SMYD2-Methylated PPARγ Facilitates Hypoxia-Induced Pulmonary Hypertension by Activating Mitophagy.

BACKGROUND: Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. METHODS: PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 (deformed epidural auto-regulatory factor-1) domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 (3-cyano-5-{2-[4-[2-(3-methylindol-1-yl)ethyl]piperazin-1-yl]-phenyl}-N-[(3-pyrrolidin-1-yl)propyl]benzamide) were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. RESULTS: We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. CONCLUSIONS: Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

Regulation of Charge Transfer Pathway in Ag-ZnIn2S4 Nanowires for Visible Photodynamic Therapy on Candida Albicans Infections.

Photodynamic therapy (PDT) is receiving extensive attention as an antimicrobial strategy that does not cause drug resistance by reactive oxygen species (ROS). Herein, hierarchical Ag-ZnIn2S4 (Ag-ZIS) core-shell nanowires are synthesized by in situ Metal-Organic Framework derived method for efficient PDT of Candida albicans (C. albicans). The core-shell structure enables spatial synergy strategy to regulate the charge transfer pathway under visible light excitation, in which the Ag nanowires are like the highway for the photogenerated electrons. The enhanced charge carrier separation efficiency greatly increased the chances for the generation of ROS. As expected, the optimized Ag-ZIS nanowires exhibit excellent performance for inactivation of C. albicans under visible light irradiation (λ ≥ 420 nm, 15 min), and the effective sterilization concentration is as high as 107CFU mL-1. Moreover, in vivo infection experiments suggested that the PDT effect of Ag-ZIS nanowires on the mouse wound healing is better than that of the clinical Ketoconazole drug. The PDT antifungal mechanism of Ag-ZIS nanowires is also investigated, and superoxide anion is found to be the predominant active species to causes C. albicans damage. This work provides a new perspective for designing novel interface structures to regulate charge transfer to achieve efficient PDT antifungal therapy.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

Gongronella sp. w5 hydrolyzes plant sucrose and releases fructose to recruit phosphate-solubilizing bacteria to provide plants with phosphorus.

The mechanisms of how plant-beneficial rhizospheric fungi interact with the soil microbial community to promote plant growth by facilitating their phosphorus acquisition are poorly understood. This work supported that a Mucoromycotina fungus, Gongronella sp. w5 (w5), could promote phosphorus uptake of Medicago truncatula by increasing the available phosphorus (P) in the soil. The abundance of phosphate-solubilizing bacteria (PSB) and the activity of alkaline phosphatase (ALP) in alfalfa rhizosphere soil increased after w5 inoculation. Further analysis showed that w5 donated a portion of ALP activity and also stimulated the PSB to secrete ALP during plant-w5-PSB interaction to help release more available P in the rhizosphere of M. truncatula. Unlike most plant-beneficial rhizospheric fungi that mainly acquire hexoses from plants, w5 gained sucrose directly from the host plant and then recruited PSB to aid P acquisition by hydrolyzing sucrose and releasing mainly fructose to induce PSB to secrete ALP. IMPORTANCE: This work supported that after absorbing plant sucrose, Gongronella sp. w5 mainly releases sucrose hydrolysis product fructose into the environment. Fructose was used as a carbon source and signaling molecules to induce PSB to co-produce higher alkaline phosphatase activity, releasing soil-available phosphorus and promoting M. truncatula growth. This is the first report that plant-beneficial fungi could directly metabolize sucrose from plants and then recruit PSB to aid P acquisition by providing fructose. Our findings revealed the diversity in pathways of plant-fungi-PSB interactions on soil P acquisition and deepened our understanding of the cooperation of growth-promoting microorganisms in plant rhizosphere.

An apoptosis-inducing factor controls programmed cell death and laccase expression during fungal interactions.

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H2O2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9. KEY POINTS: • Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions • CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense • CcAIF1 overexpression and H2O2 stimulation dramatically increase laccase production.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Sphingomicrobium clamense sp. nov., Isolated from Sediment of Clam Island Beach in China.

A Gram-stain-negative, non-flagellated, aerobic, ovoid or rod-shaped bacterium with motility, designated B8T, was isolated from the sediment of Clam Island beach, Liaoning province, China. The optimum growth of strain B8T occurred at 35 oC, pH 7.0, and in the presence of 4.0-5.0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B8T formed a distinct lineage within the genus Sphingomicrobium and was closely related to Sphingomicrobium nitratireducens O-35T (98.3% sequence similarity), Sphingomicrobium aestuariivivum KCTC 42286T (96.9%), and Sphingomicrobium astaxanthinifaciens JCM 18551T (96.5%). The digital DNA-DNA hybridization and average nucleotide identity values between strain B8T and closely related strains were lower than 21.0% and 78.0%, much lower than the cutoff values of 70.0% and 95.0%, respectively, for bacterial species delineation. The dominant respiratory quinone of strain B8T was ubiquinone-10. The major fatty acids were Sum In Feature 8 (C18:1ω7c and/or C18:1ω6c), Sum In Feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17:1ω6c, C18:1 2-OH, and C16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, glycolipids, and four unknown polar lipids. The DNA G + C content of strain B8T was 63.9%. Based on the phenotypic, phylogenetic, and chemotaxonomic analyses, strain B8T is considered a new species of Sphingomicrobium, for which the name Sphingomicrobium clamense sp. nov. is proposed. The type strain is B8T (= CGMCC 1.19486T = KCTC 92052T).

Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis.

BACKGROUND: E1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown. METHODS: Cystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53. RESULTS: Compared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243. CONCLUSION: Thus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Maribacter aquimaris sp. nov., isolated from seawater adjacent to Fildes Peninsula, Antarctica.

A novel Gram-stain-negative, aerobic, and rod-shaped bacterium with gliding motility, named strain ANRC-HE7T, was isolated from the seawater of Biological Bay adjacent to Fildes Peninsula, Antarctica. The optimal growth of this strain occurred at 28 °C, pH 7.5, and in the presence of 1.0% (w/v) NaCl. Strain ANRC-HE7T can produce amylase and harbors gene clusters involved in cellulose degradation. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ANRC-HE7T formed a distinct lineage within the genus Maribacter and was closely related to Maribacter luteus RZ05T (98.4% sequence similarity), Maribacter polysiphoniae LMG 23671T (98.3%), and Maribacter arenosus CAU 1321T (97.3%). However, digital DNA-DNA hybridization and average nucleotide identity values between strain ANRC-HE7T and closely related strains were 17.4-49.1% and 70.9-92.7%, much lower than the cutoff values of 70% and 95%, respectively. On the other hand, strain ANRC-HE7T shared characteristics with most type strains within the genus. Its respiratory quinone was MK-6. The major fatty acids were iso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and anteiso-C15:0. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, four unidentified phospholipids, and five unidentified glycolipids. The DNA G + C content of strain ANRC-HE7T was 40.1%. Based on the results of the biochemical, phylogenetic, and chemotaxonomic analyses, strain ANRC-HE7T is suggested to represent a novel species of the genus Maribacter, for which the name Maribacter aquimaris sp. nov. is proposed. The type strain is ANRC-HE7T (= MCCC 1K03787T = KCTC 72532T).

Comprehensive analysis identified a reduction in ATP1A2 mediated by ARID3A in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by abdominal aorta dilatation and progressive structural impairment and is usually an asymptomatic and potentially lethal disease with a risk of rupture. To investigate the underlying mechanisms of AAA initiation and progression, seven AAA datasets related to human and mice were downloaded from the GEO database and reanalysed in the present study. After comprehensive bioinformatics analysis, we identified the enriched pathways associated with inflammation responses, vascular smooth muscle cell (VSMC) phenotype switching and cytokine secretion in AAA. Most importantly, we identified ATPase Na+ /K+ transporting subunit alpha 2 (ATP1A2) as a key gene that was significantly decreased in AAA samples of both human and mice; meanwhile, its reduction mainly occurred in VSMCs of the aorta; this finding was validated by immunostaining and Western blot in human and mouse AAA samples. Furthermore, we explored the potential upstream transcription factors (TFs) that regulate ATP1A2 expression. We found that the TF AT-rich interaction domain 3A (ARID3A) bound the promoter of ATP1A2 to suppress its expression. Our present study identified the ARID3A-ATP1A2 axis as a novel pathway in the pathological processes of AAA, further elucidating the molecular mechanism of AAA and providing potential therapeutic targets for AAA.

ThhspA1 is involved in lacA transcriptional regulation of Trametes hirsuta AH28-2 exposed to o-toluidine.

White rot fungi, especially Trametes spp., respond to a wide range of aromatic compounds and dramatically enhance laccase activity, while the activation mechanisms remain to be elucidated. Here, we show that an Hsp70 homolog named ThhspA1 regulates the transcription of laccase LacA in Trametes hirsuta AH28-2 when confronted with o-toluidine. ThhspA1 is pulled down by lacA promoter sequence from the nuclear mixture extracted from T. hirsuta AH28-2 induced by 2 mM o-toluidine. Silencing of ThhspA1 results in a sharp decrease in lacA transcripts and laccase activity in vivo. By comparison, ThhspA1 overexpression does not affect lacA transcription, and laccase activity shows slight enhancement or remains unchanged upon induction with o-toluidine. Electrophoretic mobility shift assays suggest a direct interaction between ThhspA1 and the lacA promoter region. Further investigation shows that the integrity of ThhspA1 is critical since its substrate binding domain (SBD) and nucleotide-binding domain (NBD) are both necessary for DNA binding, with a higher affinity of SBD than NBD based on fluorescence polarization assay. Our results demonstrate that ThhspA1 functions as an aromatic-stress-related DNA binding transcriptional factor required for LacA expression.

Coprinopsis cinerea Uses Laccase Lcc9 as a Defense Strategy To Eliminate Oxidative Stress during Fungal-Fungal Interactions.

Frequently, laccases are triggered during fungal cocultivation for overexpression. The function of these activated laccases during coculture has not been clarified. Previously, we reported that Gongronella sp. w5 (w5) (Mucoromycota, Mucoromycetes) specifically triggered the laccase Lcc9 overexpression in Coprinopsis cinerea (Basidiomycota, Agaricomycetes). To systematically analyze the function of the overexpressed laccase during fungal interaction, C. cinerea mycelia before and after the initial Lcc9 overexpression were chosen for transcriptome analysis. Results showed that accompanied by specific utilization of fructose as carbohydrate substrate, oxidative stress derived from antagonistic compounds secreted by w5 appears to be a signal critical for laccase production in C. cinerea. A decrease in reactive oxygen species (ROS) in the C. cinerea wild-type strain followed the increase in laccase production, and then lcc9 transcription and laccase activity stopped. By comparison, increased H2O2 content and mycelial ROS levels were observed during the entire cocultivation in lcc9 silenced C. cinerea strains. Moreover, lcc9 silencing slowed down the C. cinerea mycelial growth, affected hyphal morphology, and decreased the asexual sporulation in coculture. Our results showed that intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Lcc9 takes part in a defense strategy to eliminate oxidative stress during the interspecific interaction with w5. IMPORTANCE The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored. Based on comparative transcriptomic analysis of C. cinerea and gene silencing of laccase Lcc9, here we show that oxidative stress derived from antagonistic compounds secreted by Gongronella sp. w5 was a signal critical for laccase Lcc9 production in Coprinopsis cinerea. Intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Ultimately, Lcc9 takes part in a defense strategy to eliminate oxidative stress and help cell growth and development during the interspecific interaction with Gongronella sp. w5. These findings deepened our understanding of fungal interactions in their natural population and communities.

A Superefficient Ochratoxin A Hydrolase with Promising Potential for Industrial Applications.

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification is the highest priority regarding OTA in food and feed industry, but currently available detoxification enzymes have relatively low effectiveness in terms of time and cost. Here we show a superefficient enzyme, ADH3, identified from Stenotrophomonas acidaminiphila that has a strong ability to transform OTA into nontoxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 µg/mL) completely degrades 50 µg/L OTA within 90 s, while the other most efficient OTA hydrolases available take several hours. The kinetic constant showed that rADH3 (Kcat/Km) catalytic efficiency was 56.7 to 35,000 times higher than those of previous hydrolases rAfOTase, rOTase, and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure allows OTA easier access to the catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase, ADH3, which can completely transform 50 µg/L OTA into ochratoxin-α within 90 s was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0 to 70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism, laying a foundation for future industrial applications.

BRD4770 functions as a novel ferroptosis inhibitor to protect against aortic dissection.

Smooth muscle cell (SMC) loss is the characteristic feature in the pathogenesis of aortic dissection (AD), and ferroptosis is a novel iron-dependent regulated cell death driven by the excessive lipid peroxidation accumulation. However, whether targeting ferroptosis is an effective approach for SMC loss and AD treatment remains unclear. Here, we found that the iron level, ferroptosis-related molecules TFR, HOMX1, ferritin and the lipid peroxidation product 4-hydroxynonenal were increased in the aorta of AD. Then, we screened several inhibitors of histone methyltransferases and found that BRD4770 had a protective effect on cystine deprivation-, imidazole ketone erastin- or RSL3-induced ferroptosis of SMCs. The classic ferroptosis pathways, System Xc--GPX4, FSP1-CoQ10 and GCH1-BH4 pathways which were inhibited by ferroptosis inducers, were re-activated by BRD4770 via inhibiting mono-, di- and tri- methylated histone H3 at lysine 9 (H3K9me1/2/3). RNA-sequencing analysis revealed that there was a positive feedback regulation between ferroptosis and inflammatory response, and BRD4770 can reverse the effects of inflammation activation on ferroptosis. More importantly, treatment with BRD4770 attenuated aortic dilation and decreased morbidity and mortality in a ß-Aminopropionitrile monofumarate-induced mouse AD model via inhibiting the inflammatory response, lipid peroxidation and ferroptosis. Taken together, our findings demonstrate that ferroptosis is a novel and critical pathological mechanism that is involved in SMC loss and AD development. BRD4770 is a novel ferroptosis inhibitor and has equivalent protective effect to Ferrostatin-1 at the optimal concentration. Translating insights into the anti-ferroptosis effects of BRD4770 may reveal a potential therapeutic approach for targeting SMC ferroptosis in AD.

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization.

BACKGROUND: A raw starch-degrading α-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. RESULTS: AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG-PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h. CONCLUSIONS: This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.

MiR155 Relieves Acute Heart Transplantation in Mice by Modulating Th1/Th17 Immune Response.

Heart transplantation is an effective method for the treatment of end-stage heart disease. Therefore, this article aimed to establish a stable and effective mouse abdominal heart transplantation model. MiR155 alleviates the acute heart transplantation response by regulating Th1 / Th17 immune cytokines. This paper used the control method of randomly selecting samples to classify 30 healthy mice that met the conditions. First, C57BL / 6 mice were used as recipients, and Balb / c mouse hearts were used as donors to establish mouse hearts as a transplantation acute reaction model. A chronic rejection model of mouse heart transplantation was established by C57BL / 6 mice as recipients and Bm12 mouse hearts as donors. The survival time of the two groups of transplanted hearts was carefully recorded. The results of the study showed that in the heart transplantation acute/chronic rejection model, the average survival time of the donor's heart in the allograft group was (7.5 ± 0.37) / (63.4 ± 4.37) days, which was the same compared with the two groups. Therefore, in-depth analysis of the experimental control results and conclusions from the experimental results of the mice, this study can better respond to the pathological changes of acute/chronic rejection and reach the standard of model establish.

Characterization of the mitogenome of Gongronella sp. w5 reveals substantial variation in Mucoromycota.

Gongronella is a genus of fungi in Mucorales (Mucoromycota). Some of its members have important biotechnological applications, but until now, not a single mitogenome has been characterized in Gongronella. Here, we present the complete mitogenome assembly of Gongronella sp. w5, a soil isolate known to interact with plants and several fungi. Its 36,593-bp circular mitogenome encodes the large and small subunit rRNAs, 14 standard mitochondrial proteins, 24 tRNAs, three free-standing ORF proteins, and the RNA subunit of RNase P (rnpB). These genes arrange in an order novel to known fungal mitogenomes. Three group I introns are present in the cob, cox1, and nad5 genes, respectively, and they are probably acquired by horizontal gene transfer. Phylogenetic analysis based on mitochondrion-encoded proteins supports the grouping of Gongronella sp. w5 with Absidia glauca, forming the Cunninghamellaceae clade within Mucoromycota. Gongronella and most other Mucoromycota species are predicted to use the standard genetic code in mitochondrial translation, rather than code 4 assigned by GenBank. A comparison among seven publicly available mitogenomes in Mucoromycota reveals the presence of the same 14 typical protein-coding genes plus rnpB, yet substantial variation in mitogenome size, intron number, gene order, and orientation. In this comparison, the uniqueness of Gongronella is evident from similarly large differences to its closest phylogenetic neighbor, A. glauca. This study promotes our understanding of fungal evolution in Mucoromycota. KEY POINTS: • This study reports the first mitogenome in Gongronella, which presents a novel gene order. • Different Mucoromycota mitogenomes show substantial variation of gene organizations. • Most Mucoromycota species use the standard genetic code to translate mitochondrial genes.

Sedimentimonas flavescens gen. nov., sp. nov., isolated from sediment of Clam Island, Liaoning Province.

A novel Gram-stain negative, aerobic and ovoid to short rod shaped bacterium with a single polar flagellum, named strain B57T, was isolated from sediment of Clam Island, Liaoning Province, China. The optimal growth of this strain was found to occur at 37 °C, pH 6-6.5, and in the presence of 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B57T forms a distinct lineage within the family Rhodobacteraceae, sharing high 16S rRNA gene sequence similarity with Sinirhodobacter populi sk2b1T (97.4%). The average amino acid identity of B57T and the closely related species were lower than the threshold level for genus delineation. The dominant respiratory quinone of strain B57T was identified as Q-10. The major fatty acids were found to be Summed Feature 8 (C18:1ω7c and/or C18:1ω6c), Summed Feature 3 (C16:1ω7c and/or C16:1ω6c) and C16: 0. The polar lipids were identified as phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, two unidentified phospholipids, one unidentified glycolipid, and one unidentified lipid. The DNA G + C content of strain B57T was determined to be 64.1 mol%. Based on the biochemical, phylogenetic and chemotaxonomic analysis, strain B57T is concluded to represent a novel species of a novel genus, for which the name Sedimentimonas flavescens gen. nov., sp. nov.is proposed. The type strain is B57T (= CGMCC1.19488T = KCTC 92053T).

Coprinopsis cinerea Galectin CGL1 Induces Apoptosis and Inhibits Tumor Growth in Colorectal Cancer Cells.

Mushroom galectins are promising anticancer agents for their low IC50 values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were thermostable at neutral pH conditions. They both existed as tetramers and shared a high affinity towards lactose. CGL1 and CGL2 strongly inhibited the cell viability of many cancer cell lines, including three colorectal cancer cells, in a dose-dependent manner by inducing mitochondria-mediated caspase-dependent apoptosis. Furthermore, CGL1 exhibited higher apoptosis-inducing ability and cytotoxicity than CGL2. In vivo cell viability experiments based on two xenograft mouse models showed that CGL1 had a more substantial inhibitory effect than CGL2 on HCT116 tumor growth (p < 0.0001), whereas only CGL1 inhibited DLD1 tumor growth (p < 0.01). This is the first study to evaluate the anti-colorectal cancer effect of mushroom lectins in vivo, and our results showed that CGL1 is a potent agent for colorectal cancer treatment.