UvHOS3-mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2 O2 . ΔUvhos3 generated significantly more ustilaginoidins. RNA-Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi.

Histone Deacetylase UvHST2 Is a Global Regulator of Secondary Metabolism in Ustilaginoidea virens.

Ustilaginoidea virens, the causal agent of rice false smut, produces a large amount of mycotoxins, including ustilaginoidins and sorbicillinoids. However, little is known about the regulatory mechanism of mycotoxin biosynthesis inU. virens. Here, we demonstrate that the NAD+-dependent histone deacetylase UvHST2 negatively regulates ustilaginoidin biosynthesis. UvHst2 knockout caused retarded hypha growth and reduced conidiation and pathogenicity inU. virens. Transcriptome analysis revealed that the transcription factor genes, transporter genes, and other tailoring genes in eight biosynthetic gene clusters (BGCs) including ustilaginoidin and sorbicillinoid BGCs were upregulated in ΔUvhst2. Interestingly, the UvHst2 deletion affects alternative splicing. Metabolomics revealed that UvHST2 negatively regulates the biosynthesis of various mycotoxins including ustilaginoidins, sorbicillin, ochratoxin B, zearalenone, and O-M-sterigmatocystin. Combined transcriptome and metabolome analyses uncover that UvHST2 positively regulates pathogenicity but negatively modulates the expression of BGCs involved in secondary metabolism. Collectively, UvHST2 functions as a global regulator of secondary metabolism inU. virens.

Histological evaluation of mouse tongue incisions after Er:YAG laser surgery with different pulse energies versus after conventional scalpel surgery.

PURPOSE: To identify the surgical instrument that allows for optimal healing of tongue incisions. METHODS: An Er:YAG laser was compared with different pulse energies to a conventional scalpel for the incision of mouse tongue tissues. Mice were sacrificed through cervical dislocation at 24, 48, and 72 h postoperatively, followed by extraction of their tongues for incision experiments. The healing of the incisions and expression of inflammation- and pain-related factors in the tongues were compared between the surgical procedure groups. RESULTS: In laser-treated mice, tongue incisions healed the fastest when the laser output energy was 60 MJ per pulse. Macrophage chemotaxis toward the incisional area was triggered on the first postoperative day for the 60-MJ group, while the time for macrophage chemotaxis to the surgical area was later in the 80-MJ group. Tumor necrosis factor-alpha expression increased and then decreased in the 80-MJ group; however, it gradually decreased in the 60-MJ and conventional scalpel groups. Prostaglandin E2 expression increased and then decreased in the 80-MJ and conventional scalpel groups but gradually decreased in the 60-MJ group. The expression of transforming growth factor beta 1 gradually decreased in the 60-MJ and 80-MJ groups but gradually increased in the conventional scalpel group. CONCLUSION: Compared with surgical procedures using conventional scalpels, those using an Er:YAG laser with appropriate pulse energies can inhibit inflammation in the incisional area and promote incision healing. The use of an Er:YAG laser with appropriate pulse energies can alleviate intraoperative and postoperative pain in the incisional area.

Influence of bone formation by composite scaffolds with different proportions of hydroxyapatite and collagen.

Composite scaffolds with different proportions of hydroxyapatite (HA) and collagen (COL) produced different bone induction results. OBJECTIVE: To examine the composite scaffolds with optimal proportion of HA and COL to achieve earlier bone induction and maximum bone formation. METHODS: Composite scaffolds with the HA/COL weight ratio of 7:3, 3:7, 5:5 and 9:1 were prepared, as HA powder was added to collagen solution at 130℃ for 48 h. Then, the composites with different proportions of HA/COL were implanted into the extraction socket of right upper central incisor of C57BL/6 J mice. The bone formation of the extraction socket was observed by Hematoxylin-eosin (HE) and Masson-trichrome (Masson) staining at 1 and 2 weeks after operation. Five weeks later, the bone formation of extraction socket was observed by micro computed tomography (micro-CT). After MC3T3-E1 cells were co-cultured with materials of different proportions for 3 days, the number of cells attached on the surface of the materials and entering the materials were counted, and the expression of osteogenic related genes (Runx2, Ocn. Osx and Alp) was detected by reverse transcription polymerase chain reaction (RT-PCR). The composite scaffolds with different proportion of HA/COL with and without mouse bone marrow mesenchymal stem cells (BMMSCs) were implanted into the back of adult mice and cultured subcutaneously for 30 days, and observed histologically by HE and Masson staining. RESULTS: After one week implantation with the composite HA/COL scaffolds with the weight ratio of 7:3, 3:7, 5:5 and 9:1, there was no new bone formation in the extraction socket in mouse. However, two weeks later, new bone was firstly observed in the tooth socket with the composite HA/COL scaffolds of 7:3. 5 weeks later, micro-CT scanning showed that the total amount of newly formed bone, trabecular width and bone mineral density of the HA/COL scaffolds of 7:3 were higher than the other HA/COL scaffolds (P < 0.05). After MC3T3-E1 cells were co-cultured with different composite HA/COL scaffolds for 3 days. The number of cells on the surface and inside of the HA/COL scaffolds of 7:3 was more than the other materials, and the difference was statistically significant (P < 0.05). The expression levels of Ocn and Osx of MC3T3-E1 cells were also the highest in the HA/COL scaffolds of 7:3 (P < 0.01). Bone formation was observed in the composite HA/COL scaffold of 7:3 with BMMSCs subcutaneously in mouse for 30 days, while only osteoid formation was observed in the same scaffold without BMMSCs. but bone formation was not detected in the other proportions of the HA/COL scaffolds. SIGNIFICANCE: Compared with other proportions of HA/COL, the composite HA/COL scaffolds of 7:3 has stronger ability to promote bone formation, recruit osteoblasts to attach and enter into the scaffolds, and promote the osteogenesis of BMMSCs.

Hepatic dysfunction induced by intestinal dysbacteriosis mainly manifests as immunologic abnormity in mice.

Currently, the potential role of the alterations occurring in the liver immune system and intestinal flora in liver injury remains unknown. Our study aimed to explore the impacts of intestinal microbial barrier damage induced by ceftriaxone on liver immunity. We developed the BALB/c mice model by administering ceftriaxone. The intestinal microbial barrier damage was observed by 16S rRNA, and the pathological changes of intestines and livers were detected by H&E or transmission electron microscope. The activation of immunocytes were tested by Flow Cytometry; the expression of LPS, ALT, AST, IL-6 and TNF-α were detected by Limulus Test or ELISA. Compared to the control, the intestinal microbes significantly decreased in ceftriaxone group. Additionally, the weight of cecum contents increased, the intestinal wall became thinner and the villus in the small intestine and cecum were damaged. The expression of LPS and the ratio of liver lymphocytes were significantly increased. H&E results indicated the structures of liver arose the pathologic changes. Meanwhile, the content of serum ALT, AST, IL-6 and TNF-α increased. Collectively, our study indicates that the damages of gut microbial barrier induced by ceftriaxone prompted activation of immunocytes and release of inflammatory cytokines, which may lead to chronic inflammation in liver.

Bmp2 and Bmp4 accelerate alveolar bone development.

Alveolar bone remodeling is a continuous process that takes place during development and in response to various physiological and pathological stimuli. However, detailed knowledge regarding the underlying mechanisms involved in alveolar bone development is still lacking. This study aims at improving our understanding of alveolar bone formation and the role of bone morphogenetic proteins (Bmps) in this process. Mice at embryonic (E) day 13.5 to postnatal (PN) day 15.5 were selected to observe the process of alveolar bone development. Alveolar bone development was found to be morphologically observable at E14.5. Molar teeth isolated from mice at PN7.5 were pretreated with Bmp2, Bmp4, Noggin, or BSA, and grafted subcutaneously into mice. The subcutaneously implanted tooth germs formed alveolar bone indicating the role of the dental follicle in alveolar bone development. Alveolar bone formation was increased after pretreatment with Bmp2 and Bmp4, but not with Noggin. Gene expression levels in dental follicle cells from murine molars were also determined by real-time RT-PCR. The expression levels of Runx2, Bsp, and Ocn were significantly higher in dental follicle cells cultured with Bmp2 or Bmp4, and significantly lower in those cultured with Noggin when compared with that of the BSA controls. Our results suggest that the dental follicle participates in alveolar bone formation and Bmp2/4 appears to accelerate alveolar bone development.

Effects of iron and phosphorus on Microcystis physiological reactions.

OBJECTIVE: To observe the effects of iron and phosphorus on Microcystis physiological reactions. METHODS: The experimental conditions were chosen as the light dark cycles of 16 h 8 h, 12 h 12 h, and 8 h 16 h. The cell change of morphology and life history, cell number, cell color, and cell area of Microcystis were analyzed quantitatively. According to the resource competition and Monod equation, Microcystis kinetics of phosphorus and iron were also examined. RESULTS: The longer light time caused more special cell division, slower growth rate, and easier change of bigger cell area. The color of alga was changed from green to brown. Ks and micromax of phosphorus absorption were 0.0352 mircomol x L(-l) and 0.493 d(-1), respectively. Those of iron absorption were 0.00323 micromol x L(-1) and 0.483 d(-1). CONCLUSION: Microcystis bloom is more dominant than other algae.

Effects of nutrients on Microcystis growth more easily forming bloom.

Different nutrient media experimentally were N, P and Fe-limited conditions and a serial of diluted BG11 media. The cell change of morphology and life history, cell number, cell color and cell area of Microcystis were analyzed quantitatively. First, the effects of nitrogen, phosphorus and iron depletion were distinctively different. Phosphorus and iron depletion caused more special division cells, slowly growth increasing, the easier change of bigger cell area. Second, the nitrogen and iron depletion could make the color of alga from green to brown. Finally, according to the resource competition and Monod equation, Microcystis kinetics of phosphorus and iron were also examined. K(s) and micro(max) of phosphorus absorption were 0.0352 micromol/L, 0.493 d(-1) respectively; iron absorption: 0.00323 micromol/L, 0.483 d(-1). In a word, some evidences of the Microcystis bloom dominance in certain nutrient conditions were indicated in the experiments. The dominances were determined as the reviving under the adverse circumstances through the special division, the various nitrogen resources, and the lower kinetics of phosphorus and iron than that of most of other algae. The conclusions provided the scientific basis for preventing and managing Microcystis bloom in freshwater.