Direct evolution of an alkaline fungal laccase to degrade tetracyclines.

A fungal laccase-mediator system capable of high effectively oxidizing tetracyclines under a wide pH range will benefit environmental protection. This study reported a directed evolution of a laccase PIE5 to improve its performance on tetracyclines oxidization at alkaline conditions. Two mutants, namely MutA (D229N/A244V) and MutB (N123A/D229N/A244V) were obtained. Although they shared a similar optimum pH and temperature as PIE5, the two mutants displayed approximately 2- and 5-fold higher specific activity toward the mediators ABTS and guaiacol at pHs 4.0 to 6.5, respectively. Simultaneously, their catalytic efficiency increased by 8.0- and 6.4-fold compared to PIE5. At a pH range of 5-8 and 28 °C, MutA or MutB at a final concentration of 2.5 U·mL-1 degraded 77 % and 81 % of 100 mg·L-1 tetracycline within 10 min, higher than PIE5 (45 %). Furthermore, 0.1 U·mL-1 MutA or MutB completely degraded 100 mg·L-1 chlortetracycline within 6 min in the presence of 0.1 mM ABTS. At pH 8.0, MutA degraded tetracycline and chlortetracycline following the routine pathways were reported previously based on LC-MS analysis.

The updated relationship between the cleft­lip and palate transmembrane protein­1­like rs401681 and lung cancer risk: A systematic review and meta­analysis.

Currently, the role of cleft-lip and palate transmembrane protein-1-like (CLPTM1L) rs401681 in various tumor types, particularly lung cancer, has garnered significant attention. However, the findings across studies have shown discrepancies. The aim of the present meta-analysis was to provide a more nuanced understanding of the involvement of CLPTM1L rs401681 in lung cancer development. Several electronic databases were systematically searched, including PubMed, Cochrane Library, Embase, Medline, Wanfang, Google Scholar and Chinese National Knowledge Infrastructure. Odds ratios (ORs) and 95% confidence intervals (CIs) were synthesized using random-effects models. Heterogeneity of included studies was assessed using the I2 statistic and Q test. Sensitivity analysis was conducted to evaluate the stability of overall estimates. Moreover, Egger's test was utilized to detect potential publication bias. The collective ORs indicated a significant association between the CLPTM1L rs401681 polymorphism and susceptibility to lung cancer across various genetic comparisons. These encompass allele T vs. allele C (OR=0.93, 95% CI=0.88-0.99, P<0.001), TT + CT vs. CC (OR=0.91, 95% CI=0.87-0.96, P<0.001), TT vs. CC + CT (OR=0.88, 95% CI=0.80-0.96, P<0.001), TT vs. CC (OR=0.84, 95% CI=0.75-0.94, P<0.001) and CT vs. CC (OR=0.84, 95% CI=0.75-0.94, P<0.001). Examination through statistical Q test and I2 statistic revealed pronounced heterogeneity across four genetic comparisons (allele T vs. allele C, TT + CT vs. CC, TT vs. CC and CT vs. CC). Ethnical distinctions emerged as the primary, if not exclusive, sources of the significant heterogeneity. Upon stratification by ethnicity, a notable reduction in heterogeneity was discernible within the Caucasian demographic. However, heterogeneity persisted within the Asian population. Furthermore, lung cancer risks were statistically significantly decreased for individuals possessing allele T through all genetic comparisons within Caucasians; whereas among Asians, significant reduction was observed solely in the TT vs. CC comparison. The present meta-analysis uncovers a significant association between the CLPTM1L rs401681 polymorphism and altered susceptibility to lung cancer.

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.

From waste to protein: a new strategy of converting composted distilled grain wastes into animal feed.

Distilled grain waste (DGW) is rich in nutrients and can be a potential resource as animal feed. However, DGW contains as much as 14% lignin, dramatically reducing the feeding value. White-rot fungi such as Pleurotus ostreatus could preferentially degrade lignin with high efficiency. However, lignin derivatives generated during alcohol distillation inhibit P. ostreatus growth. Thus, finding a new strategy to adjust the DGW properties to facilitate P. ostreatus growth is critical for animal feed preparation and DGW recycling. In this study, three dominant indigenous bacteria, including Sphingobacterium thermophilum X1, Pseudoxanthomonas byssovorax X3, and Bacillus velezensis 15F were chosen to generate single and compound microbial inoculums for DGW composting to prepare substrates for P. ostreatus growth. Compared with non-inoculated control or single microbial inoculation, all composite inoculations, especially the three-microbial compound, led to faster organic metabolism, shorter composting process, and improved physicochemical properties of DGW. P. ostreatus growth assays showed the fastest mycelial colonization (20.43 µg·g-1 ergosterol) and extension (9 mm/d), the highest ligninolytic enzyme activities (Lac, 152.68 U·g-1; Lip, 15.56 U·g-1; MnP, 0.34 U·g-1; Xylanase, 10.98 U·g-1; FPase, 0.71 U·g-1), and the highest lignin degradation ratio (30.77%) in the DGW sample after 12 h of composting with the three-microbial compound inoculation when compared to other groups. This sample was relatively abundant in bacteria playing critical roles in amino acid, carbohydrate, energy metabolism, and xenobiotic biodegradation, as suggested by metagenomic analysis. The feed value analysis revealed that P. ostreatus mycelia full colonization in composted DGW led to high fiber content retention and decreased lignin content (final ratio of 5% lignin) but elevated protein concentrations (about 130 g·kg-1 DM). An additional daily weight gain of 0.4 kg/d was shown in cattle feeding experiments by replacing 60% of regular feed with it. These findings demonstrate that compound inoculant consisting of three indigenous microorganisms is efficient to compost DGW and facilitate P. ostreatus growth. P. ostreatus decreased the lignin content of composted DGW during its mycelial growth, improving the quality of DGW for feeding cattle.

A cost-saving, safe, and highly efficient natural mediator for laccase application on aflatoxin detoxification.

Laccase mediators possess advantage of oxidizing substrates with high redox potentials, such as aflatoxin B1 (AFB1). High costs of chemically synthesized mediators limit laccase industrial application. In this study, thin stillage extract (TSE), a byproduct of corn-based ethanol fermentation was investigated as the potential natural mediator of laccases. Ferulic acid, p-coumaric acid, and vanillic acid were identified as the predominant phenolic compounds of TSE. With the assistance of 0.05 mM TSE, AFB1 degradation activity of novel laccase Glac1 increased by 17 times. The promoting efficiency of TSE was similar to ferulic acid, but superior to vanillic acid and p-coumaric acid, with 1.2- and 1.3-fold increases, respectively. After Glac1-TSE treatment, two oxidation products were identified. Ames test showed AFB1 degradation products lost mutagenicity. Meanwhile, TSE also showed 1.3-3.0 times promoting effect on laccase degradation activity in cereal flours. Collectively, a safe and highly efficient natural mediator was obtained for aflatoxin detoxification.

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.

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.

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.

The benefits of oral glucose-lowering agents: GLP-1 receptor agonists, DPP-4 and SGLT-2 inhibitors on myocardial ischaemia/reperfusion injury.

Myocardial infarction (MI) is a life-threatening cardiovascular disease that, on average, results in 8.5 million deaths worldwide each year. Timely revascularization of occluded vessels is a critical method of myocardial salvage. However, reperfusion paradoxically leads to the worsening of myocardial damage known as myocardial ischaemia/reperfusion injury (MI/RI). Therefore, reducing the size of myocardial infarction after reperfusion is critical and remains an important therapeutic goal. The susceptibility of the myocardium to MI/RI may be increased by diabetes. Currently, some traditional antidiabetic agents such as metformin reduce MI/RI by decreasing inflammation, inhibiting oxidative stress, and improving vascular endothelial function. This appears to be a new direction for the treatment of MI/RI. Recent cardiovascular outcome trials have shown that several oral antidiabetic agents, including glucagon-like peptide-1 receptor agonists (GLP-1RAs), dipeptidyl peptidase-4 inhibitors (DPP-4is), and sodium-glucose-linked transporter-2 inhibitors (SGLT-2is), not only have good antidiabetic effects but also have a protective effect on myocardial protection. This article aims to discuss the mechanisms and effects of oral antidiabetic agents, including GLP-1RAs, DPP-4is, and SGLT-2is, on MI/RI to facilitate their clinical application.

Effects of metformin, saxagliptin and repaglinide on gut microbiota in high-fat diet/streptozocin-induced type 2 diabetic mice.

INTRODUCTION: There has been increasing evidence that the gut microbiota is closely related to type 2 diabetes (T2D). Metformin (Met) is often used in combination with saxagliptin (Sax) and repaglinide (Rep) for the treatment of T2D. However, little is known about the effects of these combination agents on gut microbiota in T2D. RESEARCH DESIGN AND METHODS: A T2D mouse model induced by a high-fat diet (HFD) and streptozotocin (STZ) was employed. The T2D mice were randomly divided into six groups, including sham, Met, Sax, Rep, Met+Sax and Met+Rep, for 4 weeks. Fasting blood glucose level, serum biochemical index, H&E staining of liver, Oil red O staining of liver and microbiota analysis by 16s sequencing were used to access the microbiota in the fecal samples. RESULTS: These antidiabetics effectively prevented the development of HFD/STZ-induced high blood glucose, and the combination treatment had a better effect in inhibiting lipid accumulation. All these dosing regimens restored the decreasing ratio of the phylum Bacteroidetes: Firmicutes, and increasing abundance of phylum Desulfobacterota, expect for Met. At the genus level, the antidiabetics restored the decreasing abundance of Muribaculaceae in T2D mice, but when Met was combined with Rep or Sax, the abundance of Muribaculaceae was decreased. The combined treatment could restore the reduced abundance of Prevotellaceae_UCG-001, while Met monotherapy had no such effect. In addition, the reduced Lachnospiraceae_NK4A136_group was well restored in the combination treatment groups, and the effect was much greater than that in the corresponding monotherapy group. Therefore, these dosing regimens exerted different effects on the composition of gut microbiota, which might be associated with the effect on T2D. CONCLUSIONS: Supplementation with specific probiotics may further improve the hypoglycemic effects of antidiabetics and be helpful for the development of new therapeutic drugs for T2D.

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).

The lysine methyltransferase SMYD2 facilitates neointimal hyperplasia by regulating the HDAC3-SRF axis.

Coronary restenosis is an important cause of poor long-term prognosis in patients with coronary heart disease. Here, we show that lysine methyltransferase SMYD2 expression in the nucleus is significantly elevated in serum- and PDGF-BB-induced vascular smooth muscle cells (VSMCs), and in tissues of carotid artery injury-induced neointimal hyperplasia. Smyd2 overexpression in VSMCs (Smyd2-vTg) facilitates, but treatment with its specific inhibitor LLY-507 or SMYD2 knockdown significantly inhibits VSMC phenotypic switching and carotid artery injury-induced neointima formation in mice. Transcriptome sequencing revealed that SMYD2 knockdown represses the expression of serum response factor (SRF) target genes and that SRF overexpression largely reverses the inhibitory effect of SMYD2 knockdown on VSMC proliferation. HDAC3 directly interacts with and deacetylates SRF, which enhances SRF transcriptional activity in VSMCs. Moreover, SMYD2 promotes HDAC3 expression via tri-methylation of H3K36 at its promoter. RGFP966, a specific inhibitor of HDAC3, not only counteracts the pro-proliferation effect of SMYD2 overexpression on VSMCs, but also inhibits carotid artery injury-induced neointima formation in mice. HDAC3 partially abolishes the inhibitory effect of SMYD2 knockdown on VSMC proliferation in a deacetylase activity-dependent manner. Our results reveal that the SMYD2-HDAC3-SRF axis constitutes a novel and critical epigenetic mechanism that regulates VSMC phenotypic switching and neointimal hyperplasia.

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.

Distinct biogeographic patterns in Glomeromycotinian and Mucoromycotinian arbuscular mycorrhizal fungi across China: A meta-analysis.

Fine root endophytes, recently reclassified as Mucoromycotinian arbuscular mycorrhizal fungi (M-AMF), are now recognized as functionally important as Glomeromycotinian AMF (G-AMF). However, little is known about the biogeography and ecology of M-AMF and G-AMF communities, particularly on a large scale, preventing a systematic assessment of ecosystem diversity and functioning. Here, we investigated the biogeographic assemblies and ecological diversity patterns of both G-AMF and M-AMF, using published 18S rDNA amplicon datasets and associated metadata from 575 soil samples in six ecosystems across China. Contrasting with G-AMF, putative M-AMF were rare in natural/semi-natural sites, where their communities were a subset of those in agricultural sites characterized by intensive disturbances, suggesting different ecological niches that they could occupy. Spatial and environmental factors (e.g., vegetation type) significantly influenced both fungal communities, with soil total­nitrogen and mean-annual-precipitation being the strongest predictors for G-AMF and M-AMF richness, respectively. Both groups exhibited a strong spatial distance-decay relationship, shaped more by environmental filtering than spatial effects for M-AMF, and the opposite for G-AMF, presumably because stochasticity (e.g., drift) dominantly structured G-AMF communities; while the narrower niche breadth (at community-level) of M-AMF compared to G-AMF suggested its more susceptibility to environmental differences. Furthermore, co-occurrence network links between G-AMF and M-AMF were prevalent across ecosystems, and were predicted to play a key role in stabilizing overall communities harboring both fungi. Based on the macroecological spatial scale datasets, this study provides solid evidence that the two AMF groups have distinct ecological preferences at the continental scale in China, and also highlights the potential impacts of anthropogenic activities on distributions of AMF. These results advance our knowledge of the ecological differences between the two fungal groups in terrestrial ecosystems, suggesting the need for further field-based investigation that may lead to a more sophisticated understanding of ecosystem function and sustainable management.

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.

Improving the cellobiose hydrolysis activity of glucose-stimulating ß-glucosidase Bgl2A.

ß-Glucosidases with high catalytic activity and glucose tolerant properties possess promising applications in lignocellulose-based industries. To obtain enzymes possessing these properties, a semi-rational strategy was employed to engineer the glucose-stimulating ß-glucosidase Bgl2A for high cellobiose hydrolysis activity. A total of 18 mutants were constructed. A22S, V224D, and A22S/V224D exhibited high specific activities of 272.06, 237.60, and 239.29 U/mg toward cellobiose, which were 2.5- to 2.8-fold of Bgl2A. A22S, V224D, and A22S/V224D exhibited increased kcat values, which were 2.7- to 3.1-fold of Bgl2A. A22S and V224D maintained glucose-stimulating property, whereas A22S/V224D lost it. Using 150 g/L cellobiose as the substrate, the amount of glucose produced by A22S was the highest, yielding 129.70 g/L glucose after 3 h reaction at 35 °C. The synergistic effects of the engineered enzymes with commercial cellulase on hydrolyzing cellulose were investigated. Supplemented with the commercial cellulase and A22S, the highest glucose amount of 23.30 g/L was yielded from cellulose with hydrolysis rate of 21.02 %. Given its high cellobiose hydrolysis activity and glucose-stimulating properties, A22S can be used as a component of enzyme cocktail to match mesophilic cellulases for efficient cellulose hydrolysis.

Seryl-tRNA Synthetase Shows a Noncanonical Activity of Upregulating Laccase Transcription in Trametes hirsuta AH28-2 Exposed to Copper Ion.

The function of Seryl-tRNA synthetase in fungi during gene transcription regulation beyond translation has not been reported. Here, we report a seryl-tRNA synthetase, ThserRS, which can negatively regulate laccase lacA transcription in Trametes hirsuta AH28-2 under exposure to copper ion. ThserRS was obtained through yeast one-hybrid screening using a bait sequence of lacA promoter (-502 to -372 bp). ThserRS decreased while lacA increased at the transcription level in T. hirsuta AH28-2 in the first 36 h upon CuSO4 induction. Then, ThserRS was upregulated, and lacA was downregulated. ThserRS overexpression in T. hirsuta AH28-2 resulted in a decrement in lacA transcription and LacA activity. By comparison, ThserRS silencing led to increased LacA transcripts and activity. A minimum of a 32-bp DNA fragment containing two putative xenobiotic response elements could interact with ThserRS, with a dissociation constant of 919.9 nM. ThserRS localized in the cell cytoplasm and nucleus in T. hirsuta AH28-2 and was heterologously expressed in yeast. ThserRS overexpression also enhanced mycelial growth and oxidative stress resistance. The transcriptional level of several intracellular antioxidative enzymes in T. hirsuta AH28-2 was upregulated. Our results demonstrate a noncanonical activity of SerRS that acts as a transcriptional regulation factor to upregulate laccase expression at an early stage after exposure to copper ions. IMPORTANCE Seryl-tRNA synthetase is well known for the attachment of serine to the corresponding cognate tRNA during protein translation. In contrast, its functions beyond translation in microorganisms are underexplored. We performed in vitro and cell experiments to show that the seryl-tRNA synthetase in fungi with no UNE-S domain at the carboxyl terminus can enter the nucleus, directly interact with the promoter of the laccase gene, and negatively regulate the fungal laccase transcription early upon copper ion induction. Our study deepens our understanding of the Seryl-tRNA synthetase noncanonical activities in microorganisms. It also demonstrates a new transcription factor for fungal laccase transcription.

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.

BRD4770 inhibits vascular smooth muscle cell proliferation via SUV39H2, but not EHMT2 to protect against neointima formation.

The behavior of vascular smooth muscle cells (VSMCs) contributes to the formation of neointima. We previously found that EHMT2 suppressed autophagy activation in VSMCs. BRD4770, an inhibitor of EHMT2/G9a, plays a critical role in several kinds of cancers. However, whether and how BRD4770 regulates the behavior of VSMCs remain unknown. In this study, we evaluate the cellular effect of BRD4770 on VSMCs by series of experiments in vivo and ex vivo. We demonstrated that BRD4770 inhibited VSMCs' growth by blockage in G2/M phase in VSMCs. Moreover, our results demonstrated that the inhibition of proliferation was independent on autophagy or EHMT2 suppression which we previous reported. Mechanistically, BRD4770 exhibited an off-target effect from EHMT2 and our further study reveal that the proliferation inhibitory effect by BRD4770 was associated with suppressing on SUV39H2/KTM1B. In vivo, BRD4770 was also verified to rescue VIH. Thus, BRD4770 function as a crucial negative regulator of VSMC proliferation via SUV39H2 and G2/M cell cycle arrest and BRD4770 could be a molecule for the therapy of vascular restenosis.