Decadal soil carbon accumulation across Tibetan permafrost regions

Acknowledgements We thank the members of Peking University Sampling Teams (2001–2004) and IBCAS Sampling Teams (2013–2014) for assistance in field data collection. We also thank the Forestry Bureau of Qinghai Province and the Forestry Bureau of Tibet Autonomous Region for their permission and assistance during the sampling process. This study was financially supported by the National Natural Science Foundation of China (31670482 and 31322011), National Basic Research Program of China on Global Change (2014CB954001 and 2015CB954201), Chinese Academy of Sciences-Peking University Pioneer Cooperation Team, and the Thousand Young Talents Program.Peer reviewedPostprintPostprin

<i>Bacillus paralicheniformis</i> RP01 Enhances the Expression of Growth-Related Genes in Cotton and Promotes Plant Growth by Altering Microbiota inside and outside the Root

Plant growth-promoting bacteria (PGPB) can promote plant growth in various ways, allowing PGPB to replace chemical fertilizers to avoid environmental pollution. PGPB is also used for bioremediation and in plant pathogen control. The isolation and evaluation of PGPB are essential not only for practical applications, but also for basic research. Currently, the known PGPB strains are limited, and their functions are not fully understood. Therefore, the growth-promoting mechanism needs to be further explored and improved. The Bacillus paralicheniformis RP01 strain with beneficial growth-promoting activity was screened from the root surface of Brassica chinensis using a phosphate-solubilizing medium. RP01 inoculation significantly increased plant root length and brassinosteroid content and upregulated the expression of growth-related genes. Simultaneously, it increased the number of beneficial bacteria that promoted plant growth and reduced the number of detrimental bacteria. The genome annotation findings also revealed that RP01 possesses a variety of growth-promoting mechanisms and a tremendous growth-promoting potential. This study isolated a highly potential PGPB and elucidated its possible direct and indirect growth-promoting mechanisms. Our study results will help enrich the PGPB library and provide a reference for plant–microbe interactions

Effects of Targeted Suppression of Glutaryl-CoA Dehydrogenase by Lentivirus-Mediated shRNA and Excessive Intake of Lysine on Apoptosis in Rat Striatal Neurons

<div><p></p><p>In glutaric aciduria type 1 (GA1), glutaryl-CoA dehydrogenase (GCDH) deficiency has been shown to be responsible for the accumulation of glutaric acid and striatal degeneration. However, the mechanisms by which GA1 induces striatal degeneration remain unclear. In this study, we aimed to establish a novel neuronal model of GA1 and to investigate the effects of GCDH deficiency and lysine-related metabolites on the viability of rat striatal neurons. Thus we constructed a lentiviral vector containing short hairpin RNA targeted against the GCDH gene expression (lentivirus-shRNA) in neurons. A virus containing a scrambled short hairpin RNA construct served as a control. Addition of lysine (5 mmol/L) was used to mimic hypermetabolism. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis was assessed using Hoechst33342 staining and Annexin V-PE/7-AAD staining. The mitochondrial membrane potential (MPP) was monitored using tetramethylrhodamine methyl ester. The expression levels of caspases 3, 8, and 9 were determined by Western blotting. We found that lentivirus-shRNA induced apoptosis and decreased MMP levels in neurons, and addition of 5 mmol/L lysine enhanced this effect markedly. Lentivirus-shRNA upregulated the protein levels of caspases 3 and 9 regardless of the presence of 5 mmol/L lysine. The expression level of caspase 8 was higher in neurons co-treated with lentivirus-shRNA and 5 mmol/L lysine than in control. Benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and 5 mmol/L lysine to a great extent. These results indicate that the targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine may be a useful cell model of GA1. These also suggest that GA1-induced striatal degeneration is partially caspase-dependent.</p></div

Glutaric Acid-Mediated Apoptosis in Primary Striatal Neurons

Glutaric acid (GA) has been implicated in the mechanism of neurodegeneration in glutaric aciduria type I. In the present study, the potential cytotoxic effects of GA (0.1~50 mM for 24~96 h) were examined in cultured primary rat striatal neurons. Results showed increase in the number of cells labeled by annexin-V or with apoptotic features shown by Hoechst/PI staining and transmission electron microscopy (TEM) and upregulation of the expression of mRNA as well as the active protein fragments caspase 3, suggesting involvement of the caspase 3-dependent apoptotic pathway in GA-induced striatal neuronal death. This effect was in part suppressed by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 but not the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) antagonist 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX). Thus, GA may trigger neuronal damage partially through apoptotic pathway and via activation of NMDA receptors in cultured primary striatal neurons

Protein expression of caspases 3, 8, and 9.

<p>(A) NC; (B) lentivirus-shRNA#1; (C) NC +5 mmol/L lysine; and (D) lentivirus-shRNA#1+5 mmol/L lysine. *<i>P<</i>0.05. The protein levels of caspases 3 and 9 were significantly upregulated by lentivirus-shRNA#1, and this upregulation was intensified by 5 mmol/L lysine. Neither lentivirus-shRNA#1 nor 5 mmol/L lysine changed the expression of caspase 8 alone. Exposure to both conditions increased the protein level of caspase 8.</p

OD in the detection of neuron viability by MTT assay.

<p>Viability rate (%)  = (OD_m−OD_blank)/(OD₀−OD_blank); OD_m: The OD of each sample; OD₀: The OD of neurons with 0 mmol/L lysine group. OD_blank: The OD of the blank control (0.169±0.0252).</p>*<p><i>P<</i>0.05 <i>vs.</i> neurons with 0 mmol/L lysine group.</p

Hoechst 33342 staining of apoptotic neurons.

<p>The effects of GCDH knockdown and excess lysine on the nuclear morphological changes in rat neurons. Nuclei in uninfected neurons and neurons infected with negative control lentivirus were lightly stained blue. Apoptotic nuclei were deeply stained blue, and appeared dense and fragmented (marked with arrows). Scale bars: 20 µm. The histogram represents the percentage of apoptotic cells. *<i>P<</i>0.05.</p

Detection of apoptosis using flow cytometry.

<p>Cells were assayed for apoptosis using Annexin V-PE/7-AAD staining with flow cytometry. Cells were grouped and treated as shown to quantify the apoptosis induced by GCDH knockdown and increased lysine. Lentivirus-shRNA#1 induced apoptosis, and 5 mmol/L lysine increased the rate of apoptosis to a significantly greater extent. Z-VAD-FMK, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and increased lysine to a great extent. *<i>P<</i>0.05.</p

Efficiency of lentivirus-shRNA#1 interference as detected by Western blotting.

<p>GCDH expression in rat striatal neurons 72 h after infection with lentivirus. The lentivirus-shRNA#1 reduced the level of protein expression in GCDH by as much as 80.78% relative to the negative control lentivirus. *<i>P<</i>0.05.</p