Fluorescence Intensity Of Protein Research Articles

Effects of pectin combined with plant essential oils on water migration, myofibrillar proteins and muscle tissue enzyme activity of vacuum packaged large yellow croaker (Pseudosciaena crocea) during ice storage

The effects of pectin combined with plant essential oils on water migration, myofibrillar proteins (MPs) and muscle tissue enzyme activity of vacuum packaged large yellow croaker (Pseudosciaena crocea) during ice storage at 4±1°C were investigated. Two solutions of 2.5 % (w/v) pectin combined with 0.4 % (v/v) oregano essential oil (PO) and 0.4 % (v/v) ginger essential oil (PG) were used for coating treatments. Water holding capacity (WHC), cooking loss, Low-field nuclear magnetic resonance (LF-NMR) and Magnetic resonance imaging (MRI) analysis, carbonyl content, total sulfhydryl content, fluorescence intensity (FI) of myofibrillar protein and SDS-PAGE were analyzed, respectively. Tissue characteristics, including α-Glucosidase (AG) and β-N-acetyl-glucosaminidase (NAG) activities were measured. The results showed that compared to the CK group, LF-NMR and MRI results showed that PO and PG groups delay the transformation of mobilized water to free water and maintain better WHC during storage. PO and PG groups also had a significant protective effect on protein oxidation, such as the inhibition of carbonyl group and FI, prevention of endogenous enzyme activity, and a decrease in the sulfhydryl content. Therefore, PO and PG groups had the most optimal effect and could enhance the shelf life of large yellow croaker for another 7 days at least during ice storage.

Effects of AOT reverse micelle extraction on structure and emulsifying properties of soybean protein

AbstractThe present study aimed to investigate the forward and backward extraction efficiency, structure, and emulsifying properties of soybean proteins obtained through bis (2‐ethylhexyl) sulfosuccinate sodium salt (AOT) reverse micelle, as well as their relationship. The results showed that the extraction efficiency was the highest with the forward extraction time of 25 min, pH of 3.0, temperature of 45°C, KCl concentration of 0.0 mol L−1, and the backward extraction time of 10 min, pH of 4.0, temperature of 30°C, KCl concentration of 0.5 mol L−1. Significant changes in the secondary structure and fluorescence intensity of soybean protein after AOT reverse micelle extraction were observed. More β‐sheet structure and higher fluorescence intensity corresponded to the higher emulsion stability of soybean protein. The results indicated that reverse micelle extraction shows great application in food industry.

Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level.

Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.

Effect of solid-state fermentation by three different Bacillus species on composition and protein structure of soybean meal.

Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10mg g-1 , respectively. Bacillus licheniformis YYC4 and B. subtilis 10 160 significantly increased phenol and pyrazine content. Furthermore, B. licheniformis YYC4 fermentation could produce abundant alcohols, ketones, esters and acids. Surface hydrophobicity, sulfhydryl groups and disulfide bond contents of SBM protein were increased significantly from 98.27 to 166.13, 173.27 and 150.71, from 3.26 to 4.88, 5.03 and 4.21 μmol g-1 , and from 20.77 to 27.95, 29.53 and 25.5μmol g-1 after their fermentation. Fermentation induced red shifts of the maximum absorption wavelength (λmax ) of fluorescence spectra from 353 to 362, 376 and 361 nm, while significantly reducing the fluorescence intensity of protein, especially when B. subtilis 10 160 was used. Moreover, fermentation markedly changed the secondary structure composition of SBM protein. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscopy showed that macromolecule protein was degraded into small-sized protein or peptide during fermentation of SBM. Bacillus licheniformis YYC4 fermentation (without sterilization) improved nutrition and protein structure of SBM as B. subtilis 10 160, suggesting its potential application in the SBM fermentation industry. © 2021 Society of Chemical Industry.

Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

Construction of fungi-microalgae symbiotic system and adsorption study of heavy metal ions

Harvesting and immobilization are problem in microalgae water treatment. Here, mycelial pellets (Aspergillus fumigatus) and Synechocystis sp. PCC6803 comprised a fungi-microalgae symbiotic system (FMSS) of hypha-pellet flocculant auxiliary microalgae solidification. Optical and scanning electron microscopy (SEM) revealed that green particles were attached to fungal mycelia. A 98% adsorption and immobilization efficiency of mycelium pellets to microalgae was evident. Under the condition of 108 cells/mL logarithmic phase microalgae and no fungal culture medium, the symbiotic system had higher stability and better Cd(II) adsorption efficiency (98.89%, 37.3 mg g−1). Meanwhile, the symbiotic system prolonged the growth cycle of microalgae in the adsorption. After Cd(II) ion stimulation, the content of total extracellular polymeric substances (EPS) in the FMSS was significantly higher than that in the control group, and the change of extracellular polysaccharide was more obvious. High performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) showed that the monosaccharide composition of extracellular polysaccharides increased. Three dimensional excitation emission matrix (3D-EEM) spectrum analysis showed the peak intensity of protein fluorescence changed with the treatment of heavy metals. FTIR spectra showed that the functional groups (such as OH, CONH) of EPS participated in the adsorption process. The findings showed that the key components of EPS, extracellular polysaccharides and extracellular proteins, played an important role in the symbiotic system to cope with the stress of heavy metal and resist the toxicity of heavy metal. The preliminary data can inform development and utilization of engineering strains and gene transformation.

Thermal Reversibility and Structural Stability in Lysozyme Induced by Epirubicin Hydrochloride.

Herein we report the binding interactions between lysozyme (Lyz) and an anthracycline drug, epirubicin hydrochloride (EPR), through an extensive spectroscopic approach at both ensemble average and single molecular resolution. Our steady-state and time-resolved fluorescence spectroscopy reveals that the drug-induced fluorescence quenching of the protein proceeds through a static quenching mechanism. Isothermal titration calorimetry (ITC) and steady-state experiments reveal almost similar thermodynamic signatures of the drug-protein interactions. The underlying force that plays pivotal roles in the said interaction is hydrophobic in nature, which is enhanced in the presence of a strong electrolyte (NaCl). Circular dichroism (CD) spectra indicate that there is a marginal increase in the secondary structure of the native protein (α-helical content increases from 26.9 to 31.4% in the presence of 100 μM EPR) upon binding with the drug. Fluorescence correlation spectroscopy (FCS) was used to monitor the changes in structure and conformational dynamics of Lyz upon interaction with EPR. The individual association (Kass = 0.33 × 106 ms-1 M-1) and dissociation (Kdiss = 1.79 ms-1) rate constants and the binding constant (Kb = 1.84 × 105 M-1) values, obtained from fluctuations of fluorescence intensity of the EPR-bound protein, have also been estimated. AutoDock results demonstrate that the drug molecule is encapsulated within the hydrophobic pocket of the protein (in close proximity to both Trp62 and Trp108) and resides ∼20 Å apart from the covalently labelled CPM dye. Förster resonance energy transfer (FRET) studies proved that the distance between the donor (CPM) and the acceptor (EPR) is ∼22 Å, which is very similar to that obtained from molecular docking analysis (∼20 Å). The system also shows temperature-dependent reversible FRET, which may be used as a thermal sensor for the temperature-sensitive biological systems.

Cholesterol-lowering drugs the simvastatin and atorvastatin change the protease activity of pepsin: An experimental and computational study

In this study, the effect of long-term use drugs of cholesterol-lowering atorvastatin and simvastatin on the activity and molecular structure of pepsin as important gastric enzyme was investigated by various experimental and computational methods. Based on the results obtained from fluorescence experiments, both drugs can bond to pepsin and quench the fluorescence intensity of protein through the static quenching mechanism. Also analysis of the thermodynamic parameters of binding the drugs to pepsin showed that the main forces in the complex formation for both are hydrophobic interactions and van der Waals forces. The effects of the drugs on the enzymatic activity of pepsin were then investigated and results showed that in the presence of both drugs the catalytic activity of the enzyme was significantly increased in lower (0.3–0.6 mM) concentrations however about the atorvastatin, increasing the concentration (0.9 mM) decreased the protease activity of pepsin. Also as a result of the FTIR studies, it was found that binding of the drugs to protein did not significant alteration in the structure of the protein. In order to obtain the atomic details of drug-protein interactions, the computational calculations were performed. The results in good agreement with those obtained from the experimental for interaction; confirm that the drugs both are bind to a cleft near the active site of the protein without any change in the structure of pepsin. Overall from the results obtained in this study, it can be concluded that both simvastatin and atorvastatin can strongly bond to a location close to the active site of pepsin and the binding change the enzymatic activity of protein.

Optical characterization and tunable antibacterial properties of gold nanoparticles with common proteins

The interaction of three proteins, viz. Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) and Hen Egg White Lysozyme (HEWL) with gold nanoparticles (GNPs) is investigated using surface plasmon resonance (SPR) spectroscopy, fluorescence spectroscopy and circular dichroism (CD). Size and morphology of the samples was established using Transmission Electron Microscopy (TEM) and stability studies was established using zeta potential analysis. The stability of protein-GNP complex was found to be greater than that of individual protein as well as individual GNPs. Also HEWL-GNP complex was more stable compared to the other protein complexes. Absorbance of proteins increases with increase in gold nanoparticle concentration due to the extension of peptide strands of protein and decrease in hydrophobicity of gold nanoparticles. A ground state complex is also formed which is evident from the moderate shift observed in the absorbance peaks. Apparent association constant was also determined from the absorption spectra and was found to be maximum for HEWL and minimum for HSA. Gold nanoparticles were found to act as quenchers and reduced the protein fluorescence intensity. Binding constant and number of binding sites were found to be maximum for HEWL and minimum for HSA. The temperature dependent fluorescence studies were also performed to calculate the thermodynamic parameters and to determine the nature of interaction between the proteins and gold nanoparticles. The circular dichroism studies elucidate the reason behind the maximum binding for HEWL and minimum binding for HSA. TGA analysis determined the thermal stability of the samples. Fluorescence lifetime studies indicate static quenching of proteins. Antibacterial activity of protein-gold nanoparticles was studied against four pathogens, viz. Bacillus pumilus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. HEWL exhibits a tunable antimicrobial activity against Pseudomonas aeruginosa due to the maximum binding of HEWL with gold nanoparticles. The study proposes a novel method for adjusting the antibacterial activity of HEWL against Pseudomonas aeruginosa when the resistance of this pathogen is a major issue in the chemotherapy of many infectious diseases. Thus the combination therapy of protein-gold nanoparticles could prove to be a new approach in medical field in the near future.

Microglia-Astrocyte Communication via C1q Contributes to Orofacial Neuropathic Pain Associated with Infraorbital Nerve Injury.

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia–astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain.

Dexmedetomidine Alleviates CCI-Induced Neuropathic Pain via Inhibiting HMGB1-Mediated Astrocyte Activation and the TLR4/NF-κB Signaling Pathway in Rats.

To investigate the effects of dexmedetomidine on chronic constriction injury (CCI)-induced neuropathic pain and to further explore its mechanism. A CCI rat model was established and treatment with dexmedetomidine. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were monitored at different time points, and the effects of hematoxylin-eosin staining on the sciatic nerve morphology of rats were observed. Immunohistochemical and immunofluorescence analyses were used to detect the expression of high mobility group box-1 (HMGB1) protein and glial fibrillary acidic protein (GFAP), and protein fluorescence intensity of GFAP in spinal cord tissue, respectively. Moreover, the expression of HMGB1 and Toll-like receptor-4/nuclear factor kappa-B (TLR4/NF-κB) pathway-related proteins were detected by western blot assay. To verify whether dexmedetomidine alleviates CCI-induced neuropathic pain by inhibiting HMGB1-mediated astrocyte activation and the TLR4/NF-κB signaling pathway, the rats were further treated with an HMGB1 activator or antagonist. Dexmedetomidine was found to improve the pathological changes of the sciatic nerve and alleviate pain in the CCI rats. The expression of HMGB1, GFAP, TLR4, TRAF6, MyD88, and p-P65 were greatly downregulated in the spinal cord tissues of the CCI rats. In addition, a further study showed that an HMGB1 activator can reverse the inhibition of neuropathic pain behaviors of dexmedetomidine. Overexpression of HMGB1 downregulated the PWMT and PWTL and enhanced the astrocyte activity and the TLR4/NF-κB signaling pathway in CCI rats. These results indicated that dexmedetomidine can alleviate neuropathic pain in CCI rats by inhibiting HMGB1-mediated astrocyte activation and the TLR4/NF-κB signaling pathway.

Probing the Electron Transfer between iLOV Protein and Ag Nanoparticles.

Nanomaterials have been widely used in biomedical sciences; however, the mechanism of interaction between nanoparticles and biomolecules is still not fully understood. In the present study, we report the interaction mechanism between differently sized Ag nanoparticles and the improved light-oxygen-voltage (iLOV) protein. The steady-state and time-resolved fluorescence results demonstrated that the fluorescence intensity and lifetime of the iLOV protein decreased upon its adsorption onto Ag nanoparticles, and this decrease was dependent upon nanoparticle size. Further, we showed that the decrease of fluorescence intensity and lifetime arose from electron transfer between iLOV and Ag nanoparticles. Moreover, through point mutation and controlled experimentation, we demonstrated for the first time that electron transfer between iLOV and Ag nanoparticles is mediated by the tryptophan residue in the iLOV protein. These results are of great importance in revealing the function of iLOV protein as it applies to biomolecular sensors, the field of nano-photonics, and the interaction mechanism between the protein and nanoparticles.

A Hot Water Extract of Sideritis scardica Prolongs Life Span and Enhances Heat Shock Resistance in Caenorhabditis elegans

Sideritis scardica is a Lamiaceae plant that is endemic to the alpine zone of the Balkan Peninsula. The tea of S. scardica has been handed down as a “tea of longevity” in the Rhodope region of Bulgaria for an unknown amount of time. In this study, we prepared a hot water extract of S. scardica (SHWE) and examined its effects on both life span and stress response in living tissue using Caenorhabditis elegans and its transgenic mutants. The life span of wild-type N2 worms was prolonged by approximately 15% at the SHWE concentration of 5 µg/mL and approximately 22% at the SHWE concentration of 50 µg/mL, as compared with the control group. The effect of SHWE on the expression of heat shock protein 16.2 (HSP-16.2) under heat stress was investigated using TJ375 worms, a transgenic mutant of C. elegans. In the TJ375 worms pretreated with SHWE, the fluorescence intensity of green fluorescent protein fluorescence, which indicates the expression of HSP-16.2, was significantly increased. In the assay using TJ356 worms, the worms pretreated with SHWE did not show the translocation of DAF-16, a forkhead transcription factor class O homolog, from the cytoplasm to nucleus under heat stress. Additionally, under heat stress, the pretreatment of SHWE improved the survival rate of GR1307 worms, a knockout mutant of daf-16. These results indicate that SHWE enhances HSP-16.2 expression through a stress-response pathway (eg, HSF-1 pathway) other than the DAF-16 pathway, resulting in a prolonged life span of C. elegans under heat stress.

Phytochemical analysis of two main varieties of persimmon and kiwifruit and their antioxidative and quenching capacities

Bioactivities of persimmon and kiwifruit were studied and compared in connection with their characteristics towards human health. Based on the results of our recently published research, two fruits growing in South Korea persimmon ‘Fuyu’ and kiwifruit ‘Hayward’ were investigated and compared. Spectroscopic, fluorescence and FTIR measurements and radical scavenging reactions were used to compare the qualities of persimmon and kiwifruit. The results of above methods showed that total polyphenol and tannin substances, vitamin C and values of antioxidant capacities were higher in kiwifruit than in persimmon. Phenolics were also characterized by specific IR bands and MS spectra. Antioxidant capacities of polyphenols extracted from the studied fruits correlated with the binding abilities during their interaction with serum albumin, one of the most important proteins in human metabolism. As a result of a complex reaction between the proteins and polyphenols, the fluorescence intensity of proteins decreased and such interaction can be a model in distribution of drugs. Relationship between the most active compounds and their influence on the quality of fruits was established, based on the relatively high bioactivity and quenching properties. In conclusion, kiwifruit and persimmon according to the obtained results contain high amounts of antioxidants and can be widely consumed as fresh, processed and functional foods.

Hydrogen Peroxide-Generating Electrochemical Scaffold Activity against Trispecies Biofilms.

The antibiofilm activity of a hydrogen peroxide-generating electrochemical scaffold (e-scaffold) was determined against mono- and trispecies biofilms of methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, and Candida albicans Significant time-dependent decreases were found in the overall CFU of biofilms of all three monospecies and the trispecies forms. Confocal laser scanning microscopy showed dramatic reductions in fluorescence intensities of biofilm matrix protein and polysaccharide components of e-scaffold-treated biofilms. The described e-scaffold has potential as a novel antibiotic-free strategy for treating wound biofilms.

Intestinal Alkaline Phosphatase Exerts Anti-Inflammatory Effects Against Lipopolysaccharide by Inducing Autophagy

Intestinal alkaline phosphatase (IAP) regulates bicarbonate secretion, detoxifies lipopolysaccharide (LPS), regulates gut microbes, and dephosphorylates proinflammatory nucleotides. IAP also exhibits anti-inflammatory effects in a Toll-like Receptor-4 (TLR-4) dependent manner. However, it is not known whether IAP induces autophagy. We tested the hypothesis that IAP may induce autophagy which may mediate the anti-inflammatory effects of IAP. We found that exogenous IAP induced autophagy in intestinal epithelial cells and in macrophages. TLR4INC34 (C34), a TLR4 signaling inhibitor, suppressed IAP-induced autophagy. IAP also inhibited LPS-induced IL-1β mRNA expression and activation of NF-κB. When autophagy was blocked by 3-methyladenine (3MA) or by Atg5 siRNA, IAP failed to block LPS-mediated effects. IAP also upregulated autophagy-related gene expression in small intestine in mice. We administered either vehicle or IAP (100 U/ml) in drinking water for 14 days in C57BL/6 mice. Mice were sacrificed and ileal tissues collected. Increased expression of Atg5, Atg16, Irgm1, Tlr4, and Lyz genes was observed in the IAP treated group compared to the vehicle treated group. Increase in Atg16 protein expression and fluorescence intensity of LC3 was also observed in IAP-treated tissues compared to the vehicle-treated tissues. Thus, our study lays the framework for investigating how IAP and autophagy may act together to control inflammatory conditions.

Glucose-modified carbosilane dendrimers: Interaction with model membranes and human serum albumin.

Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucose-modified carbosilane DDMs (first-third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM2-3Glu, significantly affect the type and extent of interaction with biological structures.

Abstract TMP30: M2 Microglia Derived Exosomes Promote White Matter Repair and Long-Term Functional Recovery After Focal Cerebral Ischemia in Mice

Objectives: White matter injury aggravates neurological and cognitive impairment in experimental ischemic stroke. M2 microglia promote oligodendrocyte precursor cells survival and differentiation, and further enhance white matter repair. However, the molecular mechanism is unclear. Here, we explored the effect and mechanism of M2 microglia-derived exosomes on white matter repair after focal cerebral ischemia in mice. Methods: Microglia BV2 cells were polarized to M2 phenotype by IL-4 stimulation. Exosomes were isolated from M2 microglia (M2-Exo) and unstimulated microglia as a control (M0-Exo). M2-Exo and M0-Exo (100 μg) were intravenously injected after 90-minute middle cerebral artery occlusion in mice (n=72). Brain atrophy volume and neuro behavioral outcomes were examined in 28 days following focal cerebral ischemia. Oligodendrocyte precursor cells survival, differentiation and white matter integrity were evaluated. Exosomal miRNA and target gene were further examined to explore molecular mechanism. Results: M2-Exo treatment promoted sensorimotor and memory function recovery ( p <0.05), and further reduced brain atrophy compared to the M0-Exo control group ( p <0.001). Immunostaining showed that M2-Exo increased the number of BrdU + /Pdgfr-α + and BrdU + /adenomatous polyposis coli + cells, enhanced myelin basic protein fluorescence-intensity compared to the control ( p <0.05). M2-Exo increased oligodendrocyte precursor cell survival under OGD in vi tro , ( p <0.05) and differentiation ( p <0.05). Exosomal miRNA sequencing and PCR identified that miR-23a-5p was enriched in M2-Exo. Conclusion: Our results showed that M2-Exo treatment enhanced oligodendrocyte precursor cell survival and differentiation, further promoted white matter repair and long-term functional recovery, suggesting that M2-Exo is a novel therapeutic strategy for the white matter repair after ischemic brain injury.

MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water

Mercury ion (Hg2+ ) is a universal pollutant and its detection is crucial for public healthcare. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+ -induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40nM. The limit of detection was 0.5nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependent on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5nM concentration. However, the interference effect was more pronounced at 40nM concentration of metal ions. The measurement was completed in less than 1Min with no need for sample preparation or preincubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry.

Pretreatment-promoted sludge fermentation liquor improves biological nitrogen removal: Molecular insight into the role of dissolved organic matter

Waste activated sludge (WAS) can be used as carbon sources to support biological nutrient removal (BNR). In this study, thermal-alkaline (THALK), ozonation (OZN), electrolysis (EC) and NaClO-promoted electrolysis (EC-AOP) were investigated to facilitate WAS solubilization and production of volatile fatty acids (VFAs). EEMF-PARAFAC and FT-ICR-MS were employed to characterize the transformation of dissolved organic matter (DOM) in WAS fermentation liquors at molecular level. THALK achieved the highest fluorescence intensity of C1 protein after pretreatment. Proteins and lipids were the dominant DOM in the pretreated WAS, while the DOM shifted towards substances with higher H/C and lower O/C after fermentation. The BNR results showed that THALK (100%) and EC-AOP (96.9%) outperformed other groups (78.9–90.3%) in terms of NO3-N removal, indicating the significant impact of DOM compositions. Overall, these results demonstrated that THALK and EC-AOP effectively enhanced release of VFAs and DOM, which subsequently improved NO3-N removal efficiency.