Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

<p>Abstract</p> <p>Background</p> <p>Fumarase catalyzes the reversible hydration of fumarate to <smcaps>L</smcaps>-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of <smcaps>L</smcaps>-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.</p> <p>Results</p> <p>A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from <it>Bacteroides </it>sp. 2_1_33B and <it>Parabacteroides distasonis </it>ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in <it>E. coli </it>BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form <smcaps>L</smcaps>-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg²⁺. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: <it>K</it>_m= 0.48 mM, <it>V</it>_max= 827 μM/min/mg, and <it>k</it>_cat/<it>K</it>_m= 1900 mM/s.</p> <p>Conclusions</p> <p>We isolated a novel fumarase gene, <it>fumF</it>, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of <smcaps>L</smcaps>-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.</p

Ototoxicity of polystyrene nanoplastics in mice, HEI-OC1 cells and zebrafish

Polystyrene nanoplastics are a novel class of pollutants. They are easily absorbed by living organisms, and their potential toxicity has raised concerns. However, the impact of polystyrene nanoplastics on auditory organs remains unknown. Here, our results showed that polystyrene nanoplastics entered the cochlea of mice, HEI-OC1 cells, and lateral line hair cells of zebrafish, causing cellular injury and increasing apoptosis. Additionally, we found that exposure to polystyrene nanoplastics resulted in a significant elevation in the auditory brainstem response thresholds, a loss of auditory sensory hair cells, stereocilia degeneration and a decrease in expression of Claudin-5 and Occludin proteins at the blood-lymphatic barrier in mice. We also observed a significant decrease in the acoustic alarm response of zebrafish after exposure to polystyrene nanoplastics. Mechanistic analysis revealed that polystyrene nanoplastics induced up-regulation of the Nrf2/HO-1 pathway, increased levels of malondialdehyde, and decreased superoxide dismutase and catalase levels in cochlea and HEI-OC1 cells. Furthermore, we observed that the expression of ferroptosis-related indicators GPX4 and SLC7A11 decreased as well as increased expression of ACLS4 in cochlea and HEI-OC1 cells. This study also revealed that polystyrene nanoplastics exposure led to increased expression of the inflammatory factors TNF-α, IL-1β and COX2 in cochlea and HEI-OC1 cells. Further research found that the cell apoptosis, ferroptosis and inflammatory reactions induced by polystyrene nanoplastics in HEI-OC1 cells was reversed through the pretreatment with N-acetylcysteine, a reactive oxygen species inhibitor. Overall, our study first discovered and systematically revealed the ototoxicity of polystyrene nanoplastics and its underlying mechanism

Charge transport performance of Nafion-based redox polymer in atmosphere

The charge transport performance of the Nafion-based redox polymer films prepared under different conditions was investigated by cyclic voltammetry in the sandwiched cells. The results showed that the apparent charge transfer diffusion coefficient(D-a) of the Nafion[M(bpy)(3)(2+), PEG (polyethylene glycol)] (M=Ru or Fe) films is about 10(-6)-10(-7) cm(2) . s(-1) and the mobility of the electron or hole (mu) was about 10(-4)-10(-5) cm(2) . V-1 . s(-1). In order to decrease the resistance of the interface, a polyaniline (PANI) layer was added to the interface of indium tin oxide coated conducting glass (ITO) electrode and the redox polymer. Consequentially, the D-a could be increased to about 10(-5)-10(-6) cm(2) . s(-1) and mu to about 10(-3)-10(-4) cm(2) . V-1 . s(-1), and the operating current of the cells under 600 mV bias could be increased by almost 100 times. In addition, the D-a and mu of this redox polymer film had just reduced slightly after 30 d kept in atmosphere. Therefore, the redox polymer film is sufficiently stable

Characteristics of Stem Cells Derived from the Degenerated Human Intervertebral Disc Cartilage Endplate

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering