine secretion through SMURF2, thereby affecting the outcome of the disease.

Protocol for siRNA-mediated TET1 knockdown during differentiation of human embryonic stem cells into definitive endoderm cells.

TET1-mediated active DNA demethylation is required for endogenous retrovirus (ERV) enhancer activation during human ES differentiation into definitive endoderm (DE) cells. Here we present a protocol for siRNA-mediated TET1 knockdown during this process to decipher TET1's role in ERV activation and DE differentiation. We describe steps for inducing ES into DE cells. We then detail steps for knocking down TET1 during differentiation and for examining the effects of TET1 knockdown on LTR6B methylation, cell morphology, and gene expression. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022).1.

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination.

Aspartate ß-hydroxylase (ASPH) Accelerates Intrahepatic Cholangiocarcinoma Metastasis via Upregulating SHH Signaling Pathway.

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy with a poor prognosis. Aspartate ß-hydroxylase (ASPH) is an α-ketoglutarate-dependent dioxygenase involved in the post-translational hydroxylation of target proteins. ASPH has been demonstrated to be upregulated in ICC, yet its role remains to be elucidated. This study aimed to investigate the potential function of ASPH in ICC metastasis. Methods: Survival curves for the overall survival of pan-cancer data from The Cancer Genome Atlas (TCGA) database was depicted using the Kaplan-Meier method and compared using the log-rank test. The expression of ASPH, glycogen synthase kinase (GSK)-3ß, phosphorylation GSK-3ß (p-GSK-3ß), epithelial-mesenchymal transition (EMT) biomarkers, and sonic hedgehog (SHH) signaling elements in ICC cell lines was analyzed by western blot. Wound healing and transwell assays were conducted to examine the effects of ASPH knockdown and overexpression on cell migration and invasion. An immunofluorescence assay was conducted to evaluate the expression of glioma-associated oncogene 2 (GLI2), GSK-3ß and ASPH. The effect of ASPH on tumor in vivo was analyzed using a nude mouse xenograft model. Results: Pan-cancer data showed that expressed ASPH was significantly correlated with a poor prognosis in patients. ASPH knockdown inhibited the migration and invasion of human ICC cells lines QBC939 and RBE. ASPH overexpression contributed to an increase in the N-cadherin and Vimentin, resulting in the promotion of the EMT process. The p-GSK-3ß levels decreased in the presence of ASPH overexpression. The overexpression of ASPH led to an upregulation of the expression of SHH signaling elements GLI2 and SUFU. The results of in vivo experiments with a lung metastasis model in nude mice with ICC cell line RBE are consistent with these results. Conclusion: ASPH accelerated metastasis of ICC cells by facilitating EMT via a GSK-3ß/SHH/GLI2 axis-dependent manner, in which phosphorylation of GSK-3ß was downregulated and the SHH signaling pathway was activated.

Oxidative stress-induced TET1 upregulation mediates active DNA demethylation in human gastric epithelial cells.

The gastrointestinal (GI) tract is more vulnerable to effects by the outside environment, and experiences oxidative stress. A wide diversity of GI disorders can be partially attributed to oxidative stress. However, the mechanism of oxidative stress-caused GI pathological changes is not clear. In the present study, human gastric epithelial cells (hGECs) were treated with hydrogen peroxide (H2O2), and oxidative stress was determined. The effect of oxidative stress on the levels of some antioxidative enzymes, proliferation, nuclear DNA damage, apoptosis, expression of ten-eleven translocation (TET), and level of DNA methylation was determined in these cells. The results showed that H2O2 treatment caused oxidative stress, increased the levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), decreased the level of glutathione (GSH), inhibited proliferation, caused nuclear DNA damage and apoptosis, upregulated the expression of TET1 gene, and ultimately led to active DNA demethylation in hGECs. The present study presents a mechanism by which oxidative stress induces active DNA demethylation in hGECs. We propose that TET inhibitors can be used to restore the oxidative stress-induced DNA demethylation, and thus inhibit possible malignant transformation of GI cells.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B. mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B. mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

INTRODUCTION: Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. METHODS: In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 µg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. RESULTS: As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. CONCLUSIONS: Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Diabetes Induces Permanent Deleterious Effects in the Olfactory Bulb Associated with Increased Tyrosine Hydroxylase Expression and ERK1/2 Phosphorylation.

Diabetes mellitus type 2 (T2D) complications include brain damage which increases the risk of neurodegenerative diseases and dementia. An early manifestation of neurodegeneration is olfactory dysfunction (OD), which is also presented in diabetic patients. Previously, we demonstrated that OD correlates with IL-1ß and miR-146a overexpression in the olfactory bulb (OB) on a T2D rodent model, suggesting the participation of inflammation on OD. Here, we found that OD persists on a long-term T2D condition after the downregulation of IL-1ß. Remarkably, OD was associated with the increased expression of the dopaminergic neuronal marker tyrosine hydroxylase, ERK1/2 phosphorylation, and reduced neuronal activation on the OB of diabetic rats, suggesting the participation of the dopaminergic tone on the OD derived from T2D. Dopaminergic neurons are susceptible in neurodegenerative diseases such as Parkinson's disease; therefore further studies must be performed to completely elucidate the participation of these neurons and ERK1/2 signaling on olfactory impairment.

Pinoresinol diglucoside ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

This study is aimed to investigate the effect of pinoresinol diglucoside (PDG) in ameliorating myocardial ischemia-reperfusion injury (MIRI). Hypoxia/reperfusion (H/R)-induced H9c2 cardiomyocytes were used to establish an in-vitro ischemia-reperfusion injury model of cardiomyocytes. Cells were treated with 1 µmol/L of PDG. Reactive oxygen species (ROS) level was detected by a 2',7'-dichlorofluorescein-diacetate assay. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was examined by enzyme-linked immunosorbent assay. The viability and apoptosis of H9c2 cells were probed by MTT assay and flow cytometry. Besides this, Western blot and quantitative real-time PCR were used to detect microRNA-142-3p (miR-142-3p) and hypoxia-inducible factor 1 subunit alpha inhibitor (HIF1AN) expression levels. The binding sequence between miR-142-3p and HIF1AN 3'-untranslated region was validated by a dual-luciferase reporter gene assay. PDG treatment significantly reduced the level of ROS, LDH, and CK-MB, promoted viability, and inhibited the apoptosis of H9c2 cells. PDG treatment promoted miR-142-3p expression and inhibited HIF1AN expression in H9c2 cells. MiR-142-3p overexpression enhanced the effects of PDG on ROS, LDH, CK-MB levels, cell viability, and apoptosis in H9c2 cardiomyocytes, while overexpression of HIF1AN reversed the above effects. PDG ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

Daidzein Activates Akt Pathway to Promote the Proliferation of Female Germline Stem Cells through Upregulating Clec11a.

Female germline stem cells (FGSCs) have been successfully isolated and characterized from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we found that daidzein promoted the viability and proliferation of FGSCs. To elucidate the mechanism underlying this, we performed RNA-Sequence in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes in daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1 (Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.

Reversion of CYP450 monooxygenase-mediated acetamiprid larval resistance in dengue fever mosquito, Aedes aegypti L.

Aedes-borne diseases are on the rampant rise despite continued application of chemical insecticide-based interventions. The appearance of high degree of insecticide resistance in Aedes species and noxious effects on environment and non-targets have raised further concerns. Among new chemical interventions, neonicotinoids are considered a safe and effective approach. The present study investigated the control potency of acetamiprid and development of resistance in Aedes aegypti larvae; and the involvement of CYP450 monooxygenases in inducing resistance. The early fourth instars of Ae. aegypti parent susceptible strain (PS) were selected with acetamiprid for 15 generations (ACSF strain) increasing the resistance to 19.74-fold in ACSF-10 and 36.71-fold in ACSF-15. The ACSF-10 larvae were assayed with acetamiprid combined with piperonyl butoxide (PBO) in three different ratios (1:1, 1:5 and 1:10) and selected for next five generations with 1:10 combination. Selection with synergized acetamiprid (APSF strains) reversed as well as reduced the rate of resistance development resulting in only 1.35-fold resistance in APSF-15. The APSF strains showed %monooxygenase dependency ranging from 86.71 to 96.72%. The estimation of the monooxygenases levels in parent and selected larvae showed increased monooxygenase level in the ACSF strains by 2.42-2.87-fold. The APSF-15 strains exhibited 57.95% lower enzyme production than ACSF-15 strain. The reduction and reversion of resistance by using PBO and the elevated levels of monooxygenases in ACSF and reduction in APSF strains recommend the involvement of CYP450-mediated mechanism in the development of acetamiprid resistance in Ae. aegypti. These studies could help in devising resistance management strategies in order to preserve the efficiency of pre-existing insecticides.

Synergistic Effect of Repolarization of M2 to M1 Macrophages Induced by Iron Oxide Nanoparticles Combined with Lactate Oxidase.

Metabolic reprogramming of tumors with the accompanying reprogramming of glucose metabolism and production of lactate accumulation is required for the subsequent development of tumors. Recent evidence has indicated that tumor-secreted lactate can promote an oncolytic immune microenvironment within the tumor. Furthermore, tumor-secreted lactate directly induces polarization of tumor-supportive M2 macrophages. However, oxidized tumor-secreted lactate in the tumor microenvironment can be exploited. Iron oxide nanoparticles have shown promising anticancer potential by activating tumor-suppressing macrophages. Furthermore, lactate oxidase (LOX) generally oxidizes tumor-secreted lactate and subsequently converts to pyruvate. Particularly, the ratio of M2 macrophages to M1 macrophages corresponds with tumor growth. In this study, we present iron oxide nanoparticles with carboxylic acid combined with LOX that enhance antitumor efficacy as a synergistic effect on the repolarization of tumor-supportive M2 macrophages to tumor-suppressive M1 macrophages in a tumor microenvironment. After M2 macrophages treated with iron oxide nanoparticles were combined with LOX, the ratio of M1 macrophages was significantly greater than iron oxide nanoparticles alone or with LOX alone. It is concluded that the inhibition of cancer cell proliferation by ratio of M1 macrophages was observed. This study suggests that the iron oxide nanoparticles combined with LOX could be potentially used for potentiating immune checkpoint inhibitor therapies for cancer treatment.

Tryptophan Side-Chain Oxidase Enzyme Suppresses Hepatocellular Carcinoma Growth through Degradation of Tryptophan.

Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body's normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.

CircTET1 Inhibits Retinoblastoma Progression via Targeting miR-492 and miR-494-3p through Wnt/ß-catenin Signaling Pathway.

Purpose: Retinoblastoma (RB) is a frequent intraocular malignancy in children. Circular RNA (circRNA) plays an essential role in regulating the occurrence and development of tumors. This study aimed at investigating the function and molecular basis of hsa_circ_0093996 (circTET1) in RB.Methods: The expression of circTET1, miR-492 and miR-494-3p was examined using quantitative real-time polymerase chain reaction. Cell proliferation, cycle arrest, apoptosis, migration and invasion of RB cells were detected using Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, scratch assay and transwell analysis, respectively. The levels of matrix metalloproteinase (MMP) 2, MMP9 and Wnt/ß-catenin pathway-related proteins were measured via western blot assay. The association between circTET1 and miR-492/miR-494-3p was validated via dual-luciferase reporter assay and RNA pull-down assay. Xenograft assay was employed to analyze tumor growth in vivo.Results: CircTET1 level was reduced, while miR-492 and miR-494-3p levels were increased in RB tissues and cells. Overexpression of circTET1 inhibited proliferation, migration and invasion, and promoted apoptosis and cell cycle arrest in Y79 and WERI-Rb1 cells. Moreover, circTET1 impeded RB cell progression by sponging miR-492/miR-494-3p. Also, up-regulation of circTET1 restrained Wnt/ß-catenin pathway via regulating miR-492 and miR-494-3p. Furthermore, circTET1 suppressed tumor growth in xenograft models.Conclusion: CircTET1 inhibited RB progression by sponging miR-492/miR-494-3p and inactivating the Wnt/ß-catenin pathway, which provided new insights for RB treatment.

Enhanced konjac glucomannan hydrolysis by lytic polysaccharide monooxygenases and generating prebiotic oligosaccharides.

Konjac glucomannan (KGM) hydrolysate is a potentially valuable prebiotic that could improve gastrointestinal health by modulating the growth of probiotic bacteria and by promoting the production of short-chain fatty acids (SCFAs). In this study, we used lytic polysaccharide monooxygenases (LPMOs) to produce oligosaccharides from KGM and studied their prebiotic functions. The LPMO from Pleurotus ostreatus (PoLPMO9D) was shown to efficiently depolymerize KGM and produce a broad range of small oligomers. PoLPMO9D showed maximal activities at 50-60 °C and pH 4.0. When KGM-depolymerizing products produced by PoLPMO9D were employed as the carbon source instead of untreated KGM polymers, the growth of faecal microbiota was 2.76 times higher, a significant increase in the genus Lactococcus was observed, and the production of SCFAs increased by 14.6-fold with a significant pH decrease. This study shows that LPMOs may be a promising alternative enzyme for depolymerizing polysaccharide to prepare prebiotics from KGM.

Repolarization of M2 to M1 Macrophages Triggered by Lactate Oxidase Released from Methylcellulose Hydrogel.

Deregulated proliferation of tumors is generally associated with altered energy metabolism. A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to ∼6.7-7.2 in the tumor microenvironment and lactate accumulation. Macrophages in the tumor microenvironment can be educated by tumor cells. Tumor-derived lactate induces the polarization of M2 macrophages and promotes tumor invasion and metastasis. However, a particular challenge is to sustain lactate depletion. We propose that the repolarization of the tumor-supportive M2 macrophage to the tumor-suppressive M1 macrophage after the depletion of lactate by lactate oxidase (LOX) released from the hydrogels in the tumor microenvironment may enhance the antitumor treatment efficacy.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone.

Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less-activated hydrocarbons, by transferring peroxide-borne oxygen. We investigated a cell-free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg-1 . Although the well-known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5-epoxide of testosterone in ß-configuration and 16α-hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.