Reconstruction of TNF-α with specific isoelectric point released from SPIONs basing on variable charge to enhance pH-sensitive controlled-release.

The clinical application of tumor necrosis factor-α (TNF-α) is limited by its short half-life, subeffective concentration in the targeted area and severe systemic toxicity. In this study, the recombinant polypeptide S4-TNF-α was constructed and coupled with chitosan-modified superparamagnetic iron oxide nanoparticles (S4-TNF-α-SPIONs) to achieve pH-sensitive controlled release and active tumor targeting activity. The isoelectric point (pI) of S4-TNF-α was reconstructed to approach the pH of the tumor microenvironment. The negative-charge S4-TNF-α was adsorbed to chitosan-modified superparamagnetic iron oxide nanoparticles (CS-SPIONs) with a positive charge through electrostatic adsorption at physiological pH. The acidic tumor microenvironment endowed S4-TNF-α with a zero charge, which accelerated S4-TNF-α release from CS-SPIONs. Our studies showed that S4-TNF-α-SPIONs displayed an ideal pH-sensitive controlled release capacity and improved antitumor effects. Our study presents a novel approach to enhance the pH-sensitive controlled-release of genetically engineered drugs by adjusting their pI to match the pH of the tumor microenvironment.

Controlled SPION-Exosomes Loaded with Quercetin Preserves Pancreatic Beta Cell Survival and Function in Type 2 Diabetes Mellitus.

Introduction: Quercetin has an ideal therapeutic effect on islet function improvement in type 2 diabetes mellitus (T2DM). However, the therapeutic benefit of quercetin is hindered by its poor bioavailability and limited concentration in pancreatic islets. In this study, superparamagnetic iron oxide nanoparticle (SPION)-modified exosomes were prepared to load quercetin, hoping to endow quercetin with enhanced water solubility and active targeting capacity with the help of magnetic force (MF). Methods: Transferrin-modified SPIONs (Tf-SPIONs) were synthesized by exploiting N-hydroxysuccinimidyl (NHS) conjugation chemistry, and quercetin-loaded exosomes (Qu-exosomes) were acquired by electroporation. Tf-SPION-modified quercetin-loaded exosomes (Qu-exosome-SPIONs) were generated by the self-assembly of transferrin (Tf) and the transferrin receptor (TfR). The solubility of quercetin was determined by high-performance liquid chromatography (HPLC) analysis. The pancreatic islet targeting capacity and insulin secretagogue and antiapoptotic activities of Qu-exosome-SPIONs/MF were evaluated both in vitro and in vivo. Results: The Qu-exosome-SPIONs were well constructed and harvested by magnetic separation with a uniform size and shape in a diameter of approximately 86.2 nm. The water solubility of quercetin increased 1.97-fold when loaded into the SPION-modified exosomes. The application of SPIONs/MF endowed the Qu-exosomes with favorable targeting capacity. In vitro studies showed that Qu-exosome-SPIONs/MF more effectively inhibited or attenuated ß cell apoptosis and promoted insulin secretion in response to elevated glucose (GLC) compared with quercetin or Qu-exosome-SPIONs. In vivo studies demonstrated that Qu-exosome-SPIONs/MF displayed an ideal pancreatic islet targeting capacity, thereby leading to the restoration of islet function. Conclusion: The Qu-exosome-SPIONs/MF nano-delivery system significantly enhanced the quercetin concentration in pancreatic islets and thereby improved pancreatic islet protection.

Gradient boosting DD-MLP Net: An ensemble learning model using near-infrared spectroscopy to classify after-stroke dyskinesia degree during exercise.

This study aims to develop an automatic assessment of after-stroke dyskinesias degree by combining machine learning and near-infrared spectroscopy (NIRS). Thirty-five subjects were divided into five stages (healthy, patient: Brunnstrom stages 3, 4, 5, 6). NIRS was used to record the muscular hemodynamic responses from bilateral femoris (biceps brachii) muscles during passive and active upper (lower) limbs circular exercise. We used the D-S evidence theory to conduct feature information fusion and established a Gradient Boosting DD-MLP Net model, combining the dendrite network and multilayer perceptron, to realize automatic dyskinesias degree evaluation. Our model classified the upper limb dyskinesias with high accuracy: 98.91% under the passive mode and 98.69% under the active mode, and classified the lower limb dyskinesias with high accuracy: 99.45% and 99.63% under the passive and active modes, respectively. Our model combined with NIRS has great potential in monitoring the after-stroke dyskinesias degree and guiding rehabilitation training.

Long-distance electron transfer in a filamentous Gram-positive bacterium.

Long-distance extracellular electron transfer has been observed in Gram-negative bacteria and plays roles in both natural and engineering processes. The electron transfer can be mediated by conductive protein appendages (in short unicellular bacteria such as Geobacter species) or by conductive cell envelopes (in filamentous multicellular cable bacteria). Here we show that Lysinibacillus varians GY32, a filamentous unicellular Gram-positive bacterium, is capable of bidirectional extracellular electron transfer. In microbial fuel cells, L. varians can form centimetre-range conductive cellular networks and, when grown on graphite electrodes, the cells can reach a remarkable length of 1.08 mm. Atomic force microscopy and microelectrode analyses suggest that the conductivity is linked to pili-like protein appendages. Our results show that long-distance electron transfer is not limited to Gram-negative bacteria.

Potent in vitro and in vivo antimicrobial activity of semisynthetic amphiphilic γ-mangostin derivative LS02 against Gram-positive bacteria with destructive effect on bacterial membrane.

Semisynthetic γ-mangostin derivative LS02 is a novel cationic amphiphilic peptidomimetic antimicrobial agent containing a hydrophobic scaffold and three hydrophilic and positively charged residues of arginine. LS02 showed low in vitro toxicity, potent activities against Gram-positive bacteria including MRSA (MIC = 1.56-6.25 µg/mL) and avoidance of drug resistance. The mode of action studies indicated that LS02 killed bacteria by disrupting bacterial cell membranes. LS02 not only exhibited good water solubility, low hemolytic activity and cell cytotoxicity, but also displayed excellent in vitro and in vivo antibacterial activity, indicating its great potential of being a lead compound as a novel membrane-active antibacterial agent capable of combating bacterial resistance.

Lack of Periplasmic Non-heme Protein SorA Increases Shewanella decolorationis Current Generation.

Bacterial extracellular electron transport (EET) plays an important role in many natural and engineering processes. Some periplasmic non-heme redox proteins usually coexist with c-type cytochromes (CTCs) during the EET process. However, in contrast to CTCs, little is known about the roles of these non-heme redox proteins in EET. In this study, the transcriptome of Shewanella decolorationis S12 showed that the gene encoding a periplasmic sulfite dehydrogenase molybdenum-binding subunit SorA was significantly up-regulated during electrode respiration in microbial fuel cells (MFCs) compared with that during azo-dye reduction. The maximum current density of MFCs catalyzed by a mutant strain lacking SorA (ΔsorA) was 25% higher than that of wild strain S12 (20 vs. 16 µA/cm2). Both biofilm formation and the current generation of the anodic biofilms were increased by the disruption of sorA, which suggests that the existence of SorA in S. decolorationis S12 inhibits electrode respiration. In contrast, disruption of sorA had no effect on respiration by S. decolorationis S12 with oxygen, fumarate, azo dye, or ferric citrate as electron acceptors. This is the first report of the specific effect of a periplasmic non-heme redox protein on EET to electrode and provides novel information for enhancing bacterial current generation.

Variable cell division time and asymmetric division site lead to filament-to-rod cell cycle of Lysinibacillus varians.

All well-established cell size homeostasis paradigms are based on the researches of rod bacteria like B. subtilis and E. coli, suggesting a constant division time (timer model), division size (sizer model) or added size (adder model) before division. However, Lysinibacillus varians, a new species with regular filament-to-rod cell cycle, is inconsistent with existing models. In this study, the cell size parameters of the type strain GY32, were investigated by combing multiple microscopy techniques and single-cell approach. Our results showed that the filaments of strain GY32 were unicellular cells with multiple nucleoids. The division time of GY32 cells was variable and their daughter cells produced by asymmetric binary fission had different birth sizes, which were proportional to their elongation rates, resulting in high heterogeneity among the sister cells. Furthermore, the added size from birth to division was significantly shorter than birth size (p < 0.01) and decreased along generations. The results above revealed that the asymmetric division site and varied cell size parameters resulted in filament-to-rod cell cycle of L. varians and cell size homeostasis could be a more complex and dynamic process than previously assumed. These findings would be helpful in elucidating the open questions in cell division and cell size heterogeneity.

Effects of NADH Availability on 3-Phenyllactic Acid Production by Lactobacillus plantarum Expressing Formate Dehydrogenase.

It is well known that cofactors play a key role in the production of different compounds in bioconversion processes, while the high cost of cofactors limits their usage in industrial applications. In the present study, a NADH regeneration system was successfully developed in Lactobacillus plantarum by expressing the fdh gene coding for formate dehydrogenase (FDH) from Candida boidinii. Results indicated that the FDH was expressed with the highest activity of 0.82 U/mg of protein when cells entered early stationary phase. In addition, the expression of FDH increased the intracellular level of NADH and NADH/NAD+ ratio in L. plantarum, and therefore, enhanced the NADH-dependent production of 3-phenyllactic acid (PLA) in repeated and fed-batch bioconversions. In brief, the results demonstrate that the NADH regeneration by expressing FDH is a promising strategy for producing NADH-dependent microbial metabolites in L. plantarum.

Selenium Stimulates Cadmium Detoxification in Caenorhabditis elegans through Thiols-Mediated Nanoparticles Formation and Secretion.

Antagonism between heavy metal and selenium (Se) could significantly affect their biotoxicity, but little is known about the mechanisms underlying such microbial-mediated antagonistic processes as well as the formed products. In this work, we examined the cadmium (Cd)-Se interactions and their fates in Caenorhabditis elegans through in vivo and in vitro analysis and elucidated the machinery of Se-stimulated Cd detoxification. Although the Se introduction induced up to 3-fold higher bioaccumulation of Cd in C. elegans than the Cd-only group, the nematode viability remained at a similar level to the Cd-only group. The relatively lower level of reactive oxygen species in the Se & Cd group confirms a significantly enhanced Cd detoxification by Se. The Cd-Se interaction, mediated by multiple thiols, including glutathione and phytochelatin, resulted in the formation of less toxic cadmium selenide (CdSe)/cadmium sulfide (CdS) nanoparticles. The CdSe/CdS nanoparticles were mainly distributed in the pharynx and intestine of the nematodes, and continuously excreted from the body, which also benefitted the C. elegans survival. Our findings shed new light on the microbial-mediated Cd-Se interactions and may facilitate an improved understanding and control of Cd biotoxicity in complicated coexposure environments.

Core-shell red silica nanoparticles based immunochromatographic assay for detection of Escherichia coli O157:H7.

In this paper, a new type immunochromatographic assay (ICA) based on core-shell red silica nanoparticles (core-shell red SiO2NPs) was proposed and used to detect Escherichia coli O157:H7 (E. coli O157:H7). This is the first report of qualitative ICA for detecting E. coli O157:H7 in phosphate buffer saline (PBS) and food sample using core-shell red SiO2NPs. Monodispersed red SiO2NPs were synthesized in the aqueous solution by modifying amino silane and C.I Reactive Red 136 on unmodified silica nanoparticles. The limit of detection (LOD) of this core-shell red SiO2NPs based ICA for E. coli O157:H7 was 4.5 × 105 CFU/mL in sterile PBS within 20 min. The LOD of this ICA strip for E. coli O157:H7 in milk and pork samples both were 4.5 × 106 CFU/mL. The core-shell red SiO2NPs based ICA for detection of E. coli O157:H7 has no cross activity with other bacteria. All these results show that this new kind of core-shell colored SiO2NPs is promising for the practical applications in ICA and other rapid detection fields.

Copy number of ArsR reporter plasmid determines its arsenite response and metal specificity.

The key component in bacteria-based biosensors is a transcriptional reporter employed to monitor induction or repression of a reporter gene corresponding to environmental change. In this study, we made a series of reporters in order to achieve highly sensitive detection of arsenite. From these reporters, two biosensors were developed by transformation of Escherichia coli DH5α with pLHPars9 and pLLPars9, consisting of either a high or low copy number plasmid, along with common elements of ArsR-luciferase fusion and addition of two binding sequences, one each from E. coli and Acidithiobacillus ferrooxidans chromosome, in front of the R773 ArsR operon. Both of them were highly sensitive to arsenite, with a low detection limit of 0.04 µM arsenite (~ 5 µg/L). They showed a wide dynamic range of detection up to 50 µM using high copy number pLHPars9 and 100 µM using low copy number pLLPars9. Significantly, they differ in metal specificity, pLLPars9 more specific to arsenite, while pLHPars9 to both arsenite and antimonite. The only difference between pLHPars9 and pLLPars9 is their copy numbers of plasmid and corresponding ratios of ArsR to its binding promoter/operator sequence. Therefore, we propose a working model in which DNA bound-ArsR is different from its free form in metal specificity.

Hormetic effect and mechanism of imidazolium-based ionic liquids on the nematode Caenorhabditis elegans.

In the present study, we used Caenorhabditis elegans assay system to investigate in hormetic effects of imidazolium-based bromide Ionic Liquids (ILs) and explored the possible underlying mechanism. Firstly, C. elegans was treated with ILs with different alkyl chain lengths at different concentrations. We found that exposure to ILs at 0.01 mg/L extended the mean lifespan of C. elegans and the ILs with longer alkyl chain showed more obvious effects. To investigate the possible mechanism, the nematodes were exposed to the three ILs at 0.01 mg/L for 2, 5, 7, 9 and 11 days. The levels of reactive oxygen species (ROS) in C. elegans increased significantly when treated for 2 days and then declined gradually compared to those of respective controls as time went on. After exposure for 11 days, the ROS levels and liposuscin accumulation were significantly lower in the treated groups than those of control group. Meanwhile, the expression of aging-related genes sod-5 and daf-16 were both massively up-regulated for the three ILs examined. Our results show that low concentration of ILs exert hormetic effect on C. elegans. ROS generation and expression of aging-related genes may play important roles in the IL-induced hormetic effect on C. elegans.

Effects of culture conditions on the kinetic behavior of 1,3-propanediol fermentation by Clostridium butyricum with a kinetic model.

The effects of culture conditions on the kinetic behavior of 1,3-propanediol (PD) fermentation were investigated with a kinetic model. First, with initial glycerol concentration (S0) increasing, µmax and PD inhibition increased. Glycerol assimilation was harder and a little glycerol was consumed on cell maintenance at high S0. Second, with yeast extract concentration increasing, PD inhibition decreased. However, µmax decreased and glycerol assimilation became harder. It seems that the stimulus effect of yeast extract resulted from decreased PD inhibition. Glycerol amount consumed on cell maintenance also decreased. Third, with temperature decreasing, µmax and PD inhibition decreased. Glycerol assimilation was harder and a little more glycerol was consumed on cell maintenance at low temperature. Fourth, with pH increasing, µmax and PD inhibition decreased. Glycerol assimilation was harder and much more glycerol was consumed on cell maintenance at pH 6.5 and 7.5 than 7.0. This work facilitates further fermentation process optimization.

Complete genome sequence of Lysinibacillus varians GY32, a bacterium with filament-to-rod cell cycle.

The complete genome sequence of Lysinibacillus varians GY32 was determined to be 4,662,822 base pairs in a single circular chromosome. Genes in cell division, cell cycle, surface layer and cell wall synthesis are foundation of its unique cell morphology. The genome contains multiple clusters of transcriptional regulator, two-component system and sigma factors, providing the organism with the ability to regulate a filament-to-rod cell cycle progression. L. varians GY32 was, to our knowledge, the first bacterium with a filament-to-rod cell cycle to be sequenced and its annotated genome might provide new insights into our understanding of bacterial cell cycle regulation.

Lysinibacillus varians sp. nov., an endospore-forming bacterium with a filament-to-rod cell cycle.

Six Gram-stain-positive, motile, filamentous and/or rod-shaped, spherical spore-forming bacteria (strains GY32(T), L31, F01, F03, F06 and F07) showing polybrominated diphenyl ether transformation were investigated to determine their taxonomic status. After spore germination, these organisms could grow more than one hundred microns long as intact single cells and then divide into rod cells and form endospores in 33 h. The cell-wall peptidoglycan of these strains was type A4α, the predominant menaquinone was MK-7 and the major fatty acids were iso-C(16:0), iso-C(15:0) and C(16:1)ω7C. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were detected in the polar lipid profile. Analysis of the 16S rRNA gene sequences indicated that these strains should be placed in the genus Lysinibacillus and they were most closely related to Lysinibacillus sphaericus DSM 28(T) (99% 16S rRNA gene sequence similarity). The gyrB sequence similarity and DNA-DNA relatedness between strain GY32(T) and L. sphaericus JCM 2502(T) were 81% and 52%, respectively. The G+C content of the genomic DNA of strain GY32(T) was 43.2 mol%. In addition, strain GY32(T) showed differences in nitrate reduction, starch and gelatin hydrolysis, carbon resource utilization and cell morphology. The phylogenetic distance from its closest relative measured by DNA-DNA relatedness and DNA G+C content, and its phenotypic properties demonstrated that strain GY32(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus varians sp. nov. is proposed. The type strain is GY32(T) ( = NBRC 109424(T) = CGMCC 1.12212(T) = CCTCC M 2011307(T)).