Deletion of TECRL promotes skeletal muscle repair by up-regulating EGR2.

Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.

Zyxin inhibits the epithelial-mesenchymal transition process in gastric cancer by upregulating SIRT1.

Tumor development relies on the stemness of cancer stem cells, which is regulated by environmental cues. Previous studies have shown that zyxin can inhibit the expression of genes for embryonic stem cell status. In the present study, the expression levels of zyxin protein in cancer tissues and adjacent noncancerous tissues from 73 gastric cancer patients with different clinical stages were analyzed by Western blot. We showed that the relative expression levels of zyxin in gastric cancer tissues (cancer tissues/adjacent tissues) were significantly downregulated in advanced clinical stages. Overexpression of zyxin inhibited the stemness and epithelial-mesenchymal transition (EMT) processes in gastric cancer cells. Zyxin also inhibited the proliferation, migration, and invasion but increased the sensitivity of cancer cells to drugs. Overexpression of zyxin in MKN45 cells inhibited tumor growth in nude mice. We show that the interactions between zyxin and SIRT1 led to the upregulation of SIRT1, reduced acetylation levels of histone H3 K9 and K23, decreased transcription levels of SNAI 1/2, and inhibition of the EMT process. This study demonstrated that zyxin negatively regulates the progression of gastric cancer by inhibiting the stemness of cancer stem cells and EMT. Our findings shed new light on the pathogenesis of gastric cancer.

Loss of CRY2 promotes regenerative myogenesis by enhancing PAX7 expression and satellite cell proliferation.

The regenerative capacity of skeletal muscle is dependent on satellite cells. The circadian clock regulates the maintenance and function of satellite cells. Cryptochrome 2 (CRY2) is a critical component of the circadian clock, and its role in skeletal muscle regeneration remains controversial. Using the skeletal muscle lineage and satellite cell-specific CRY2 knockout mice (CRY2scko), we show that the deletion of CRY2 enhances muscle regeneration. Single myofiber analysis revealed that deletion of CRY2 stimulates the proliferation of myoblasts. The differentiation potential of myoblasts was enhanced by the loss of CRY2 evidenced by increased expression of myosin heavy chain (MyHC) and myotube formation in CRY2-/- cells versus CRY2+/+ cells. Immunostaining revealed that the number of mononucleated paired box protein 7 (PAX7+) cells associated with myotubes formed by CRY2-/- cells was increased compared with CRY2+/+ cells, suggesting that more reserve cells were produced in the absence of CRY2. Loss of CRY2 leads to the activation of the ERK1/2 signaling pathway and ETS1, which binds to the promoter of PAX7 to induce its transcription. CRY2 deficient myoblasts survived better in ischemic muscle. Therefore, CRY2 is essential in regulating skeletal muscle repair.

Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments.

In the current study, we investigated nitrite-dependent anaerobic methane oxidation (N-DAMO) as a potential methane sink in the Hangzhou Bay and the adjacent Zhoushan sea area. The potential activity of the N-DAMO process was primarily observed in Hangzhou Bay by means of (13)C-labeling experiments, whereas very low or no potential N-DAMO activity could be detected in the Zhoushan sea area. The measured potential N-DAMO rates ranged from 0.2 to 1.3 nmol (13)CO2 g(-1) (dry sediment) day(-1), and the N-DAMO potentially contributed 2.0-9.4 % to the total microbial methane oxidation in the examined sediments. This indicated that the N-DAMO process may be an alternative pathway in the coastal methane cycle. Phylogenetic analyses confirmed the presence of Candidatus Methylomirabilis oxyfera-like bacteria in all the examined sediments, while the group A members (the dominant bacteria responsible for N-DAMO) were found mainly in Hangzhou Bay. Quantitative PCR showed that the 16S rRNA gene abundance of Candidatus M. oxyfera-like bacteria varied from 5.4 × 10(6) to 5.0 × 10(7) copies g(-1) (dry sediment), with a higher abundance observed in Hangzhou Bay. In addition, the overlying water NO3 (-) concentration and salinity were identified as the most important factors influencing the abundance and potential activity of Candidatus M. oxyfera-like bacteria in the examined sediments. This study showed the evidence of N-DAMO in coastal environments and indicated the importance of N-DAMO as a potential methane sink in coastal environments.

Improvement of the trace metal composition of medium for nitrite-dependent anaerobic methane oxidation bacteria: Iron (II) and copper (II) make a difference.

Nitrite-dependent anaerobic methane oxidation (n-damo) is a potential bioprocess for treating nitrogen-containing wastewater. This process uses methane, an inexpensive and nontoxic end-product of anaerobic digestion, as an external electron donor. However, the low turnover rate and slow growth rate of n-damo functional bacteria limit the practical application of this process. In the present study, the short- and long-term effects of variations in trace metal concentrations on n-damo bacteria were investigated, and the concentrations of trace metal elements of medium were improved. The results were subsequently verified by a group of long-term inoculations (90 days) and were applied in a sequencing batch reactor (SBR) (84 days). The results indicated that iron (Fe(II)) and copper (Cu(II)) (20 and 10 µmol L(-1), respectively) significantly stimulated the activity and the growth of n-damo bacteria, whereas other trace metal elements, including zinc (Zn), molybdenum (Mo), cobalt (Co), manganese (Mn), and nickel (Ni), had no significant effect on n-damo bacteria in the tested concentration ranges. Interestingly, fluorescence in situ hybridization (FISH) showed that a large number of dense, large aggregates (10-50 µm) of n-damo bacteria were formed by cell adhesion in the SBR reactor after using the improved medium, and to our knowledge this is the first discovery of large aggregates of n-damo bacteria.

Nitrogen removal from wastewater by anaerobic methane-driven denitrification in a lab-scale reactor: heterotrophic denitrifiers associated with denitrifying methanotrophs.

Nitrite-dependent anaerobic methane oxidation (n-damo) is a newly discovered bioprocess that reduces nitrite to dinitrogen with methane as electron donor, which has promising potential to remove nitrogen from wastewater. In this work, a lab-scale sequencing batch reactor (SBR) was operated for 609 days with methane as the sole external electron donor. In the SBR, nitrite in synthetic wastewater was removed continuously; the final volumetric nitrogen removal rate was 12.22±0.02 mg N L(-1) day(-1) and the percentage of nitrogen removal was 98.5 ± 0.2 %. Microbial community analysis indicated that denitrifying methanotrophs dominated (60-70 %) the population of the final sludge. Notably, activity testing and microbial analysis both suggested that heterotrophic denitrifiers existed in the reactor throughout the operation period. After 609 days, the activity testing indicated the nitrogen removal percentage of heterotrophic denitrification was 17 ± 2 % and that of n-damo was 83 ± 2 %. A possible mutualism may be developed between the dominated denitrifying methanotrophs and the associated heterotrophs through cross-feed. Heterotrophs may live on the microbial products excreted by denitrifying methanotrophs and provide growth factors that are required by denitrifying methanotrophs.

Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 µM for methane and 8.7 ± 1.5 µM for nitrite.

The short- and long-term effects of environmental conditions on anaerobic methane oxidation coupled to nitrite reduction.

Anaerobic oxidation of methane coupled to nitrite reduction (n-damo) plays an important role in global carbon and nitrogen cycles and also is a potential bioprocess in wastewater treatment. In this work, the effects of environmental conditions ­ temperature, pH and salinity ­ on the metabolic activity and growth rate of n-damo bacteria were investigated by short-term batch test and long-term bacterial incubation. Quantitative PCR and 16S rRNA and pmoA gene sequencing were applied to detect the microbial community in the long-term incubation. The results indicated that all the three environmental factors significantly affected the metabolic activity and growth rate of n-damo bacteria and the optimum temperature, pH and salinity were 35 °C, 7.6 and 0 g NaCl L⁻¹, respectively. Notably, salinity adaption of n-damo bacteria was first observed under salinity stress of 20 g NaCl L⁻¹. It's predicted that n-damo process might occur in saline environments and future work could focus on this.

Evidence for the cooccurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in a flooded paddy field.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N2 g(-1) (dry weight) day(-1) and the potential n-damo rates varied from 0.2 to 2.1 nmol CO2 g(-1) (dry weight) day(-1) in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10(5) to 2.0 × 10(6) copies g(-1) (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10(5) to 6.1 × 10(6) copies g(-1) (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with "Candidatus Brocadia" and "Candidatus Kuenenia" and n-damo bacteria related to "Candidatus Methylomirabilis oxyfera" were present in the soil cores. It is estimated that a total loss of 50.7 g N m(-2) per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH4 m(-2) per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.

Cultivation of nitrite-dependent anaerobic methane-oxidizing bacteria: impact of reactor configuration.

Nitrite-dependent anaerobic methane oxidation (n-damo) is mediated by bacteria that anaerobically oxidize methane coupled with nitrite reduction and is a potential bioprocess for wastewater treatment. In this work, the effect of reactor configuration on n-damo bacterial cultivation was investigated. A magnetically stirred gas lift reactor (MSGLR), a sequencing batch reactor (SBR), and a continuously stirred tank reactor (CSTR) were selected to cultivate the bacteria. Microbial community was monitored by using quantitative PCR, 16S rRNA gene sequencing, pmoA gene sequencing, and fluorescence in situ hybridization (FISH). The effects of substrate inhibition, methane mass transfer, and biomass washout in the three reactors were focused on. The results indicated that the MSGLR had the best performance among the three reactor systems, with the highest total and specific n-damo activities. Its maximum volumetric nitrogen removal rate was up to 76.9 mg N L(-1) day(-1), which was higher than previously reported values (5.1-37.8 mg N L(-1) d(-1)).

Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Here, evidence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable isotope experiments confirmed the occurrence of n-damo in the examined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and particulate methane monooxygenase genes demonstrated that M. oxyfera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10(7) gene copies per gram of dry soil. It is estimated that ∼0.51 g of CH4 m(-2) per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents previously unidentified confirmation that the n-damo process is a previously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution.

Mdodeling a nitrite-dependent anaerobic methane oxidation process: parameters identification and model evaluation.

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered process that is intermediated by n-damo bacteria that oxidize methane with nitrite to generate nitrogen gas. In this work, a kinetic model based on Monod type kinetics and diffusion-reaction model was developed to describe the bioprocess. Some key kinetic parameters needed in the model were obtained from a series of batch activity tests and a sequencing batch reactor (SBR) operation over 100 days. The growth rate, decay rate, methane affinity constant, nitrite affinity constant and inhibition constant were 0.0277±0.0022 d(-1), 0.00216±0.00010 d(-1), 0.092±0.005 mmol L(-1), 0.91±0.09 mmol L(-1) and 4.1±0.5 mmol L(-1) for n-damo bacteria at 30 °C, respectively. The results showed that the model could simulate actual performance of the SBR in the first 76 days, that methane was not a limiting factor at atmospheric pressure for its high affinity, and that the optimum nitrite concentration was 1.92 mmol L(-1).