Polysaccharides from Gracilaria lemaneiformis promote the HaCaT keratinocytes wound healing by polarised and directional cell migration.

In this study, we evaluated the wound healing activity of polysaccharides fractions from Gracilaria lemaneiformis. Three purified fractions, namely, GLP-1, GLP-2 and GLP-3 were obtained from anion-exchange chromatography and were evaluated for their cell proliferation activity. Among the three fractions, the fraction GLP-2 promoted cell proliferation at a concentration of 100 µg/mL. Further, chemical and structural analysis of GLP-2 revealed it to be a homogenous and repeating structure of alternating 4-linked 3,6-anhydro-α-L-galactopyranosyl and 3-linked ß-d-galactopyranosyl units with sulfate residues. Further, we analysed the wound healing activities of the fraction GLP-2 and its underlying mechanisms. The results showed that GLP-2 promotes cell proliferation and migration through activation of PI3 K/aPKC signaling during human keratinocytes wound healing. Based on the findings in this study, we concluded that the wound healing activities of the GLP-2 fraction can provide the scientific basis for the development of G. lemaneiformis polysaccharides based product for wound management.

Dynamic equilibrium of endogenous selenium nanoparticles in selenite-exposed cancer cells: a deep insight into the interaction between endogenous SeNPs and proteins.

Elemental selenium (Se) was recently found to exist as endogenous nanoparticles (i.e., SeNPs) in selenite-exposed cancer cells. By sequestrating critical intracellular proteins, SeNPs appear capable of giving rise to multiple cytotoxicity mechanisms including inhibition of glycolysis, glycolysis-dependent mitochondrial dysfunction, microtubule depolymerization and inhibition of autophagy. In this work, we reveal a dynamic equilibrium of endogenous SeNP assembly and disassembly in selenite-exposed H157 cells. Endogenous SeNPs are observed both in the cytoplasm and in organelles. There is an increase in endogenous SeNPs between 24 h and 36 h, and a decrease between 36 h and 72 h according to transmission electron microscopy results and UV-Vis measurements. These observations imply that elemental Se in SeNPs could be oxidized back into selenite by scavenging superoxide radicals and ultimately re-reduced into selenide; then the assembly and disassembly of SeNPs proceed simultaneously with the sequestration and release of SeNP high-affinity proteins. There is also a possibility that the reduction of elemental Se to selenide pathway may lie in selenite-exposed cancer cells, which results in the assembly and disassembly of endogenous SeNPs. Genome-wide expression analysis results show that endogenous SeNPs significantly altered the expression of 504 genes, compared to the control. The endogenous SeNPs induced mitochondrial impairment and decreasing of the annexin A2 level can lead to inhibition of cancer cell invasion and migration. This dynamic flux of endogenous SeNPs amplifies their cytotoxic potential in cancer cells, thus provide a starting point to design more efficient intracellular self-assembling systems for overcoming multidrug resistance.

Effects of ivabradine on cardiac electrophysiology in dogs with age-related atrial fibrillation.

BACKGROUND: Ivabradine is an inhibitor of mixed Na+-K+ current that could combine with HCN channels to reduce the transmembrane velocity of funny current (If), heart rate, and cardiac efficiency, and thus be used for the treatment of cardiovascular diseases such as chronic heart failure. As an ion channel blocker, Ivabradine is also a potential antiarrhythmic agent. MATERIAL/METHODS: Twelve aging dogs (8-10 years old) underwent rapid atrial pacing for 2 months to induce age-related AF in this study. The dogs were randomly divided into the Ivabradine group and aging-AF group. The effects of Ivabradine on the electrophysiological parameters, including the effective refractory period (ERP) of the pulmonary veins and atrium, duration of AF, and inducing rate of AF, were investigated. RESULTS: As compared to the aging-AF group, the ERPs of the left superior pulmonary vein (139.00±4.18 ms vs. 129.00±4.08 ms, P=0.005) and left auricle (135.00±3.53 ms vs. 122.00±4.47 ms, P=0.001) were significantly increased, while the duration of AF (46.60±5.07 s vs. 205.40±1.14 s, P=0.001) and inducing rate of AF (25% vs. 60%, P=0.001) were significantly decreased. CONCLUSIONS: Ivabradine could effectively reduce the inducing rate of AF, and thus be used as an upstream drug for the prevention of age-related AF.

Selenite-induced toxicity in cancer cells is mediated by metabolic generation of endogenous selenium nanoparticles.

Selenite has been a touted cancer chemopreventative agent but generates conflicting outcomes. Multiple mechanisms of selenite cytotoxicity in cancer cells are thought to be induced by metabolites of selenite. We observed that intracellular metabolism of selenite generates endogenous selenium nanoparticles (SeNPs) in cancer cells. Critical proteins that bind with high affinity to elemental selenium during SeNPs self-assembly were identified through proteomics analysis; these include glycolytic enzymes, insoluble tubulin, and heat shock proteins 90 (HSP90). Sequestration of glycolytic enzymes by SeNPs dramatically inhibits ATP generation, which leads to functional and structural disruption of mitochondria. Transcriptome sequencing showed tremendous down-regulation of mitochondrial respiratory NADH dehydrogenase (complex I), cytochrome c oxidase (complex IV), and ATP synthase (complex V) in response to glycolysis-dependent mitochondrial dysfunction. Sequestration of insoluble tubulin led to microtubule depolymerization, altering microtubule dynamics. HSP90 sequestration led to degradation of its downstream effectors via autophagy, ultimately resulting in a cell-signaling switch to apoptosis. Additionally, the surface effects of SeNPs generated oxidative stress, thus contributing to selenite cytotoxicity. Herein, we reveal that the multiple mechanisms of selenite-induced cytotoxicity are caused by endogenous protein-assisted self-assembly of SeNPs and suggest that endogenous SeNPs could potentially be the primary cause of selenite-induced cytotoxicity.

Achieving nitritation and phosphorus removal in a continuous-flow anaerobic/oxic reactor through bio-augmentation.

The feasibility of achieving nitritation and phosphorus removal using bio-augmentation was investigated in a continuous-flow anaerobic/oxic (A/O) reactor treating sewage. The results indicated that nitritation could be quickly start-up, and reconstructed with an increase in the nitrite accumulation rate (NAR) from 1% to 89% within 15 days by using bio-augmentation and controlling DO at 0.96 mg/L. Biological phosphorus removal could be achieved with the average phosphorus removal efficiency of 96.43% when the NAR was maintained above 78.60%. Meanwhile, sludge settleablity was good with a sludge volume index (SVI) of between 62 and 102 mL/g even under high NAR. After nitritation and biological phosphorus removal were achieved, this A/O reactor has the potential to supply appropriate influent for the anammox UASB reactor.

CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.

Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing.

Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efficient, thereby limiting the genetic manipulation of the recalcitrant species. In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell walls were weakened by adding glycine (Gly) and DL-threonine (DL-Thr), and the cell-membrane fluidity was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response surface methodology (RSM), the transformation efficiency reached 1.13 ± 0.34 × 10(7) cfu/µg syngeneic pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of the host restriction enzyme, a transformation efficiency of 8.94 ± 0.77 × 10(5) cfu/µg DNA was achieved with xenogeneic shuttle plasmids, a 10(3)-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study. This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difficult to be transformed using conventional methods.