Aptamer regulated peroxidase-like activity of cobalt oxyhydroxide nanosheets for colorimetric detection of kanamycin.

A straightforward label-free colorimetric aptasensor utilizing the aptamer-enhanced peroxidase-like activity of cobalt oxyhydroxide (CoOOH) nanosheets has been established for kanamycin detection. In the kanamycin-free state, aptamers adsorb onto the CoOOH surface through electrostatic forces, enhancing the peroxidase-like activity of CoOOH and thereby resulting in a strong absorption signal and a yellow hue in 3,3',5,5'-tetramethylbenzidine (TMB) upon termination of the reaction with a stop solution. Conversely, upon the introduction of kanamycin, aptamers and CoOOH nanosheets compete for binding to kanamycin, resulting in a significant decrease in the number of aptamers bound to CoOOH. As a result, the activity of CoOOH diminishes, leading to a corresponding reduction in coloration and absorbance of the solution. Hence, the quantitative determination of kanamycin could be realized by analyzing the absorbance variations. Under optimal conditions, the aptasensor demonstrated high sensitivity and specificity, with a linear detection range from 500 nM to 5 µM and a detection limit as low as 54.6 nM. Moreover, the aptasensor effectively identified kanamycin in river water samples, achieving a recovery rate between 91.7% and 102.1%. This approach offers good practicability and provides a novel platform for kanamycin detection in environmental samples.

A novel vessel segmentation algorithm for pathological en-face images based on matched filter.

The vascular information in fundus images can provide important basis for detection and prediction of retina-related diseases. However, the presence of lesions such as Coroidal Neovascularization can seriously interfere with normal vascular areas in optical coherence tomography (OCT) fundus images. In this paper, a novel method is proposed for detecting blood vessels in pathological OCT fundus images. First of all, an automatic localization and filling method is used in preprocessing step to reduce pathological interference. Afterwards, in terms of vessel extraction, a pore ablation method based on capillary bundle model is applied. The ablation method processes the image after matched filter feature extraction, which can eliminate the interference caused by diseased blood vessels to a great extent. At the end of the proposed method, morphological operations are used to obtain the main vascular features. Experimental results on the dataset show that the proposed method achieves 0.88 ± 0.03, 0.79 ± 0.05, 0.66 ± 0.04, results in DICE, PRECISION and TPR, respectively. Effective extraction of vascular information from OCT fundus images is of great significance for the diagnosis and treatment of retinal related diseases.

A label-free aptasensor for clenbuterol detection based on fluorescence resonance energy transfer between graphene oxide and rhodamine B.

A novel label-free aptasensor for the specific detection of clenbuterol was developed through the fluorescence resonance energy transfer (FRET) mechanism by using an aptamer as the target recognition element, rhodamine B (RhoB) as the fluorescence probe and graphene oxide (GO) as the fluorescence quencher. In the absence of clenbuterol, the aptamer was adsorbed on the surface of GO, preventing the interaction between RhoB and GO, and a high fluorescence signal was obtained. In the presence of clenbuterol, the aptamer specially bound to clenbuterol with a high affinity and detached from the surface of GO, while positively charged rhodamine B could be electrostatically adsorbed onto the surface of GO, thus quenching the fluorescence. By comparing the fluorescence intensity before and after the addition of clenbuterol, a simple and fast fluorescence assay for clenbuterol was established with a detection range of 100-700 nM and a detection limit of 9.6 nM. Moreover, the proposed method was successfully applied in the determination of clenbuterol in pork samples with recoveries in the range of 96.75-104.91% and a relative standard deviation of less than 5.67%. Because of its easy operation, fast response, low cost and competitive analytical performance, this method is a promising candidate for the detection of clenbuterol and can be extended to the detection of other targets by changing the corresponding aptamers.

Recognition Method for Broiler Sound Signals Based on Multi-Domain Sound Features and Classification Model.

In view of the limited number of extracted sound features, the lack of in-depth analysis of applicable sound features, and the lack of in-depth study of the selection basis and optimization process of classification models in the existing broiler sound classification or recognition research, the author proposes a recognition method for broiler sound signals based on multi-domain sound features and classification models. The implementation process is divided into the training stage and the testing stage. In the training stage, the experimental area is built, and multiple segments of broiler sound signals are collected and filtered. Through sub-frame processing and endpoint detection, the combinations of start frames and end frames of multiple sound types in broiler sound signals are obtained. A total of sixty sound features from four aspects of time domain, frequency domain, Mel-Frequency Cepstral Coefficients (MFCC), and sparse representation are extracted from each frame signal to form multiple feature vectors. These feature vectors are labeled manually to build the data set. The min-max standardization method is used to process the data set, and the random forest is used to calculate the importance of sound features. Then, thirty sound features that contribute more to the classification effect of the classification model are retained. On this basis, the classification models based on seven classification algorithms are trained, the best-performing classification model based on k-Nearest Neighbor (kNN) is obtained, and its inherent parameters are optimized. Then, the optimal classification model is obtained. The test results show that the average classification accuracy achieved by the decision-tree-based classifier (abbreviated as DT classifier) on the data set before and after min-max standardization processing is improved by 0.6%, the average classification accuracy achieved by the DT classifier on the data set before and after feature selection is improved by 3.1%, the average classification accuracy achieved by the kNN-based classification model before and after parameter optimization is improved by 1.2%, and the highest classification accuracy is 94.16%. In the testing stage, for a segment of the broiler sound signal collected in the broiler captivity area, the combinations of the start frames and end frames of multiple sound types in the broiler sound signal are obtained through signal filtering, sub-frame processing, endpoint detection, and other steps. Thirty sound features are extracted from each frame signal to form the data set to be predicted. The optimal classification model is used to predict the labels of each piece of data in the data set to be predicted. By performing majority voting processing on the predicted labels of the data combination corresponding to each sound type, the common labels are obtained; that is, the predicted types are obtained. On this basis, the definition of recognition accuracy for broiler sound signals is proposed. The test results show that the classification accuracy achieved by the optimal classification model on the data set to be predicted is 93.57%, and the recognition accuracy achieved on the multiple segments of the broiler sound signals is 99.12%.

A simple and sensitive AuNPs-based colorimetric aptasensor for specific detection of azlocillin.

A new colorimetric biosensor for specific detection of azlocillin was developed by using DNA aptamer as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric indicator. In the absence of azlocillin, the AuNPs were protected by the aptamer and stabilized at high NaCl concentrations, displaying a red solution. In the presence of azlocillin, the aptamer reacts specifically with azlocillin, resulting in the aggregation of AuNPs and an apparent red to blue color change. The characteristic change can be easily observed by the naked eye and quantitatively detected by an ultraviolet-visible (UV-Vis) spectrometer. Under the optimal conditions, the absorbance variation at 522 nm (ΔA522) of AuNPs changed proportionally with increasing concentration of azlocillin, which exhibited a linear relationship in the concentration range of 50 nM to 500 nM, with a detection limit of 11.6 nM. Furthermore, the aptasensor was successfully used to detect azlocillin in milk and tap water samples, with recoveries ranging from 97.64% to 102.21% and a relative standard deviation (RSD) less than 3.81%.

Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif.

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.

Structure of the GAT domain of the endosomal adapter protein Tom1.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain's association to Tollip's Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.

Tom1 Modulates Binding of Tollip to Phosphatidylinositol 3-Phosphate via a Coupled Folding and Binding Mechanism.

Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking.

Heat shock protein 90 mediates the apoptosis and autophage in nicotinic-mycoepoxydiene-treated HeLa cells.

Heat shock protein 90 (Hsp90) is a fascinating target for cancer therapy due to its significant role in the crossroad of multiple signaling pathways associated with cell proliferation and regulation. Hsp90 inhibitors have the potential to be developed into anti-cancer drugs. Here, we identified nicotinic-mycoepoxydiene (NMD), a structurally novel compound as Hsp90 inhibitor to perform the anti-tumor activity. The compound selectively bound to the Hsp90 N-terminal domain, and degraded the Hsp90 client protein Akt. The degradation of Akt detained Bad in non-phosphorylation form. NMD-associated apoptosis was characterized by the formation of fragmented nuclei, poly(ADP-ribose) polymerase cleavage, cytochrome c release, caspase-3 activation, and the increased proportion of sub-G1 phase cells. Interestingly, the apoptosis was accompanied with autophagy, by exhibiting the increased expression of LC-3 and the decrease of lysosome pH value. Our findings provide a novel cellular mechanism by which Hsp90 inhibitor adjusts cell apoptosis and autophagy in vitro, suggesting that NMD not only has a potential to be developed into a novel anti-tumor pharmaceutical, but also exhibits a new mechanism in regulating cancer cell apoptosis and autophagy via Hsp90 inhibition.

Biophysical and molecular-dynamics studies of phosphatidic acid binding by the Dvl-2 DEP domain.

The Wnt-dependent, ß-catenin-independent pathway modulates cell movement and behavior. A downstream regulator of this signaling pathway is Dishevelled (Dvl), which, among other multiple interactions, binds to the Frizzled receptor and the plasma membrane via phosphatidic acid (PA) in a mechanism proposed to be pH-dependent. While the Dvl DEP domain is central to the ß-catenin-independent Wnt signaling function, the mechanism underlying its physical interaction with the membrane remains elusive. In this report, we elucidate the structural and functional basis of PA association to the Dvl2 DEP domain. Nuclear magnetic resonance, molecular-dynamics simulations, and mutagenesis data indicated that the domain interacted with the phospholipid through the basic helix 3 and a contiguous loop with moderate affinity. The association suggested that PA binding promoted local conformational changes in helix 2 and ß-strand 4, both of which are compromised to maintain a stable hydrophobic core in the DEP domain. We also show that the Dvl2 DEP domain bound PA in a pH-dependent manner in a mechanism that resembles deprotonation of PA. Collectively, our results structurally define the PA-binding properties of the Dvl2 DEP domain, which can be exploited for the investigation of binding mechanisms of other DEP domain-interacting proteins.

A rapid procedure to isolate isotopically labeled peptides for NMR studies: application to the Disabled-2 sulfatide-binding motif.

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [¹5N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at -80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production.

Enteric duplication cyst of the pancreas associated with chronic pancreatitis and pancreatic cancer.

Pancreas-associated enteric duplication cysts are rare developmental anomalies that communicate with the main pancreatic duct and may be associated with recurrent acute and chronic abdominal pain in children. In adults, these lesions may masquerade as pancreatic pseudocysts or pancreatic cystic neoplasms. An adult patient with a pancreas-associated enteric duplication is described which represents the first reported instance of association with both chronic calcific pancreatitis and pancreatic cancer. The clinical spectrum of pancreas-associated enteric duplication cyst, including diagnostic and therapeutic options, is reviewed.

The enigmatic role of sulfatides: new insights into cellular functions and mechanisms of protein recognition.

Sulfatides are sphingolipids commonly found at the surface of most of eukaryotic cells. Sulfatides are not just structural components of the plasma membrane but also participate in a wide range of cellular processes including protein trafficking, cell adhesion and aggregation, axon-myelin interactions, neural plasticity, and immune responses, among others. The intriguing question is how can sulfatides trigger such cellular processes? Their dynamic presence and specific localization at plasma membrane sites may explain their multitasking role. Crystal and NMR structural studies have provided the basis for understanding the mechanism of binding by sulfatide-interacting proteins. These proteins generally exhibit a hydrophobic cavity that is responsible for the interaction with the sulfatide acyl chain, whereas the hydrophilic, negatively charged moiety can be found either buried in the hydrophobic cavity of the protein or exposed for additional intermolecular associations. Since sulfatides vary in their acyl chain composition, which are tissue-dependent, more emphasis on understanding acyl chain specificity by sulfatide-binding proteins is warranted. Importantly, changes in cellular sulfatide levels as well as circulating sulfatides in serum directly impact cardiovascular and cancer disease development and progress. Therefore, sulfatides might prove useful as novel biomarkers. The scope of this review is to overview cell functions and mechanisms of sulfatide recognition to better understand the role of these lipids in health and disease.

Gambogic acid protects from endotoxin shock by suppressing pro-inflammatory factors in vivo and in vitro.

OBJECTIVE: Gambogic acid (GBA) targeted Heat shock protein 90 (Hsp90) and prohibited TNF-α/NF-κB signaling pathway. It can be inferred that the anti-inflammatory activity of GBA results from inhibiting the cytokine production via NF-κB signaling pathway. We used the RAW264.7 cell line and the endotoxin shock mouse model to confirm the hypothesis that GBA protects mice from endotoxin shock by suppressing cytokine synthesis. METHOD: RAW264.7 cells were cultured and the endotoxin shocked mice model was constructed. ELISA was employed to evaluate the change of cytokine secretion levels. The effects of GBA on the activation of NF-κB signaling pathway were also determined by western blot and immune-fluorescent analysis. Cell viability was determined by MTT assay, and the cell migration was tested by wound healing assay. RESULT: Our results demonstrated that GBA significantly inhibited the LPS-induced release of pro-inflammatory factors both in cell lines and mice serum, thereby protecting mice from endotoxin shock. Furthermore, we observed that the reduction of inflammatory cytokines interleukin 1-beta, interleukin 6 and TNF-α resulted from the Hsp90's client protein IKK degradation and the suppression of NF-κB pathway. Moreover, GBA suppressed the migration of LPS-induced RAW264.7 cells. CONCLUSION: Our results indicate that GBA has a potential both as an antitumor and anti-inflammatory therapeutic agent.

Structure, sulfatide binding properties, and inhibition of platelet aggregation by a disabled-2 protein-derived peptide.

Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α(IIb)ß(3) integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.

Guanacastane-type diterpenoids with cytotoxic activity from Coprinus plicatilis.

Four new guanacastane-type diterpenoids (1-4), together with the known compound, guanacastepene E (5), were isolated from a basidiomycete of the macro-fungi, Coprinus plicatilis 82. Their structures were elucidated on the basis of extensive spectroscopic analyses, including FT-ICR-MS, UV, IR and 1D and 2D NMR experiments. The in vitro cytotoxic activities of all compounds against the human cancer cell lines HepG2, HeLa, MDA-MB-231, BGC-823, HCT 116, and U2OS were evaluated, only compound 1 exhibited significant cytotoxicities with IC(50) values ranging from 1.2 to 6.0 µM.

Hsp90 interacts with AMPK and mediates acetyl-CoA carboxylase phosphorylation.

Heat shock protein 90 (Hsp90) serves to stabilise and correctly fold multiple significant client proteins associated with cell proliferation and cell survival. However, little is known about the Hsp90 client proteins that regulate cell metabolism. Here, we describe a unique ability of Hsp90 to regulate the stability and activity of AMP-activated kinase (AMPK), a key sensor of cellular energy status. Hsp90 is found to interact with AMPK and to maintain its AMP-activated kinase activity, which in turn is required for the phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), the key enzyme in fatty acid metabolism. Our binding analysis reveals that both the γ subunit and the α subunit of AMPK bind to Hsp90 with a high affinity. We demonstrate that Hsp90 inhibitors, including geldanamycin (GA) and mycoepoxydiene (MED), can induce the dissociation of AMPK from Hsp90, and cause a significant decrease in phosphorylation of AMPK and ACC. Furthermore, we demonstrate that shRNAs of Hsp90 can efficiently suppress the activation of AMPK. These findings not only establish a novel interaction between Hsp90 and AMPK but also suggest a new mechanism for regulating tumour cell fatty acid metabolism.

The structure and assembly model of the third transmembrane domain of Slc11a1 in SDS micelles revealed by NMR study of the Leu-substituted peptide.

Slc11a1 is an integral membrane protein with 12 putative transmembrane domains (TMDs) and functions as a pH-coupled divalent metal cation transporter. The conservation of three negatively charged residues in the TMD3 of Slc11 protein family implies the important role of this domain in the function of the proteins. However, aggregation of the transmembrane peptide in micelles prevents structural study of the peptide in these membrane-mimetic environments by NMR spectroscopy. Here, we characterized the structure, position, and assembly model of Slc11a1-TMD3 (Lys128-Ile151) in SDS micelles by the NMR study of its Leu-substituted peptide. It was found that the two-site substitutions of Ala for Leu residues at positions 136 and 140 of TMD3 disrupt the aggregation without altering the secondary structure of the peptide. The Leu-substituted peptide folds as an α-helix spanning from Leu133 to Gly144 and embedded in the micelles. A Leu zipper is suggested to account for the self-assembly of the wild-type peptide in SDS micelles.

Folding and assembly of TMD 6-related segments of DMT 1 in trifluoroethanol aqueous solution.

Divalent metal-ion transporter 1 (DMT1) belongs to a large class of metal-ion transporters that drive the translocation of a wide range of divalent metal substrates across membranes toward the cytosol with couple of protons. Two highly conserved histidines in the sixth transmembrane domain (TMD6) are essential for metal transport activity in DMT1. In the present study, we determine the high-resolution structures of three 25-residue peptides, corresponding to TMD6 of the wildtype DMT1 (the segment 255-279) and its H267A and H272A mutants, in 30% TFE-d(2) aqueous solution by the combined use of circular dichroism (CD) and NMR spectroscopies. The wildtype peptide forms an 'α-helix-extended segment-α-helix' structure with two helices spanning over Gly258-Ala262 and Met265-Lys277 linked by a hinge at residues Val263-Ile264. The H267A mutation reduces the hinge to one residue (Ile264), while the H272A mutation extends the flexible region of the central part from Val263 to His267. Diffusion-ordered spectroscopy (DOSY) study demonstrates that all the peptides are self-assembly as trimer in 30% TFE-d(2) aqueous solution. The H272A substitution decreases the intermolecular interaction whereas the H267A substitution may enhance the intermolecular interaction. The specific structure of the discontinuous helix and the self-assembly feature of DMT1-TMD6 may be crucial for its biological function. The changes in conformation and intermolecular interaction induced by histidine substitution may be correlated with the deficiency of DMT1 in metal-ion permeation.

Identification of an "alpha-helix-extended segment-alpha-helix" conformation of the sixth transmembrane domain in DMT1.

DMT1 (divalent metal ion transporter 1) is one member of a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions. A pair of mutation-sensitive and highly conserved histidines in the sixth transmembrane domain (TM6) of DMT1 was found to be important for proton-metal ion cotransport. In the present work, we investigate the structures and locations of the peptides from TM6 of DMT1 and its H267A and H272A mutants in SDS micelles by CD and NMR methods. The circular dichroism studies show that the alpha-helix is a predominant conformation for the wildtype peptide and H267A mutant in SDS micelles, whereas the helicity is evidently decreased for H272A mutant. The pH value has little effect on the alpha-helical contents of the three peptides. The NMR studies indicate that the wildtype peptide in SDS micelles forms an "alpha-helix-extended segment-alpha-helix" structure in which the His267 locates near the central part of the extended segment, while the His272 is involved in the alpha-helical folding. Both histidines are buried in SDS micelles as evidenced by their pK(a) values. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of an alpha-helix from the C-terminus to the central part and continuous turns in the residual part. The extended structure in the central part of the wildtype peptide is abolished by H267A mutation. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. The specific "alpha-helix-extended segment-alpha-helix" structure of TM6 may have an important implication for the binding of the transporter to H(+) and metal ions and the conformation change induced by the mutations of two highly conserved histidines may be correlated to the deficiency of the transport activity of DMT1.