Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.

Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. To test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species. IMPORTANCE Rhodanobacter denitrificans is capable of denitrification and is also resistant to toxic heavy metals and low pH. Accordingly, the presence of Rhodanobacter species at a particular environmental site is considered an indicator of nitrate and uranium contamination. These characteristics suggest its future potential application in bioremediation of nitrate or concurrent nitrate and uranium contamination in groundwater ecosystems. Due to the lack of genetic tools in this organism, the mechanisms of low-pH and heavy metal resistance in R. denitrificans strains remain elusive, which impedes its use in bioremediation strategies. Here, we developed a genome editing method in two R. denitrificans strains. This work marks a crucial step in developing Rhodanobacter as a model for studying the diverse mechanisms of low-pH and heavy metal resistance associated with denitrification.

Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway.

BACKGROUND AND AIM: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, which is mainly caused by inflammation. Inhibiting inflammation can relieve PAH. Grape seed procyanidin (GSP) possesses remarkable anti-inflammatory property and vascular protective function. In this experiment, we verified the anti-inflammatory property of GSP in cigarette smoke-exposed PAH rats and revealed its molecular mechanism. METHODS AND RESULTS: In vivo, 45 Sprague Dawley (SD) rats were divided into 5 groups randomly, treated with normoxia/cigarette smoke (CS)/GSP + CS/CS + solvent/GSP. After GSP + CS administration, a decrease in mPAP, PVR, RVHI, WT%, and WA% was detected in the rats as compared to those treated with CS. In vitro, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) caused by cigarette smoke extract (CSE) was effectively attenuated with GSP + CSE administration. Furthermore, GSP significantly increased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) together with the lowered expression level of cyclooxygenase 2 (COX-2) in PASMCs co-incubated with CSE. CONCLUSION: These findings indicate that GSP ameliorates inflammation by the PPAR-γ/COX-2 pathway and finally inhibits the proliferation of PASMCs, which leads to pulmonary vascular remodeling.

[Clinical effects of high frequency repeated transcranial magnetic stimulation therapy on dyskinesia in patients with incomplete spinal cord injury:a Meta-analysis].

OBJECTIVE: To systematically evaluate the clinical effect of high frequency repeated transcranial magnetic stimulation(HF rTMS)therapy on dyskinesia in patients with incomplete spinal cord injury. METHODS: Randomized controlled trials(RCTs) about HF rTMS therapy on patients with motor incomplete spinal cord injury were searched electronically in PubMed, Google scholar, Cochrane library, Clinical trial, Medline, Web of science, CNKI, VIP, and Wanfang database before October 2016. Two reviewers independently screened the literatures according to the inclusion and exclusion criteria, as well as extracted the data and assessed the methodological quality. The observed outcomes included ASIA motor score, ASIA lower extremities motor score(LEMS), Modified Ashworth score (MAS), Ten-meter walking test (10MWT) and Walking index for SCI II(WISCI II), and the outcomes were analyzed using RevMan5.2 software provided by the Cochrane information management system. RESULTS: Five RCTs involved 103 patients were included, and 61 patients(experimental group) accepted real rTMS and physical rehabilitation care for SCI, 51 patients(control group) accepted only physical rehabilitation care. There were significant differences in ASIA motor score, LEMS and 10MWT between two groups after HF rTMS therapy (statistics were Z=2.96, P=0.003; Z=3.04, P=0.002; Z=2.16, P=0.03; respectively). When stimulating the leg motor cortex, there was significant difference in MAS between two groups(Z=2.79, P=0.005), and when stimulating the vertex, there was no significant difference(Z=0.09, P=0.93). There was no significant difference in WISCI IIscore after HF rTMS therapy between two groups(Z=0.90, P=0.37). CONCLUSIONS: HF rTMS can raise motor score in patients with incomplete spinal cord injury, improve the spasticity of the lower extremities, and increase the motor ability.