Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458?nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E _app). The E _app closely matches the value of true enantioselectivity (E _true) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.     

包埋法固定化对硫氧化微生物菌群结构和功能的影响

【目的】为探讨包埋法固定化过程对硫氧化菌群硫化物去除能力及菌群微生物群落结构的影响,【方法】以聚乙烯醇-海藻酸钠-活性炭为载体,对硫氧化菌群进行了固定化,并采用富含硫化物的无机盐培养基,对比固定化与非固定化硫氧化菌群对硫化物的氧化去除能力。同时,利用PCR-DGGE技术,探讨硫氧化菌群在固定化前后以及在硫化物氧化去除过程中微生物群落结构变化。【结果】在对硫氧化菌群进行固定化之后,12 h之内对硫化物的最大去除能力从1000 mg/L下降为600 mg/L。硫氧化菌群的微生物群落结构发生了明显变化,但菌群中的硫氧化菌Catenococcus thiocycli未受影响,硫氧化菌Thioclava pacifica在菌群中的地位反而得到了强化。【结论】受制于底物在载体材料中的扩散迁移效率,硫氧化菌群对硫化物的氧化去除能力在固定化之后有所下降。由于不同微生物对固定化形成的微环境的适应能力以及对载体附着能力的不同,固定化对硫氧化菌群的微生物群落结构产生较大影响。     

The Physiological Effects of Ethrel on Seedlings of Second Season Rice

Spray of ethrel (1000 ppm)to seedlings of double-cropping second season rice at 5-leaf stage can control plant height and root length, and consequently makes seedlings strong and tough. The ABA content and ethylene released by young seedling were significantly higher than the control. However, there were some changes: cell elongation inhibited, leaf area decreased, and leaf color became dark green, photosynthetic rate increased, translocation of photosynthetate in leaf sheath enhanced, leaf emergence was rapid. The growth of root system and root vigor were though temporarily inhibited, but slightly increased after transplantation. All these are beneficial to seedling quality, the plant growth and development after transplantation, which subsequently bring about positive effects on stimulating early heading and on increasing rice yield.     

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

Background

The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy.

Methods

We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model.

Results

Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions.

Conclusions

Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

     

TRIM46 activates AKT/HK2 signaling by modifying PHLPP2 ubiquitylation to promote glycolysis and chemoresistance of lung cancer cells

The incidence of lung cancer is increasing worldwide. Although great progress in lung cancer treatment has been made, the clinical outcome is still unsatisfactory. Tripartite motif (TRIM)-containing proteins has been shown to be closely related to tumor progression. However, the function of TRIM46 in lung cancer is largely unknown. Here, TRIM46 amplification was found in lung adenocarcinoma (LUAD) tissues and TRIM46 amplification was significantly associated with a poor survival rate. Overexpression of wild type TRIM46 increased the proliferation of LUAD cells and glycolysis, promoted xenografts growth, and enhanced cisplatin (DDP) resistance of LUAD cells via increased ubiquitination of pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) and upregulation of p-AKT. In contrast, overexpression of RING-mutant TRIM46 did not show any effects, suggesting the function of TRIM46 was dependent on the E3 ligase activity. Furthermore, we found that TRIM46 promoted LUAD cell proliferation and DDP resistance by enhancing glycolysis. PHLPP2 overexpression reversed the effects of TRIM46 overexpression. Amplification of TRIM46 also promoted LUAD growth and enhanced its DDP resistance in a patient-derived xenograft (PDX) model. In conclusion, our data highlight the importance of TRIM46/PHLPP2/AKT signaling in lung cancer and provide new insights into therapeutic strategies for lung cancer.Subject terms: Cancer, Biomarkers     

Crystal structure of R22L trichosanthin

Ｔｒｉｃｈｏｓａｎｔｈｉｎ(ＴＣＳ)ｉｓａｎｉｍｐｏｒｔａｎｔｍｅｍｂｅｒｏｆｒｉｂｏｓｏｍｅｉｎａｃｔｉｖａｔｉｎｇｐｒｏｔｅｉｎｓ[1].ＩｔｐｏｓｓｅｓｓｅｓＮｇｌｙｃｏｓｉｄａｓｅａｃｔｉｖｉｔｙｒｅｍｏｖｉｎｇａｄｅｎｉｎｅ(ＡＤＥ)ａｔｐｏｓｉｔｉｏｎＡ4324ｏｆ28ＳｒＲＮＡ[2].ＴｈｅａｃｔｉｖｅｐｏｃｋｅｔｏｆＮｇｌｙｃｏｓｉｄａｓｅｈａｓｂｅｅｎｅｓｔａｂｌｉｓｈｅｄｔｈｒｏｕｇｈｔｈｅｃｒｙｓｔａｌｓｔｒｕｃｔｕｒｅｓｏｆＴＣＳ,αＭＭＣａｎｄｒｉｃｉｎａｎｄａｓｓａｙｏｆｍｕｔａｎｔｓ…     

Bioactive thin film of acidic fibroblast growth factor fabricated by layer-by-layer assembly

A new class of bioactive thin films using growth factors as building blocks has been fabricated via layer-by-layer assembly (LBL) technique. Acid fibroblast growth factor (aFGF) in the presence of heparin was used as negatively charged polyelectrolytes, while poly(ethyleneimine) (PEI) was chosen as a positively charged counterpart. The self-deposition process and surface morphology of the resultant multilayers were monitored and detected by UV-vis absorbance spectra, advanced contact angle measurements, and scanning force microscopy (SFM) observations. Cell culture was performed to assess the efficiency of the growth factors. The fibroblasts proliferated faster on the surface assembled with five bilayers of (aFGF/heparin)/PEI with apparent higher cytoviability than on those surfaces modified by one bilayer of (aFGF/heparin)/PEI, five bilayers of aFGF/PEI, or five bilayers of heparin/PEI, and tissue culture polystyrene. Enhanced secretion of collagen type I and interleukin 6 (IL-6) by the fibroblasts seeded on the five bilayers of (aFGF/heparin)/PEI was also verified by immunohistochemical examination. The bioactivity of the (aFGF/heparin)/PEI multilayers could be largely preserved when stored at -20 degrees C.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.