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High-performance determination of pyrophosphate ions (PPi) and pH is an important goal in biological systems. In this work, Cu-doped carbon dots (Cu-CDs) were synthesized rapidly and simply via a one-pot hydrothermal method. The as-obtained Cu-CDs, with an average size of 2.55 nm, exhibit an excitation-independent fluorescence emission and possess desirable functional groups of carboxyl and amine, which can be served as fluorescence nanoprobes for detection of PPi based on surface passivation. Under the optimal condition, the linear range for detection of PPi was 0.05 - 20 µM, and the corresponding limit of detection (LOD) was 0.013 µM, indicative of a promising assay for the PPi. Moreover, the fluorescent intensity of the Cu-CDs is linear against pH value from 6 to 8.7 in buffer solution, suggesting the feasibility as a pH sensor. The synthesized Cu-CDs coated fluorescent paper indeed can monitor pH in urine with satisfaction by naked eyes through ultraviolet irradiation. The successful detection of PPi and the visual detection of pH value suggest a highly promising application of Cu-CDs in the field of biosensing.

Herein, a simple and facile one-step hydrothermal carbonization synthesis procedure for the fabrication of N, Cu-doped carbon quantum dots (N, Cu-CQDs) as a peroxidase-mimicking enzyme was reported. The peroxidase-like performance of N, Cu-CQDs was assessed based on the oxidative coupling reaction of phenol with 4-aminoantipyrine (4-AAP) in the presence of hydrogen peroxide (H2O2). The N, Cu-CQDs/4-AAP/H2O2 system was applied to sensing phenol based on double signals of absorption spectra (or colorimetric visualization) as well as fluorescence spectra. The obtained limits of detection (LODs) were as low as 0.12 μM and 0.02 μM, respectively. Moreover, the proposed method was successfully applied to the determination of phenol in sewage with satisfactory recovery. Our results demonstrate that the N, Cu-CQDs/4-AAP/H2O2/phenol sensing system has a great potential prospect for applications in environmental chemistry and biotechnology.

Biosurfactants have been put into applications in breadmaking industry, while the effects of mannosylerythritol lipid-A (MEL-A) on gluten network of frozen dough, bread quality and microbial spoilage were firstly investigated in this study. Rheology and differential scanning calorimetry (DSC) analysis showed that MEL-A significantly improved the rheological properties of frozen dough and reduced the content of frozen water. Further experiments showed that MEL-A promoted the formation of aggregates by interacting with gluten protein, and strengthened the gluten network through molecular weight distribution measurement and microstructure observation, effectively avoiding the destruction of ice crystals. A series of bread assessments illustrated MEL-A improved the loaf volume, gas retention ability and textural property. In addition, MEL-A (1.5%) killed 99.97% of the vegetative cells of Bacillus cereus and 75.54% of the spores, and at the same time had a slight inactivation effect on yeast. These results indicate that MEL-A has broad application prospects in the baking industry and the storage stage of flour products.

According to Traditional Chinese Medicine (TCM), Aconiti Radix Cocta (AC) is clinically employed to expel wind, remove dampness, and relieve pain. We evaluated the antirheumatoid arthritis (RA) activities and underlying mechanisms of AC. The chemical constituents of AC were analyzed by high-performance liquid chromatography (HPLC) using three reference compounds (benzoylaconitine, benzoylmesaconine, and benzoylhypacoitine). The anti-RA effects of AC were evaluated in adjuvant-induced arthritis (AIA) rats by hind paw volume and histopathological analysis. The effects of AC on inflammatory cytokines (IL-1β and IL-17A) were determined by enzyme-linked immunosorbent assay. The regulation of cyclooxygenases (COX-1 and/or COX-2) was determined by Western blot and real-time quantitative reverse transcription polymerase chain reaction analyses. AC significantly reduced paw swelling, attenuated the inflammation and bone destruction in joint tissues, and reduced IL-1β and IL-17A in the serum. Moreover, AC downregulated the expression of COX-1 and COX-2 in the synovial tissues. We also identified that AC possesses significant anti-RA activities on AIA, which may be ascribed to the regulation of inflammatory cytokines IL-1β and IL-17, as well as to the inhibition of arachidonic acid signaling pathways. Our findings provide theoretical support for AC as an effective nature-derived therapeutic agent for RA treatment.

Glycolipid biosurfactants are natural amphiphiles and have gained particular interest recently in their biodegradability, diversity, and bioactivity. Microbial infection has caused severe morbidity and mortality and threatened public health security worldwide. Glycolipids have played an important role in combating many diseases as therapeutic agents depending on the self-assembly property, the anticancer and anti-inflammatory properties, and the antimicrobial properties, including antibacterial, antifungal, and antiviral effects. Besides, their role has been highlighted as scavengers in impeding the biofilm formation and rupturing mature biofilm, indicating their utility as suitable anti-adhesive coating agents for medical insertional materials leading to a reduction in vast hospital infections. Notably, glycolipids have been widely applied to the synthesis of novel antimicrobial materials due to their excellent amphipathicity, such as nanoparticles and liposomes. Accordingly, this review will provide various antimicrobial applications of glycolipids as functional ingredients in medical therapy.

Mannosylerythritol lipids-A (MEL-A) is a novel biosurfactant with excellent surface activity and potential biomedical applications. In this study, we explored the antibacterial activity and the underlying mechanisms of MEL-A against the important food-borne pathogen Listeria monocytogenes. The bacterial growth and survival assays revealed a remarkable antibacterial activity of MEL-A. Since MEL-A is a biosurfactant, we examined the cell membrane integrity and morphological changes of MEL-A-treated bacteria by biochemical assays and flow cytometry analysis and electron microscopes. The results showed obvious damaging effects of MEL-A on the cell membrane and morphology. To further explore the antibacterial mechanism of MEL-A, a transcriptome analysis was performed, which identified 528 differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed that the gene categories of membrane, localization and transport were enriched among the DEGs, and the analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways demonstrated significant changes in the maltodextrin ABC transporter system and stress response system. Furthermore, the growth of L. monocytogenes could also be significantly inhibited by MEL-A in milk, a model of a real food system, suggesting that MEL-A could be potentially applied as an natural antimicrobial agent to control food-borne pathogens in the food industry.

Mannosylerythritol lipids (MELs) are novel biosurfactants performing excellent physical-chemical properties as well as bioactivities. This study is aimed to explore the antibacterial and antibiofilm activity of mannosylerythritol lipids against foodborne gram-positive Staphylococcus aureus. The results of growth curve and survival rate revealed the significant inhibitory effect of MELs against S. aureus. The visualized pictures by scanning electron microscope and transmission electron microscope exposed apparent morphological and ultrastructure changes of MEL-treated cells. Furthermore, flow cytometry confirmed that MELs have promoted cell apoptosis and damaged the cell membrane. Notably, MEL-A also exhibited outstanding antibiofilm activity against S. aureus biofilm on different material surfaces including polystyrene, glass, and stainless steel, verified by confocal laser scanning microscope. These findings suggest that the antimicrobial activity of MELs is related to inhibit planktonic cells and biofilm of S. aureus, indicating that it has potential to be an alternative to antibacterial agents and preservatives applied into food processing. Key Points • MELs have strong antibacterial activity against Staphylococcus aureus. • MELs mainly damage the cell membrane of Staphylococcus aureus. • Mannosylerythritol lipids inhibit the bacterial adhesion to remove biofilm.

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen–deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1–R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

Non-enzymatic deamidation of asparagine and glutamine in peptides and proteins is a frequent modification both in vivo and in vitro. The biological effect is not completely understood, but it is often associated with protein degradation and loss of biological function. Here we describe the deamidation of CsgA, the major protein subunit of curli, which are important proteinaceous components of biofilms. CsgA has a high content of Asn and Gln, a feature seen in a few proteins that self-aggregate. We have implemented an approach to monitor deamidation rapidly by following the globally centroid mass shift, providing guidance for studies at the residue level. From the global mass measurement, we identified, using LC-MS/MS, extensive deamidation of several Asn residues and discovered three “Asn–Gly” sites to be the hottest spots for deamidation. The fibrillization of deamidated CsgA was measured using thioflavin T (ThT) fluorescence, circular dichroism (CD), and a previously reported hydrogen–deuterium exchange (HDX) platform. Deamidated proteins exhibit a longer lag phase and lower final ThT fluorescence, strongly suggesting slower and less amyloid fibril formation. CD spectra show that extensively deamidated CsgA remains unstructured and loses its ability to form amyloids. Mass-spectrometry-based HDX also shows that deamidated CsgA aggregates more slowly than wild-type CsgA. Taken together, the results show that deamidation of CsgA slows its fibrillization and disrupts its function, suggesting an opportunity to modulate CsgA fibrillization and affect curli and biofilm formation.

Zinc finger protein X-linked (ZFX) is frequently upregulated in multiple human malignancies and also plays a critical role in the maintenance of self-renewal in embryonic stem cells. However, the role of ZFX in liver cancer stem cells (CSCs) remains obscure. We observed that the elevated expression of both ZFX and epithelial cell adhesion molecule (EpCAM) was associated with aggressive clinicopathological features and indicated poor prognosis in patients with hepatocellular carcinoma (HCC). ZFX was commonly enriched in liver EpCAM+ CSCs. Knockdown of ZFX decreased the proportion of EpCAM+ CSCs in HCC cells and suppressed their expression of stemness-related genes, self-renewal capacity, chemoresistance, metastatic potential, and tumorigenicity. Conversely, upregulation of ZFX in CSCs rescued these inhibitory effects and enhanced stem-like properties. Mechanistically, depletion of ZFX reduced nuclear translocation and transactivation of β-catenin, thereby inhibiting the self-renewal capacity of EpCAM+ CSCs. Moreover, knockdown of β-catenin attenuated the self-renewal of EpCAM+ HCC cells stably expressing ZFX, further indicating that β-catenin is required for ZFX-mediated expansion and maintenance of EpCAM+ CSCs. Taken together, our findings indicate that ZFX activates and maintains EpCAM+ liver CSCs by promoting nuclear translocation and transactivation of β-catenin. Furthermore, combination of ZFX and EpCAM may serve as a significant indicator for prognosis of patients with HCC.

In this work, using Saccharomyces cerevisiae as a model, we showed that BetA could inhibitcell proliferation and lead to lethal cytotoxicity accompanying programmed cell death (PCD).Interestingly, it was found that vacuolar protease Pep4p played a pivotal role in BetA‐induced S.cerevisiae PCD. The presence of Pep4p reduced the damage of BetA‐induced cells. This work impliedthat BetA may induce cell death of S. cerevisiae through mitochondria‐mediated PCD, and thedeletion of Pep4 gene possibly accelerated the effect of PCD. The present investigation provided thepreliminary research for the complicated mechanism of BetA‐induced cell PCD regulated by vacularprotease Pep4p and lay the foundation for understanding of the Pep4p protein in an animal model.

As a novel natural compound delivery system, liposomes are capable of incorporating lipophilic bioactive compounds with enhanced compound solubility, stability and bioavailability, and have been successfully translated into real-time clinical applications. To construct the soy phosphatidylcholine (SPC)&ndash, cholesterol (Chol) liposome system, the optimal formulation was investigated as 3:1 of SPC to Chol, 10% mannosylerythritol lipid-A (MEL-A) and 1% betulinic acid. Results show that liposomes with or without betulinic acid or MEL-A are able to inhibit the proliferation of HepG2 cells with a dose-effect relation remarkably. In addition, the modification of MEL-A in liposomes can significantly promote cell apoptosis and strengthen the destruction of mitochondrial membrane potential in HepG2 cells. Liposomes containing MEL-A and betulinic acid have exhibited excellent anticancer activity, which provide factual basis for the development of MEL-A in the anti-cancer applications. These results provide a design thought to develop delivery liposome systems carrying betulinic acid with enhanced functional and pharmaceutical attributes.

Vacuolar proteinase A (Pep4p) is required for the post-translational precursor maturation of vacuolar proteinases in Saccharomyces cerevisiae, and important for protein turnover after oxidative damage. The presence of proteinase A in brewing yeast leads to the decline of beer foam stability, thus the deletion or inhibition of Pep4p is generally used. However, the influence of Pep4p deletion on cell metabolism in Saccharomyces cerevisiae is still unclear. Herein, we report the identification of differentially down-regulated metabolic proteins in the absence of Pep4p by a comparative proteomics approach. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) presented that the number of significantly up-regulated spots (the Pep4p-deficient species versus the wild type) was 183, whereas the down-regulated spots numbered 111. Among them, 35 identified proteins were differentially down-regulated more than 10-fold in the Pep4p-deficient compared to the wild-type species. The data revealed that Pep4p was required for the synthesis and maturation of several glycolytic enzymes and stress proteins, including Eno2p, Fba1p, Pdc1p, Tpi1p, Ssa1, Hsp82p, and Trr1p. The transcription and post-translational modifications of glycolytic enzymes like Eno2p and Fba1p were sensitive to the absence of Pep4p, whereas the depletion of the pep4 gene had a negative impact on mitochondrial and other physiological functions. The finding of this study provides a systematic understanding that Pep4p may serve as a regulating factor for cell physiology and metabolic processes in S. cerevisiae under a nitrogen stress environment.

Co-culturing two or more species has recently been used in fermenting foods for product flavor or safety control. Ethyl carbamate (EC) is a potential carcinogenic compound presented in Chinese yellow rice wine which can be reduced by fermentation starters modification. In this work, the effect of Lactobacillus brevis (L. brevis) on EC in yellow rice wine fermentation with Saccharomyces cerevisiae (S. cerevisiae) was studied and an optimal mixed-culture fermentation method was constructed according to comparative analyses of EC concentration and fundamental quality. EC concentration could be further reduced by 68.4% at most through adding 108 cfu/L L. brevis during S. cerevisiae fermentation. L. brevis also had beneficial influence on hedonic quality of yellow rice wine. Meanwhile, the effect of gallic acid on EC formation in this mixed-culture fermentation system was elucidated. Unfortunately, the coexistence of gallic acid and L. brevis attenuated their inhibitory effect on EC formation.

Graphical abstract, Methicillin-resistant Staphylococcus aureus (MRSA) is drug-resistant and biofilm-forming pathogenic bacteria with severe morbidity and mortality, and has been continuously detected in food products in recent years. Mannosylerythritol lipids (MELs) are novel biosurfactants and perform antibacterial property against gram-positive bacteria. Ultrasound has been applied into food sterilization as non-thermal techniques and has advantage of maintaining food nutrition and flavor over heat pasteurization. In this work, the synergistic treatment of ultrasound and MEL-A was used to combat planktonic cells and biofilm of MRSA. As a result, the combined treatment has exhibited remarkable antibacterial effect proved by enumeration of viable microbes. Furthermore, flow cytometry, scanning electron microscopy and transmission electron microscopy revealed ultrasound has enhanced the inhibitory effect of MEL-A through exacerbating cell membrane damage. On the other hand, the collaborating working modes to eradicate MRSA biofilm were disturbing cell adhesion to surface by MEL-A and destructing mature biofilm mechanically by ultrasound, reaching to over 90% of clearance rate. The findings of this study illustrated the synergistic antimicrobial mechanism of ultrasound and MEL-A treatments, and offered theoretical basis for their potential applications in food preservation.

Objectives To examine the expression of ALDOB in gastric cancer (GC) tissue and to reveal its potential clinicopathological and prognostic significance. Materials and methods We screened for genes that were differentially expressed between GC and nontumor tissues using a microarray, specifically the Affymetrix U133 Plus 2.0 Array platform. We then verified the transcriptional and translational levels of ALDOB by performing quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). In addition, a merged data set based on the Gene Expression Omnibus was generated and a survival analysis performed. Results The microarray analysis revealed that ALDOB was downregulated (more than sevenfold) in GC compared with nontumor tissue. Both qRT-PCR and IHC validated the decrease of ALDOB in GC tissue. Moreover, we found that the expression of ALDOB was significantly related to tumor-invasion depth, lymph-node metastasis, distant metastasis, and TNM stage. The survival analysis, based on the IHC and merged data set, indicated that the overall survival was better in patients with high ALDOB expression. The Cox regression analysis showed that ALDOB expression was an independent prognostic factor for GC. Conclusion The expression of ALDOB in GC tissue was significantly related to the clinicopathological features and prognosis of the disease, thus suggesting that ALDOB could act as a novel molecular marker for GC.

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal “tails” were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections., IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson’s disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.

This study aimed to investigate the antibacterial activity and underlying mechanism of mannosylerythritol lipids (MELs) against vegetative cells and spores of Gram-positive Bacillus cereus. The minimum inhibitory concentration and minimum bactericidal concentration of MELs was 1.25 mg/mL and 2.50 mg/mL, respectively. The growth curve assay and survival rate also showed that MELs had great antibacterial effect. The analysis data of intracellular constituent leakage and cell viability demonstrated MELs damaged the integrity of cell membrane. By scanning electron microscope and transmission electron microscope, the treated bacterial cells exhibited apparent morphological and ultrastructural changes, further affirming the disruption of cell membrane and the unbalance of cellular environments. Furthermore, MELs has showed good ability to inhibit the germination of B. cereus spores. The findings suggested that MELs acted its antibacterial effect primarily by means of membrane disruption, and indicating it will be alternatives to antibacterial agents and preservatives applied into food processing.

Several chitosan sodium tripolyphosphate (TPP) nanoparticles embedded with Torreya grandis aril essential oils (TEOs) were synthesized using an emulsion-ionic gelation technique. Mannosylerythritol lipid A (MEL-A), a type of biosurfactant, was selected as the emulsifier. In order to replace acetic acid, an ionic liquid (IL) was employed to dissolve chitosan. The physical properties, diameters, morphology, embedding rate, and antibacterial effects of those essential oil loaded chitosan (CS) nanoparticles were characterized. The results demonstrated that chitosan nanoparticles can be successfully prepared in an ionic liquid containing system and the diameters for nanoparticles in acetic acid and ionic liquid solutions are 144.1 ± 1.457 and 219.0 ± 4.045 nm. After loading with essential oils, the size increased to 349.6 ± 10.55 and 542.9 ± 16.74 nm, respectively. Antibacterial properties were investigated by the observation of the inhibition zone against S. aureus. The results revealed that TEO loaded nanoparticles synthesized in acid and IL aqueous systems have stronger antibacterial activities than CS nanoparticles.

The integrated mechanisms of dynamic signaling of sodium channels involved in clinical pain are still not yet clear. In this study, a new rat inflammatory pain model was developed by using the unilateral intraplantar injection of BmK I, a receptor site 3-specific modulator of sodium channels from the venom of scorpion Buthus martensi Karsch (BmK). It was found that BmK I could induce several kinds of inflammatory pain-related behaviors including spontaneous pain companied with unique episodic paroxysms, primary thermal hypersensitivity, and mirror-image mechanical hypersensitivity with different time course of development, which could be suppressed by morphine, indomethacin, or bupivacaine to a different extent. The dramatic attenuation by pretreatment with resiniferatoxin (RTX), an ultrapotent analog of capsaicin, on BmK I-induced pain-related behaviors, paw edema, and spinal L4-L5 c-Fos expression demonstrated that capsaicin-sensitive primary afferent neurons played important roles in pain induced by BmK I. Furthermore, the electrophysiological recordings showed that BmK I persistently increased whole-cell and tetrodotoxin-resistant (TTX-R) peak sodium currents and significantly delayed the inactivation phase of whole-cell sodium currents but could not enhance capsaicin-evoked inward currents, in acute isolated small dorsal root ganglion neurons of rat. The results strongly suggested that the dynamic modulation of BmK I on sodium channels located in peripheral primary afferent neurons, especially in capsaicin-sensitive neurons, mediated pain sensation. Thus, BmK I may be a valuable pharmacological tool to understand the sodium channel-involved pain mechanisms.

We reported the in situ synthesis and use of porous polymer monolith (PPM) columns in an integrated multilayer PDMS/glass microchip for microvalve-assisted on-line microextraction and microchip electrophoresis for the first time. Under the control of PDMS microvalves, the grafting of the microchannel surface and in situ photopolymerization of poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolith in a defined zone were successfully achieved. Different factors including the surface grafting, polymerization time, PDMS elastic properties (ratio of oligomer/curing reagent) and UV intensity that affect the monolith synthesis in the PDMS microchannel were investigated and optimized. Dopamine, a model analyte, has been online microextracted, eluted, electrophoresized and electrochemically detected in the microchip, with a mean concentration enrichment factor of 80 (n=3). The results demonstrated that the PPM could be synthesized successfully in the PDMS microchip with a homogeneous structure and excellent mechanical properties. Furthermore, owing to the intrinsic character using PDMS in large-scale integrated microsystems, the implantation of PPM pretreatment units in PDMS microchips would make it possible to deal with complicated analytical processes in a high-throughput way.

The three-dimensional structure of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of spider Selenocosmia huwena with a unique disulfide bond linkage as I-III, II-V, and IV-VI, has been determined using 2D (1)H-NMR. The resulting structure of HWTX-II contains two beta-turns (C4-S7 and K24-W27) and a double-stranded antiparallel beta-sheet (W27-C29 and C34-K36). Although the C-terminal double-stranded beta-sheet cross-linked by two disulfide bonds (II-V and IV-VI in HWTX-II, II-V and III-VI in the ICK molecules) is conserved both in HWTX-II and the ICK molecules, the structure of HWTX-II is unexpected absence of the cystine knot because of its unique disulfide linkage. It suggests that HWTX-II adopts a novel scaffold different from the ICK motif that is adopted by all other spider toxin structures elucidated thus far. Furthermore, the structure of HWTX-II, which conforms to the disulfide-directed beta-hairpin (DDH) motif, not only supports the hypothesis that the ICK is a minor elaboration of the more ancestral DDH motif but also suggests that HWTX-II may have evolved from the same structural ancestor.

Martentoxin as a 37-residue peptide was capable of blocking large-conductance Ca(2+)-activated K(+) (BK) channels in adrenal medulla chromaffin cells. This study investigated the pharmacological discrimination of martentoxin on BK channel subtypes. The results showed that the iberiotoxin-insensitive neuronal BK channels (alpha+beta4) could be potently blocked by martentoxin (IC(50) = approximately 80 nM). In contrast, the iberiotoxin-sensitive BK channel consisting of only alpha-subunit was less sensitive to martentoxin. Distinctively, martentoxin inhibited neuronal BK channels (alpha+beta4) with a novel interaction mode. Two possible interaction sites of neuronal BK channels (alpha+beta4) might be responsible for the binding with martentoxin: one for trapping and the other located at the pore region for blocking. In addition, the inhibition of martentoxin on neuronal BK channels (alpha+beta4) depended on cytoplasmic Ca(2+) concentration. On the other hand, in vivo experiments from EEG recordings suggested that neuronal BK channels (alpha+beta4) were the primary target of martentoxin. Therefore, this research not only sheds light on a unique ligand for neuronal BK channels (alpha+beta4), but also highlights a novel model approach for the interaction between K(+) channels and specific-ligands.

An effectual method is presented to determine the profiles of a tungsten (W) layer, such as the density, the thickness and the roughness in the multilayer structures, using the x-ray reflectivity technique. To avoid oxidation effects of tungsten, a B4C capping layer is deposited onto to the W layer. To observe the profiles of the tungsten layer with different thicknesses, three groups of W/B4C bilayers with different thicknesses are prepared by using ultra high vacuum dc magnetron sputtering and measured by an x-ray diffractometer. A type of genetic algorithm called the differential evolution is used to simulate the measurement data so as to obtain the parameters of bilayers. According to the simulation, it is shown that the W layer density varies from 95.26% to 97.51% compared to the bulk. In our experiment, the deposition rate is 0.044 nm/s, and the thickness is varied in the range of 9.8–19.4 nm.

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called Curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we first determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS we observed an interesting structural rearrangement that occurs during the oligomerization of CsgE.

Valproic acid is a well-tolerated anticonvulsant that has been identified recently as a histone deacetylase inhibitor. To evaluate the antitumor efficacy and mechanisms of action of valproic acid in medulloblastoma and supratentorial primitive neuroectodermal tumor (sPNET), which are among the most common malignant brain tumors in children with poor prognosis, two medulloblastoma (DAOY and D283-MED) and one sPNET (PFSK) cell lines were treated with valproic acid and evaluated with a panel of in vitro and in vivo assays. Our results showed that valproic acid, at clinically safe concentrations (0.6 and 1 mmol/L), induced potent growth inhibition, cell cycle arrest, apoptosis, senescence, and differentiation and suppressed colony-forming efficiency and tumorigenicity in a time- and dose-dependent manner. The medulloblastoma cell lines were more responsive than the sPNET cell line and can be induced to irreversible suppression of proliferation and significantly reduced tumorigenicity by 0.6 and 1 mmol/L valproic acid. Daily i.p. injection of valproic acid (400 mg/kg) for 28 days significantly inhibited the in vivo growth of DAOY and D283-MED s.c. xenografts in severe combined immunodeficient mice. With Western hybridization and real-time reverse transcription-PCR, we further showed that the antitumor activities of valproic acid correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, and suppression of TP53, CDK4, and CMYC expression. In conclusion, valproic acid possesses potent in vitro and in vivo antimedulloblastoma activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, valproic acid may represent a novel therapeutic option in medulloblastoma treatment. [Mol Cancer Ther 2005;4(12):1912–22]

Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous (beta/alpha)8-barrel enzyme crucial for purine metabolism and normal immune competence. In this study, it was observed that loss of enzyme activity of murine ADA (mADA) precedes the global secondary and tertiary structure transition when the protein is exposed to denaturant. The structural mechanism for this phenomenon was probed using site-specific 19F NMR spectroscopy in combination with [6-19F]tryptophan labeling and inhibitor binding. There are four tryptophan residues in mADA and all are located more than 12 A from the catalytic site. The 19F NMR spectra of [6-19F]Trp-labelled mADA show that the urea-induced chemical shift change of 19F resonance of W161, one of the four tryptophan 19F nuclei, correlates with the loss of enzyme activity. The urea-induced chemical shift change of another 19F resonance of W117 correlates with the change of the apparent rate constant for the binding of transition-state analogue inhibitor deoxycoformycin to the enzyme. On the other hand, the chemical environment of the local region around W264 does not change significantly, as a consequence of perturbation by low concentrations of urea or substrate analog. The results indicate that different regions of mADA have different local stability, which controls the activity and stability of the enzyme. The results provide new insights into the relationship between the function of a protein and its conformational flexibility as well as its global stability. This study illustrates the advantage of 19F NMR spectroscopy in probing site-related conformational change information in ligand binding, enzymatic activity and protein folding.

We have isolated a highly potent neurotoxin from the venom of the Chinese bird spider, Selenocosmia huwena. This 4.1-kDa toxin, which has been named huwentoxin-IV, contains 35 residues with three disulfide bridges: Cys-2-Cys-17, Cys-9-Cys-24, and Cys-16-Cys-31, assigned by a chemical strategy including partial reduction of the toxin and sequence analysis of the modified intermediates. It specifically inhibits the neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel with the IC(50) value of 30 nm in adult rat dorsal root ganglion neurons, while having no significant effect on the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel. This toxin seems to be a site I toxin affecting the sodium channel through a mechanism quite similar to that of TTX: it suppresses the peak sodium current without altering the activation or inactivation kinetics. The three-dimensional structure of huwentoxin-IV has been determined by two-dimensional (1)H NMR combined with distant geometry and simulated annealing calculation by using 527 nuclear Overhauser effect constraints and 14 dihedral constraints. The resulting structure is composed of a double-stranded antiparallel beta-sheet (Leu-22-Ser-25 and Trp-30-Tyr-33) and four turns (Glu-4-Lys-7, Pro-11-Asp-14, Lys-18-Lys-21 and Arg-26-Arg-29) and belongs to the inhibitor cystine knot structural family. After comparison with other toxins purified from the same species, we are convinced that the positively charged residues of loop IV (residues 25-29), especially residue Arg-26, must be crucial to its binding to the neuronal tetrodotoxin-sensitive voltage-gated sodium channel.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DSer), can be observed in the Cys cycles during Edman degradation of native HWTXII. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4‐Cys18, Cys8‐Cys29 and Cys23‐ Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n 2 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.

Three different types of isolated nerve–synapse preparations, guinea pig ileum, rat vas deferens and toad heart, were used to investigate the physiological activity of Huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena. The twitch response of isolated guinea pig ileum induced by electrical stimulus can be inhibited by HWTX-I. After blockage, contraction of the ileum can be induced by exogenously applied acetylcholine. HWTX-I caused the inhibition of the twitch response to electrical nerve stimulation in the rat vas deferens. After the twitch was completely inhibited, noradrenaline triggered rhythmic contraction of the vas deferens. The inhibitory effect on heart of toad induced by stimulating sympathetic-vagus nerve can be reversed by HWTX-I, although exogenously applied acetylcholine still acts as an effective inhibitor. All of these results support the conclusion that HWTX-I has the presynaptic activity that effects the release of neurotransmitter from the nerve endings of both the cholinergic synapse and the adrenergic synapse.

Huwentoxin-I, a neurotoxic peptide with 33 amino acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/ denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4 degrees C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.

A neurotoxic peptide, huwentoxin-II (HWTX-II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED50 of 127 +/- 54 microg/g. HWTX-II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin-I. The complete amino sequence of HWTX-II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/Gln) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX-II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.

Ni films are deposited by using ultra high vacuum dc magnetron sputtering onto silicon substrates at room temperature, and the high-quality and high-density films are prepared. The parameters, such as thickness, density and surface roughness, are obtained by using small-angle x-ray diffraction (XRD) analyses with the Marquardt gradient-expansion algorithm. The deposition rate is calculated and the Ni single layer can be fabricated precisely. Based on the fitting results, we can find that the surface roughness of the Ni films is about 0.7 nm, the densities of Ni films are around 97% and the deposition rate is 0.26 nm/s. The roughness of the surface is also characterized by using an atomic force microscope (AFM). The changing trend of the surface roughness in the simulation of XRD is in good agreement with the AFM measurement.

In this work, we demonstrate the immunocapture and on-line fluorescence immunoassay of protein and virus based on porous polymer monoliths (PPM) in microfluidic devices. Poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)] monoliths were successfully synthesized in the polydimethylsiloxane (PDMS) microfluidic channels by in situ UV-initiated free radical polymerization. After surface modification, PPM provides a high-surface area and specific affinity 3D substrate for immunoassays. Combining with well controlled microfluidic devices, the direct immunoassay of IgG and sandwich immunoassay of inactivated H1N1 influenza virus using 5 μL sample has been accomplished, with detection limits of 4 ng mL(-1) and less than 10 pg mL(-1), respectively. The enhanced detection sensitivity is due to both high surface area of PPM and flow-through design. The detection time was obviously decreased mainly due to the shortened diffusion distance and improved convective mass transfer inside the monolith, which accelerates the reaction kinetics between antigen and antibody. This work provides a novel microfluidic immunoassay platform with high efficiency thereby enabling fast and sensitive immunoassay.

Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS) substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF). For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05). Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection.

Highly sensitive detection of proteins offers the possibility of early and rapid diagnosis of various diseases. Microchip-based immunoassay integrates the benefits from both immunoassays (high specificity of target sample) and microfluidics (fast analysis and low sample consumption). However, direct capture of proteins on bare microchannel surface suffers from low sensitivity due to the low capacity of microsystem. In this study, we demonstrated a microchip-based heterogeneous immunoassay using functionalized SiO(2) nanoparticles which were covalently assembled on the surface of microchannels via a liquid-phase deposition technique. The formation of covalent bonds between SiO(2) nanoparticles and polydimethylsiloxane substrate offered sufficient stability of the microfluidic surface, and furthermore, substantially enhanced the protein capturing capability, mainly due to the increased surface-area-to-volume ratio. IgG antigen and FITC-labeled anti-IgG antibody conjugates were adopted to compare protein-enrichment effect, and the fluorescence signals were increased by ~75-fold after introduction of functionalized SiO(2) nanoparticles film. Finally, a proof-of-concept experiment was performed by highly efficient capture and detection of inactivated H1N1 influenza virus using a microfluidic chip comprising highly ordered SiO(2) nanoparticles coated micropillars array. The detection limit of H1N1 virus antigen was 0.5 ng mL(-1), with a linear range from 20 to 1,000 ng mL(-1) and mean coefficient of variance of 4.71%.

C13H9Cl2N3O2, monoclinic, P121/c1 (no. 14), a = 8.0607(1) A, b = 10.7551(2) A, c = 15.6323(2) A, ' = 90.894(1)°, V = 1355.1 A, Z = 4, Rgt(F) = 0.034, wRref(F) = 0.094, T = 296 K. Source of material Pyridine-4-carboxylic acid hydrazide (1 mmol, 0.137 g) was dissolved in anhydrous ethanol (8 ml), the mixture was stirred for several minitutes at room temperature, 3,5-dichloro-2-hydroxybenzaldehyde (1 mmol, 0.191 g) in ethanol (4 ml) was added dropwise, and themixturewas stirred at refluxing temperature for 2 h. The product was separated and recrystallised in ethanol, brown single crystals of the title compound were obtained after 3 d. Experimental details H1 bound to O1was located from the difference Fourier map and refined freely. The other hydrogen atoms were positioned geometrically and allowed to ride on their parent atomswith d(C—H) = 0.93 A, d(N—H) = 0.86 A and Uiso(H) = 1.2 Ueq(C,N). Discussion Hydrazones derived from the reactions of aldehyde and hydrazides show a great deal of interest due to their potential biological properties [1,2]. Untill now, there are only a few examples of hydrazones which have been synthesized and structurallycharacterized [3,4]. The asymmetric unit of the title crystal structure is build up of one molecule, which is not planar. The pyridine ring and the benzene ring form a dihedral angle of 10.26(7)°. The C7/N1/N2/C8 group is slightly twisted with respect to the benzene ring, making a dihedral angle of 2.43(7)°. An intramolecular O–H···N interation is observed involving the hydroxylmoiety and the nitrogen atom. In the crystal, the intermolecular N–H···N hydrogen bonds help to stablize the molecular structure. Z. Kristallogr. NCS 226 (2011) 63-64 / DOI 10.1524/ncrs.2011.0031 63 © by Oldenbourg Wissenschaftsverlag, Munchen Crystal: brown block, size 0.18 × 0.21 × 0.24 mm Wavelength: Mo Kα radiation (0.71073 A) +: 4.83 cm−1 Diffractometer, scan mode: Bruker SMART CCD, $ 2)max: 55.26° N(hkl)measured, N(hkl)unique: 10988, 3131 Criterion for Iobs, N(hkl)gt: Iobs > 2 &(Iobs), 2580 N(param)refined: 186 Programs: SHELXS-97, SHELXL-97, SHELXTL [5] Table 1. Data collection and handling. H(2A) 4e −0.3468 1.0497 −0.1359 0.050 H(7A) 4e −0.1766 0.8885 −0.0935 0.049 H(5A) 4e 0.0021 0.7170 −0.0443 0.048 H(3A) 4e 0.1049 0.6613 0.2043 0.053 H(10A) 4e −0.6195 1.3748 −0.0539 0.057 H(11A) 4e −0.7192 1.5045 −0.1581 0.065 H(13A) 4e −0.4116 1.1692 −0.2359 0.061 H(12A) 4e −0.5130 1.3081 −0.3349 0.067 H(1A) 4e −0.235(3) 1.017(2) 0.098(2) 0.076(7) Table 2. Atomic coordinates and displacement parameters (in A). Atom Site x y z Uiso

Adenosine deaminase (ADA) is a key enzyme in purine metabolism and crucial for normal immune competence. It is a 40 kDa monomeric TIM-barrel protein containing a tightly bound Zn(2+), which is required for activity. In this study, we have investigated the role of Zn(2+) with respect to ADA structure and stability. After removing Zn(2+), the crystallographic structure of the protein remains highly ordered and similar to that of the holo protein with structural changes limited to regions capping the active site pocket. The stability of the protein, however, is decreased significantly in the absence of Zn(2+). Denaturation with urea shows the midpoint to be about 3.5 M for the apo enzyme, compared with 6.4 M for the holo enzyme. ADA contains four tryptophan residues distant from the Zn(2+) site. (19)F NMR studies in the presence and absence of Zn(2+) were carried out after incorporation of 6-(19)F-tryptophan. Chemical shift differences were observed for three of the four tryptophan residues, suggesting that, in contrast to the X-ray data, Zn(2+)-induced structural changes are propagated throughout the protein. Changes throughout the structure as suggested by the NMR data may explain the lower stability of the Zn(2+)-free protein. Real-time (19)F NMR spectroscopy measuring the loss of Zn(2+) showed that structural changes correlated with the loss of enzymatic activity.