METTL14 mediates m6a modification on osteogenic proliferation and differentiation of bone marrow mesenchymal stem cells by regulating the processing of pri-miR-873.

N6-methyl-adenosine (m6a) is involved in the occurrence and development of various diseases such as autogenic immune disease and tumors. Methyltransferases regulate primary (pri)-microRNA (miRNA/miR) processing by mediating m6a modifications, consequently affecting pathological processes including immune-related diseases by regulating both innate and adaptive immune cells. However, the roles of m6a on the biological functions of bone marrow mesenchymal stem cells (BMSCs) remain to be elucidated. The relative expression levels of methyltransferase-like 14 (METTL14) and other methyltransferases, demethylases, and miR-873 in bone samples from patients with osteoporosis and from normal individuals were measured by reverse transcription-quantitative PCR. Cell Counting Kit-8 assay was used to examine the proliferation of BMSCs. Co-immunoprecipitation (Co-IP) was used to investigate the binding of METTL14 to DiGeorge syndrome critical region 8 (DGCR8). RNA immunoprecipitation (RIP) was used to examine the binding of METTL14 to pri-miR-873. METTL14 and m6a modifications were highly detected in patients with osteoporosis compared with the controls. Co-IP results indicated that silencing of METTL14 reduced METTL14 and m6a modification levels in BMSCs. Downregulation of METTL14 significantly promoted the proliferation of BMSCs. RIP results suggested that METTL14/m6a methylation modification promoted the processing of pri-miR-873 by binding to DGCR8 in BMSCs. Furthermore, overexpression of miR-873 inhibited the proliferation of BMSCs. The results also showed that miR-873 mimics significantly inhibited the proliferation in small interfering (si)-METTL14 transfected BMSCs; however, miR-873 inhibitors markedly promoted the proliferation of si-METTL14 transfected BMSCs. METTL14 and m6a modifications were upregulated in osteoporosis samples. METTL14 promoted the processing of pri-miR-873 into mature miR-873 by regulating m6a modification. Furthermore, overexpression of miR-873 significantly inhibited the proliferation of BMSCs. Therefore, the METTL14/m6a/miR-873 axis may be a potential target for the treatment of osteoporosis.

An Improved Technique for Trimethylamine Detection in Animal-Derived Medicine by Headspace Gas Chromatography-Tandem Quadrupole Mass Spectrometry.

Animal-derived medicines have distinctive characteristics and significant curative effects, but most of them have an obvious fishy odor, resulting in the poor compliance of clinical patients. Trimethylamine (TMA) is one of the key fishy odor components in animal-derived medicine. It is difficult to identify TMA accurately using the existing detection method due to the increased pressure in the headspace vial caused by the rapid acid-base reaction after the addition of lye, which causes TMA to escape from the headspace vial, stalling the research progress of the fishy odor of animal-derived medicine. In this study, we proposed a controlled detection method that introduced a paraffin layer as an isolation layer between acid and lye. The rate of TMA production could be effectively controlled by slowly liquefying the paraffin layer through thermostatic furnace heating. This method showed satisfactory linearity, precision experiments, and recoveries with good reproducibility and high sensitivity. It provided technical support for the deodorization of animal-derived medicine.

Astaxanthin promotes mitochondrial biogenesis and antioxidant capacity in chronic high-intensity interval training.

PURPOSE: Reactive oxygen and nitrogen species are required for exercise-induced molecular adaptations; however, excessive exercise may cause cellular oxidative distress. We postulate that astaxanthin (ASX) can neutralize oxidative distress and stimulate mitochondrial biogenesis in high-intensity exercise-trained mice. METHODS: Six-week-old mice (n = 8/group) were treated with ASX (10 mg/kg BW) or placebo. Training groups participated in 30 min/day high-intensity interval training (HIIT) for 6 weeks. Gastrocnemius muscle was collected and assayed following the exercise training period. RESULTS: Compared to the HIIT control mice, the ASX-treated HIIT mice reduced malonaldehyde levels and upregulated the expression of Nrf2 and FOXO3a. Meanwhile, the genes NQO1 and GCLC, modulated by Nrf2, and SOD2, regulated by FOXO3a, and GPx4, were transcriptionally upregulated in the ASX-treated HIIT group. Meanwhile, the expression of energy sensors, AMPK, SIRT1, and SIRT3, increased in the ASX-treated HIIT group compared to the HIIT control group. Additionally, PGC-1α, regulated by AMPK and SIRT1, was upregulated in the ASX-treated HIIT group. Further, the increased PGC-1α stimulated the transcript of NRF1 and Tfam and mitochondrial proteins IDH2 and ATP50. Finally, the ASX-treated HIIT mice had upregulations in the transcript level of mitochondrial fusion factors, including Mfn1, Mfn2, and OPA1. However, the protein level of AMPK, SIRT1, and FOXO3a, and the transcript level of Nrf2, NQO1, PGC-1α, NRF1, Mfn1, Mfn2, and OPA1 decreased in the HIIT control group compared to the sedentary control group. CONCLUSION: Supplementation with ASX can reduce oxidative stress and promote antioxidant capacity and mitochondrial biogenesis during strenuous HIIT exercise in mice.

p-Hydroxybenzyl Alcohol Prevents Memory Deficits by Increasing Neurotrophic Factors and Decreasing Inflammatory Factors in a Mice Model of Alzheimer's Disease.

p-hydroxybenzyl alcohol (HBA) is one of the major components of Gastrodia elata Blume (GEB) phenolic compound. HBA has been reported to have a protective effect on amyloid-ß (Aß) induced cell death. However, the systemic effects and the detail molecular mechanism of HBA in Alzheimer's disease (AD) animal models is not clear. In this study, we revealed the protective effects and the potential mechanisms of HBA on the impairments of cognitive function induced by soluble Aß oligomers. Our results showed that HBA prevented neuronal cells death in a dose-dependent manner. The working memory and the spatial memory were significantly lower in AD model mice. HBA treatment prevented the memory deficits of the AD mice. HBA treatment significantly prevented the decreased spine density and decreased expression levels of synaptic proteins induced by Aß42. In addition, both mRNA levels and protein levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in the Aß42-treated mice were decreased, the decreases were prevented by HBA treatment. The expression levels of TNF-α and IL-1ß were increased by Aß42 treatment and the increase can be prevented by the HBA treatment. Moreover, HBA prevents the decreases in the level of nuclear erythroid 2 p45-related factor 2 (Nrf2) induced by Aß42 in hippocampal. Thus, we predict that HBA might prevent Aß42 oligomer-induced synapse and cognitive impairments through multiple targets including increasing Nrf2, increasing neurotrophic factors and decreasing inflammatory factors. Our study provided novel insights into the cellular mechanisms for the protective effects of HBA on AD.

Taxifolin prevents ß-amyloid-induced impairments of synaptic formation and deficits of memory via the inhibition of cytosolic phospholipase A2/prostaglandin E2 content.

Taxifolin is a potent flavonoid with anti-inflammatory activity. Taxifolin has been reported to decrease the accumulation of ß-amyloid (Aß), and reduce Aß-induced neurotoxicity. However, the detail molecular mechanism of taxifolin against Aß-induced neurotoxicity is largely unknown. In this study, we revealed the protective effects and the underlying mechanisms of taxifolin on the impairments of cognitive function and synapse formation induced by soluble Aß oligomers. Our results showed that taxifolin prevented neuronal cell death in a concentration-dependent manner. The recognition memory in novel object recognition tasks and the spatial memory in Morris water maze tests are significantly lower in the Alzheimer's disease (AD) model mice induced by hippocampal injection of Aß42. Taxifolin treatment prevented the recognitive and spatial memory deficits of the AD mice. 10 mg/kg taxifolin treatment also significantly prevented the decreased expression levels of PSD 95 induced by Aß42. Live cell imaging study showed that 2 h pre-treatment of taxifolin prevented the decrease in the number of filopodium and spine induced by Aß42 oligomers. Aß42 oligomers significantly increased the production of cytosolic phospholipase A2 (cPLA2), a crucial enzyme of pro-inflammatory mediator, and prostaglandin E2 (PGE2), a neuroinflammatory molecule. Taxifolin significantly reduced the content of cPLA2 and PGE2 induced by Aß42 both in the primary hippocampal neurons and hippocampal tissues. These results indicated that taxifolin might prevent Aß42 oligomer-induced synapse and cognitive impairments through decreasing cPLA2 and PGE2. Our study provided novel insights into the cellular mechanisms for the protective effects of taxifolin on AD.

HAL2 overexpression induces iron acquisition in bdf1Δ cells and enhances their salt resistance.

The yeast Saccharomyces cerevisiae is capable of responding to various environmental stresses, such as salt stress. Such responses require a complex network and adjustment of the gene expression network. The goal of this study is to further understand the molecular mechanism of salt stress response in yeast, especially the molecular mechanism related to genes BDF1 and HAL2. The Bromodomain Factor 1 (Bdf1p) is a transcriptional regulator, which is part of the basal transcription factor TFIID. Cells lacking Bdf1p are salt sensitive with an abnormal mitochondrial function. We previously reported that the overexpression of HAL2 or deletion of HDA1 lowers the salt sensitivity of bdf1Δ. To better understand the mechanism behind the HAL2-related response to salt stress, we compared three global transcriptional profiles (bdf1Δ vs WT, bdf1Δ + HAL2 vs bdf1Δ, and bdf1Δhda1Δ vs bdf1Δ) in response to salt stress using DNA microarrays. Our results reveal that genes for iron acquisition and cellular and mitochondrial remodeling are induced by HAL2. Overexpression of HAL2 decreases the concentration of nitric oxide. Mitochondrial iron-sulfur cluster (ISC) assembly also decreases in bdf1Δ + HAL2. These changes are similar to the changes of transcriptional profiles induced by iron starvation. Taken together, our data suggest that mitochondrial functions and iron homeostasis play an important role in bdf1Δ-induced salt sensitivity and salt stress response in yeast.

[Study on RhBMP-2 induced osteoporosis rat BMSCs in vitro osteogenesis and VEGF expression].

OBJECTIVE: To observe the impact of bone morphogenetic protein-2 (rhBMP-2) on bone marrow stromal cells (BMSCs) osteogenesis in vitro and vascular endothelial growth factors (VEGF) expression in bone osteoporotic to prevent and treat the osteoporosis. METHODS: Twenty 6-month-old female SD rats weighted (300±20) g underwent bilateral ovariectomized. At 3 months after operation, dual-energy X-ray absorptiometry was used to measure bone mineral density of rats,the values were compared with preoperative to ensure the model successfully, and the osteoporosis rats' BMSCs were cultured by bone marrow adherent cultured and the BMSCs morphology was observed under a phase contrast microscope upside down. The osteoporosis rats' BMSCs at the 2nd generation (p2) were randomly divided into experimental and control groups and were added complete medium (containing rhBMP-2) and osteogenic induced liquid, respectively. Two weeks later, the formation of cell calcium nodules were detected by Alizarin red staining method,alkaline phosphatase activity was measured by enzyme standard instrument and the expression of VEGF was detected by RT-PCT method. RESULTS: (1)Whole body bone mineral density of rats before and after operation were (0.179±0.007), (0.158±0.006) g/cm2,there was statistically significant (t=4.180,P< 0.05). (2)Alizarin red staining at 2 weeks after osteogenesis induced by BMSCs (P2) in the experimental group had more strong dyeing effect than the control group obviously. (3)Alkaline phosphatase activity at 2 weeks after osteogenesis induced by BMSCs (P2) of the experimental group (15.62±1.27) ug/gprot was significantly higher than that of the control group (8.62±0.93) ug/gprot,there was statistically significant (t=7.709, P<0.01). (4)The expression of VEGF at 2 weeks after osteogenesis induced by BMSCs (P2) of the experimental group 3.723±0.143 was significantly higher than that of the control group 0.950±0.072, there was statistically significant (t=29.462, P<0.01). CONCLUSION: RhBMP-2 can improve the in-vitro osteogenesis ability of ovary osteoporosis rat BMSCs, promote the VEGF expression of osteogenesis factor. Regulating the VEGF expression may be one of the mechanisms of BMP-2 to participate in bone metabolism.

The yeast BDF1 regulates endocytosis via LSP1 under salt stress.

Bromodomain-containing transcription factor, a kind of important regulating protein, can recognize and bind to acetylated histone. The homologous genes, BDF1 and BDF2, in Saccharomyces cerevisiae, respectively, encode a bromodomain-containing transcription factor. Previously study has demonstrated that both BDF1 and BDF2 participate in yeast salt stress response. Bdf1p deletion cells are sensitive to salt stress and this phenotype is suppressed by its homologue BDF2 in a dosage-dependent manner. In this study, we show that the histone deacetylase SIR2 over-expression enhanced dosage-dependent compensation of BDF2. SIR2 over-expression induced a global transcription change, and 1959 gene was down-regulated. We deleted some of the most significant down-regulated genes and did the spot assay. The results revealed that LSP1, an upstream component of endocytosis pathway, and CIN5, a transcription factor that mediates cellular resistance to stresses, can enhance salt resistance of bdf1∆. Further analysis demonstrated that under salt stress the endocytosis is over-activated in bdf1∆ but was recovered in bdf1∆ lsp1∆. To our best knowledge, this is the first report that the transcription factor Bdf1p regulates endocytosis under salt stress via LSP1, a major component of eisosomes that regulate the sites of endocytosis.

Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought-stress conditions.

A transgenic gene-silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down-regulated ACC synthases (ACSs), enzymes that catalyse the rate-limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought-stress and rain-fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58 Mg/ha (9.3 bushel/acre) increase after a flowering period drought stress. A (genotype × transgene) × environment interaction existed among the events, highlighting the need to better understand the context in which the down-regulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis-silking interval and a concomitant increase in kernel number/ear in transgene-positive events versus nulls. Selected events were also field tested under a low-nitrogen treatment, and the best event was found to have a significant 0.44 Mg/ha (7.1 bushel/acre) yield increase. This set of extensive field evaluations demonstrated that down-regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.

Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae.

Improving the cellulase secretion is beneficial for Saccharomyces cerevisiae used in consolidated bioprocessing (CBP) of cellulosic ethanol. In this study, protein secretory pathway, including protein folding, disulfide bond formation, and protein trafficking and sorting, was modified in S. cerevisiae. The effects of these modifications on the secretion of cellobiohydrolase I (Tr-Cel7A) with its native signal peptide from Trichoderma reesei were investigated. The results showed that overexpression of the protein disulfide isomerase Sc-PDI1 and the plasma membrane targeting soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sc-SSO1, and disruption of the sorting receptor Sc-VPS10 and a Ca(2+)/Mn(2+) ATPase Sc-PMR1, improved respectively the extracellular Tr-Cel7A activities. Among them, disruption of Sc-PMR1 showed better improvement of 162% in the extracellular activity and decreased the glycosylation of Tr-Cel7A. Multiple modifications generally resulted in higher activities. The extracellular activities of the quadruple-modified strain (vps10Δ/pmr1Δ/SSO1/PDI1/cel7AF) using p-nitrophenyl-ß-d-cellobioside (pNPC) and phosphoric acid swollen cellulose (PASC) as the substrates, respectively, were 3.9-fold and 1.3-fold higher than that of the reference strain cel7AF. The results indicated that engineering of the protein secretory pathway is an effective approach to improve the Tr-Cel7A secretion in S. cerevisiae.

Hal2p functions in Bdf1p-involved salt stress response in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Bdf1p associates with the basal transcription complexes TFIID and acts as a transcriptional regulator. Lack of Bdf1p is salt sensitive and displays abnormal mitochondrial function. The nucleotidase Hal2p detoxifies the toxic compound 3' -phosphoadenosine-5'-phosphate (pAp), which blocks the biosynthesis of methionine. Hal2p is also a target of high concentration of Na(+). Here, we reported that HAL2 overexpression recovered the salt stress sensitivity of bdf1Δ. Further evidence demonstrated that HAL2 expression was regulated indirectly by Bdf1p. The salt stress response mechanisms mediated by Bdf1p and Hal2p were different. Unlike hal2Δ, high Na(+) or Li(+) stress did not cause pAp accumulation in bdf1Δ and methionine supplementation did not recover its salt sensitivity. HAL2 overexpression in bdf1Δ reduced ROS level and improved mitochondrial function, but not respiration. Further analyses suggested that autophagy was apparently defective in bdf1Δ, and autophagy stimulated by Hal2p may play an important role in recovering mitochondrial functions and Na(+) sensitivity of bdf1Δ. Our findings shed new light towards our understanding about the molecular mechanism of Bdf1p-involved salt stress response in budding yeast.

Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast.

Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

[Effect of controlled overexpression of xylulokinase by different promoters on xylose metabolism in Saccharomyces cerevisiae].

OBJECTIVE: To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome. METHODS: Based on S. cerevisiae CEN. PK 113-5D, we constructed xylose-metabolizing strains where the promoter of xylulokinase gene XKS1 was replaced by TEF1 promoter, PGK1 promoter and HXK2 promoter on the chromosome. We quantitated the transcriptional level of XKS1 gene (accumulated mRNA) and measured the activity of xylulokinase in each stains. Furthermore, we also determined the intracellular level of ATP and evaluated the xylose-fermenting abilities of the engineered strains. RESULTS: The engineered strains exhibited higher expression of xylulokinase than the parental strain at both transcription and enzyme activity levels. The highest xylulokinase activity was observed in the strain whose XKS1 was controlled by PGKlp, and was decreasingly followed by the strains whose XKS1 was controlled by TEF1p, HXK2p and native promoter. The expression level of xylulokinase negatively correlated with intracellular level of ATP and positively correlated with ability of ethanol production from xylose. The highest ethanol yield was 0.35 g/g consumed sugars while the lowest xylitol yield, which was 0.18 g/g consumed xylose, was observed. CONCLUSION: By promoter replacement, xylulokinase was overexpressed at different levels. In this work, higher expressional level of xylulokinase improved the conversion of xylose to ethanol.

Bdf1p deletion affects mitochondrial function and causes apoptotic cell death under salt stress.

The Saccharomyces cerevisiae BDF1 gene encodes a bromodomain-containing transcription factor. We previously reported that deletion of Bdf1p in yeast cells resulted in increased sensitivity to NaCl stress. In this paper, we show that the function of Bdf1p in salt tolerance is not directly linked with the Ena1p-mediated Na(+) extrusion system, and a number of other well-characterized stress-response pathways. Interestingly, however, our data demonstrate that, under the NaCl stress, the absence of Bdf1p leads to mitochondrial dysfunction, including decreasing of mitochondrial membrane potential (Delta Psi) and accumulation of reactive oxygen species, and chromatin fragmentation and condensation. These results indicate that the bromodomain-containing protein, Bdf1p, is involved in the regulation of apoptosis in yeast cells.

Characterization of cyclopropane fatty-acid synthase from Sterculia foetida.

Cyclopropane synthase from Sterculia foetida developing seeds catalyzes the addition of a methylene group from S-adenosylmethionine to the cis double bond of oleic acid (Bao, X., Katz, S., Pollard, M., and Ohlrogge, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7172-7177). To understand this enzyme better, differential expression in leaf and seed tissues, protein properties, and substrate preferences of plant cyclopropane synthase were investigated. Immunoblot analysis with antibodies raised to recombinant S. foetida cyclopropane synthase (SfCPA-FAS) revealed that SfCPA-FAS is expressed in S. foetida seeds, but not in leaves, and is a membrane protein localized to microsomal fractions. Transformed tobacco cells expressing SfCPA-FAS were labeled in vivo with L-[methyl-(14)C]methionine and assayed in vitro with S-adenosyl-L-[methyl-(14)C]methionine. These kinetic experiments demonstrated that dihydrosterculate was synthesized from oleic acid esterified at the sn-1 position of phosphatidylcholine (PC). Furthermore, analysis of acyl chains at sn-1 and sn-2 positions that accumulated in PC from S. foetida developing seeds and from tobacco cells expressing SfCPA-FAS also demonstrated that greater than 90% of dihydrosterculate was esterified to the sn-1 position. Thus, we conclude that SfCPA-FAS is a microsomal localized membrane protein that catalyzes the addition of methylene groups derived from S-adenosyl-L-methionine across the double bond of oleic acid esterified to the sn-1 position of PC. A survey of plant and bacterial genomes for sequences related to SfCPA-FAS indicated that a peptide domain with a putative flavin-binding site is either fused to the methyltransferase domain of the plant protein or is often found encoded by a gene adjacent to a bacterial cyclopropane synthase gene.

Carbocyclic fatty acids in plants: biochemical and molecular genetic characterization of cyclopropane fatty acid synthesis of Sterculiafoetida.

Fatty acids containing three-member carbocyclic rings are found in bacteria and plants. Bacteria synthesize cyclopropane fatty acids (CPA-FAs) only by the addition of a methylene group from S-adenosylmethionine to the cis-double bond of monoenoic phospholipid-bound fatty acids. In plants CPA-FAs are usually minor components with cyclopropene fatty acids (CPE-FAs) more abundant. Sterculia foetida seed oil contains 65-78% CPE-FAs, principally sterculic acid. To address carbocyclic fatty acid synthesis in plants, a cDNA library was constructed from developing seeds during the period of maximum oil deposition. About 0.4% of 5,300 expressed sequence tags were derived from one gene, which shared similarities to the bacterial CPA-FA synthase. However, the predicted protein is twice as large as the bacterial homolog and represents a fusion of an FAD-containing oxidase at the N terminus and a methyltransferase at the C terminus. Functional analysis of the isolated full-length cDNA was conducted in tobacco suspension cells where its expression resulted in the accumulation of up to 6.2% dihydrosterculate of total fatty acids. In addition, the dihydrosterculate was specifically labeled by [methyl-(14)C]methionine and by [(14)C]oleic acid in the transgenic tobacco cells. In in vitro assay of S. foetida seed extracts, S-adenosylmethionine served as a methylene donor for the synthesis of dihydrosterculate from oleate. Dihydrosterculate accumulated largely in phosphatidylcholine in both systems. Together, a CPA-FA synthase was identified from S. foetida, and the pathway in higher plants that produce carbocyclic fatty acids was defined as by transfer of C(1) units, most likely from S-adenosylmethionine to oleate.