Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Urolithin A alleviates early brain injury after subarachnoid hemorrhage by regulating the AMPK/mTOR pathway-mediated autophagy.

OBJECTIVE: Unfavorable outcomes in patients with subarachnoid hemorrhage (SAH) are mainly attributed to early brain injury (EBI). Reduction of neuronal death can improve the prognosis in SAH patients. Autophagy and apoptosis are critical players in neuronal death. Urolithin A (UA) is a natural compound produced by gut bacteria from ingested ellagitannins and ellagic acid. Here, we detected the role of UA in EBI post-SAH. METHODS: We established an animal model of SAH in rats by endovascular perforation, with administration of UA, 3-methyladenine (3-MA) and Compound C. SAH grading, neurological function, brain water content, western blotting analysis of levels of proteins related to apoptosis, autophagy and pathways, blood-brain barrier (BBB) integrity, TUNEL staining, and immunofluorescence staining of LC3 were evaluated at 24h after SAH. RESULTS: SAH induction led to neurological dysfunctions, BBB disruption, and cerebral edema at 24h post-SAH in rats, which were relieved by UA. Additionally, cortical neuronal apoptosis in SAH rats was also attenuated by UA. Moreover, UA restored autophagy level in SAH rats. Mechanistically, UA activated the AMPK/mTOR pathway. Furthermore, inhibition of autophagy and AMPK limited UA-mediated protection against EBI post-SAH CONCLUSION: UA alleviates neurological deficits, BBB permeability, and cerebral edema by inhibiting cortical neuronal apoptosis through regulating the AMPK/mTOR pathway-dependent autophagy in rats following SAH.

Human Brain-Derived Neurotrophic Factor Gene-Modified Bone Marrow Mesenchymal Stem Cells Combined With Erythropoietin Can Improve Acute Spinal Cord Injury.

OBJECTIVE: To assess the effect as well as mechanism of bone marrow mesenchymal stem cells (BMSCs) modified by the human brain-derived neurotrophic factor gene combined with erythropoietin (EPO) in the treatment of acute spinal cord injury (SCI) in rats. METHODS: The Brain-derived neurotrophic factor (BDNF) gene was transected by a virus vector. Rats with SCI were randomly split into following groups: The normal saline (NS) group, the EPO group, The Basso, Beattie, and Bresnahan scores, messenger RNA BDNF expression, and apoptosis rates were compared between the 4 groups at 1, 3, 7, 14, and 21 days after SCI. RESULTS: At 7, 14, and 21 days after operation, the expression of the BDNF gene in the other 3 groups was higher than that of the NS group, and the difference was statistically significant (P < .05). The apoptosis rate in the combined group was less than that of NS, EPO, and BDNF/BMSC groups, and the differences were statistically significant (P < .05). CONCLUSION: Brain-derived neurotrophic factor gene-modified BMSC transplantation combined with EPO can promote the repair of nerve function after SCI in rats.

XB130 enhances invasion and migration of human colorectal cancer cells by promoting epithelial­mesenchymal transition.

The expression of XB130 is associated with invasion and migration of many tumor cells, but its roles in human colorectal cancer (CRC) remains unknown. To investigate this, protein expression levels of XB130 in numerous human CRC cell lines were compared with a normal colorectal mucosa cell line by western blotting. Knockdown of XB130 using small interfering (si)RNA was performed to assess the effects on cell invasion and migration in a Transwell assay and a scratch test. Western blotting was also used to quantify the levels of proteins associated with epithelial­mesenchymal transition (EMT), including E­cadherin, vimentin, phosphorylated (p)­protein kinase B (AKT), p­forkhead homeobox type O 3a (FOXO3a) and zinc finger E­box­binding homeobox 1 (ZEB­1). The relative expression of XB130 protein was significantly higher in CRC cells compared with control cells (P<0.01). Knockdown of XB130 using siRNA significantly decreased the invasive and migratory responses of CRC cells (P<0.01). In addition, levels of E­cadherin were increased, while vimentin, p­AKT, p­FOXO3a and ZEB­1 were decreased (P<0.01). In conclusion, the present study demonstrated that the expression of XB130 is elevated in CRC cells. Loss of XB130 was associated with decreased invasion and migration of CRC cells, possibly as a result of EMT inhibition. Thus, upregulation of XB130 may underlie some of the tumorigenic events observed in human CRCs. XB130 may be a promising target for CRC therapy in humans; further mechanistic studies exploring the function of XB130 in CRC cells are warranted.

[Analysis of genetic diversity of Sichuan indigenous chicken breeds using microsatellite markers].

Thirty microsatellite markers with medium or high polymorphisms were selected to detect the genetic diversity of 8 indigenous chicken breeds in Sichuan. According to the allele frequencies of 30 microsatellite sites, mean heterozygosity (H), polymorphism information content (PIC) and DA genetic distances were calculated for each breeds. The results showed that 24 of 30 microsatellite sites were highly polymorphic, so the 24 microsatellite markers were effective markers for analysis of genetic relationship among chicken breeds. The mean heterozygosity of 8 chicken breeds was all over 0.5. The highest was the Luning chicken (0.681), and the lowest was the Jiuyuan Dark chicken. The high diversity of 8 chicken breeds might be caused by the traffic obstruction(geographic isolation). The results of the heterozygosity were consistent with that of PIC. UPGMA tree was completed through analysis of DA genetic distances. Emei Dark chicken, Miyi chicken, Luning chicken and Jiuyuan Dark chicken were the first group: Miyi chicken and Luning chicken were grouped firstly, then Emei Dark chicken were grouped with them in shorter time distances, and Jiuyuan Dark chicken were grouped with them at last. Shimiancao Ke chicken Xingwen Silky chicken and Muchuan Silky were the second group: Xingwen Silky chicken and Muchuan Silky were grouped firstly, and then Shimiancao Ke chicken was grouped with them. Liangshangya Ying chicken had its own branch. The result of UPGM was consistent with the genesis, breeding history, differentiation and location of 8 chicken breeds.

Modulation of cyclophilin gene expression by N-4-(hydroxyphenyl)retinamide: association with reactive oxygen species generation and apoptosis.

To explore the mechanisms underlying the pro-apoptotic effects of the synthetic retinoid N-4-(hydroxyphenyl)retinamide (4-HPR) on LNCaP human prostate cancer cells, we used the differential display-polymerase chain reaction (DD-PCR) technique to identify 4-HPR-responsive genes. RNA extracted from LNCaP cells that had been treated for 24 h with 4-HPR at a dose (2.5 microM) optimal for apoptosis induction was used for DD-PCR analysis using random primers. A differentially expressed 115 bp fragment was cloned and sequenced and then identified in GenBank as having a high degree of homology with several members of the cyclophilin gene family. Northern blot analyses using specific probes for cyclophilin A, cyclophilin D, and the cloned 115-bp fragment were performed on RNA extracted from LNCaP cells and MCF-7 human breast cancer cells treated with 4-HPR, N-acetylcysteine (NAC, an anti-oxidant), 4-HPR plus NAC, cyclosporin A, R-1881 (a synthetic androgen), dehydroepiandrosterone, all-trans retinoic acid, or prednisone. 4-HPR downregulated the transcript detected by the 115-bp fragment. Expression patterns detected by the 115-bp fragment and cyclophilin D probes were identical in response to each treatment; none of these treatments affected cyclophilin A expression. Furthermore, expression of mRNA transcripts detected by the 115-bp fragment and cyclophilin D probes correlated with the generation of reactive oxygen species (ROS), as detected by measurement of 2,7-dichlorofluorescein oxidation. Therefore, members of the cyclophilin gene family, such as cyclophilin D (a component of the mitochondrial permeability transition pore previously linked with oxidative stress and apoptosis), may play a role in the ROS-mediated apoptotic effects of 4-HPR.