A trajectory outlier detection method based on variational auto-encoder.

Trajectory outlier detection can identify abnormal phenomena from a large number of trajectory data, which is helpful to discover or predict potential traffic risks. In this work, we proposed a trajectory outlier detection model based on variational auto-encoder. First, the model encodes the trajectory data as parameters of distribution functions based on the statistical characteristics of urban traffic. Then, an auto-encoder network is built and trained. The training goal of the auto-encoder network is to maximize the generation probability of original trajectories when decoding. Once the model training is completed, we can detect the trajectory outlier by the difference between a trajectory and the trajectory generated by the model. The advantage of the proposed model is that it only needs to compute the difference between the original trajectory and the trajectory generated by the model when detecting the trajectory outlier, which greatly reduces the amount of calculation and makes the model very suitable for real-time detection scenarios. In addition, the distance threshold between the abnormal trajectory and the normal trajectory can be set by referring to the proportion of the abnormal trajectory in the training data set, which eliminates the difficulty of setting the threshold manually and makes the model more convenient to be applied in different actual scenes. In terms of effect, the proposed model has achieved more than 95% in accuracy, which is better than the two typical density-based and classification-based detection methods, and also better than the methods based on machine learning in recent years. In terms of efficiency, the model has good convergence in the training phase and the training time increases slowly with the data scale, which is better than or as the same as the comparison methods.

Noninvasive prenatal testing for ß-thalassemia by targeted nanopore sequencing combined with relative haplotype dosage (RHDO): a feasibility study.

Noninvasive prenatal testing (NIPT) for single gene disorders remains challenging. One approach that allows for accurate detection of the slight increase of the maternally inherited allele is the relative haplotype dosage (RHDO) analysis, which requires the construction of parental haplotypes. Recently, the nanopore sequencing technologies have become available and may be an ideal tool for direct construction of haplotypes. Here, we explored the feasibility of combining nanopore sequencing with the RHDO analysis in NIPT of ß-thalassemia. Thirteen families at risk for ß-thalassemia were recruited. Targeted region of parental genomic DNA was amplified by long-range PCR of 10 kb and 20 kb amplicons. Parental haplotypes were constructed using nanopore sequencing and next generation sequencing data. Fetal inheritance of parental haplotypes was classified by the RHDO analysis using data from maternal plasma DNA sequencing. Haplotype phasing was achieved in 12 families using data from 10 kb library. While data from the 20 kb library gave a better performance that haplotype phasing was achieved in all 13 families. Fetal status was correctly classified in 12 out of 13 families. Thus, targeted nanopore sequencing combined with the RHDO analysis is feasible to NIPT for ß-thalassemia.

Validation of fetal DNA fraction estimation and its application in noninvasive prenatal testing for aneuploidy detection in multiple pregnancies.

OBJECTIVE: To analyze the fetal fraction, fetal sex, and chromosomal aneuploidy in multiple pregnancies using noninvasive prenatal testing (NIPT). METHOD: A total of 362 pregnant women including 203 singleton pregnancies, 69 twins, and 90 higher-order multiple pregnancies were recruited. Fetal fractions estimated by size ratio-based and Y chromosome-based approaches in singleton pregnancies with male fetus were used as source data to establish the model. The model was then applied to multiple pregnancies for fetal fraction estimation. By comparing the fetal fractions estimated by size ratio to those estimated by Y chromosome or autosomal chromosomes, fetal sex and chromosomal aneuploidy can be analyzed. RESULTS: The size ratio-based approach has been well established in estimating fetal fractions for twin and higher-order multiple pregnancies. Fetal fraction had a positive correlation with gestational age in twin and triplet pregnancies. Fetal sex was determined with accuracies of 98.6% (95% CI, 92.19%-99.96%) in twins and 97.6% (95% CI, 91.76%-99.71%) in triplet pregnancies. Four trisomy 21, one trisomy 18, and one trisomy 13 cases were detected by NIPT. Two trisomy 21 singleton pregnancies and one trisomy 21 twin pregnancy were confirmed by karyotyping. CONCLUSION: Fetal sex and chromosomal aneuploidy in multiple pregnancies can be determined using NIPT.

Roux-en-Y gastric bypass enhances insulin secretion in type 2 diabetes via FXR-mediated TRPA1 expression.

OBJECTIVE: Roux-en-Y gastric bypass surgery (RYGB) improves the first phase of glucose-stimulated insulin secretion (GSIS) in patients with type 2 diabetes. How it does so remains unclear. Farnesoid X receptor (FXR), the nuclear receptor of bile acids (BAs), is implicated in bariatric surgery. Moreover, the transient receptor potential ankyrin 1 (TRPA1) channel is expressed in pancreatic ß-cells and involved in insulin secretion. We aimed to explore the role of BAs/FXR and TRPA1 in improved GSIS in diabetic rats after RYGB. METHODS: RYGB or sham surgery was conducted in spontaneous diabetic Goto-Kakizaki (GK) rats, or FXR or TRPA1 transgenic mice. Gene and protein expression of islets were assessed by qPCR and western blotting. Electrophysiological properties of single ß-cells were studied using patch-clamp technique. Binding of FXR and histone acetyltransferase steroid receptor coactivator-1 (SRC1) to the TRPA1 promoter, acetylated histone H3 (ACH3) levels at the TRPA1 promoter were determined using ChIP assays. GSIS was measured using enzyme-linked immunosorbent assays or intravenous glucose tolerance test (IVGTT). RESULTS: RYGB increases GSIS, particularly the first-phase of GSIS in both intact islets and GK rats in vivo, and ameliorates hyperglycemia of GK rats. Importantly, the effects of RYGB were attenuated in TRPA1-deficient mice. Moreover, GK ß-cells displayed significantly decreased TRPA1 expression and current. Patch-clamp recording revealed that TRPA1-/- ß-cells displayed a marked hyperpolarization and decreased glucose-evoked action potential firing, which was associated with impaired GSIS. RYGB restored TRPA1 expression and current in GK ß-cells. This was accompanied by improved glucose-evoked electrical activity and insulin secretion. Additionally, RYGB-induced TRPA1 expression involved BAs/FXR-mediated recruitment of SRC1, promoting ACH3 at the promoter of TRPA1. CONCLUSIONS: The BAs/FXR/SRC1 axis-mediated restoration of TRPA1 expression plays a critical role in the enhanced GSIS and remission of diabetes in GK rats after RYGB.

GLP-1 signaling suppresses menin's transcriptional block by phosphorylation in ß cells.

Both menin and glucagon-like peptide 1 (GLP-1) pathways play central yet opposing role in regulating ß cell function, with menin suppressing, and GLP-1 promoting, ß cell function. However, little is known as to whether or how GLP-1 pathway represses menin function. Here, we show that GLP-1 signaling-activated protein kinase A (PKA) directly phosphorylates menin at the serine 487 residue, relieving menin-mediated suppression of insulin expression and cell proliferation. Mechanistically, Ser487-phosphorylated menin gains increased binding affinity to nuclear actin/myosin IIa proteins and gets sequestrated from the Ins1 promoter. This event leads to reduced binding of repressive epigenetic histone modifiers suppressor variegation 3-9 homologue protein 1 (SUV39H1) and histone deacetylases 1 (HDAC1) at the locus and subsequently increased Ins1 gene transcription. Ser487 phosphorylation of menin also increases expression of proproliferative cyclin D2 and ß cell proliferation. Our results have uncovered a previously unappreciated physiological link in which GLP-1 signaling suppresses menin function through phosphorylation-triggered and actin/myosin cytoskeletal protein-mediated derepression of gene transcription.

Fabrication of Porous ZnO/Co3O4 Composites for Improving Cycling Stability of Supercapacitors.

To tackle the issue of poor cycling stability for metal oxide nanoparticles as supercapacitor electrode, porous ZnO/Co3O4 composites were fabricated via solid-state thermolysis of [CoZn(BTC)(NO3)](2H2O)(0.5DMF) under air atmosphere. The results demonstrate that the products are mesoporous polyhedron structure with the diameter of about 10 µm, which are constructed by many interconnected nanocrystals with the sizes of around 20 nm. ZnO/Co3O4 composites as supercapacitor electrode exhibited excellent cyclic stability capacity, showing a maximum specific capacitance of 106.7 F g-1 and a capacity retention of 102.7 F · g-1 after 1000 cycles at 0.5 A · g-1. The superior electrochemical performance was contributed to ZnO/Co3O4 composites with porous structures and small size, which shortened the route of electronic transmission as well as ions insertion and desertion processes. Additionally, the synergetic effect of bimetallic oxides improved the electrochemical stability.

Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg2+/ADP complex in the overall GCL structure. L51P mutant spread distortion on the ß-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a ß-sheet. The results confirm that introduction of proline located at the middle of the ß-sheet or at the N- or C-terminal between α-helix and ß-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus.

Clustered regularly interspaced short palindromic repeats, associated proteins (CRISPR/Cas), has been developed into a powerful, targeted genome-editing tool in a wide variety of species. Here, we report an extensive investigation of the type II CRISPR/Cas9 system for targeted gene editing in Streptomyces rimosus. S. rimosus is used in the production of the antibiotic oxytetracycline, and its genome differs greatly from other species of the genus Streptomyces in the conserved chromosome terminal and core regions, which is of major production and scientific research value. The genes zwf2 and devB were chosen as target genes, and were edited separately via single-site mutations, double-site mutations and gene fragment disruptions. The single-site mutation guided by sgRNA-1 or sgRNA-2, respectively, involved GG changing to CA, GC changing to AT, and GG changing to CC. The double-site mutations guided by sgRNA-1 and sgRNA-2 included deletions and/or point mutations. Consistently, all mutations occurred in the gRNA sequence regions. Deletion mutations were characterized by the absence of eight bases, including three bases upstream of the PAM (protospacer adjacent motif) sequence, the PAM sequence itself and two bases downstream of the PAM sequence. A mutant (zwf2-devB-) with a high yield of oxytetracycline was successfully obtained, whose oxytetracycline level was increased by 36.8 % compared to the original strain. These results confirm that CRISPR/Cas9 can successfully serve as a useful targeted genome editing system in S. rimosus.

Squamosamide Derivative FLZ Protects Pancreatic ß-Cells from Glucotoxicity by Stimulating Akt-FOXO1 Pathway.

Chronic hyperglycemia increases apoptosis and reduces glucose-stimulated insulin secretion. Although protective agents have been searched extensively, none has been found so far. Here we tested FLZ, a synthetic derivative of squamosamide from a Chinese herb, as a potential candidate for antiglucotoxicity in INS-1E cells and mouse islets. Chronic culture of ß-cells in 30 mM glucose caused progressive reduction of cell viability, accompanied with increased apoptosis and reduced insulin secretion. These effects on apoptosis and insulin were reversed by FLZ in a dose-dependent manner. FLZ treatment also increased forkhead box O1 protein phosphorylation and reduced its nuclear location. On the contrary, FLZ increased pancreatic and duodenal homeobox-1 expression and its nuclear localization, an effect mediated by increased p-Akt. Consistently, Akt selective inhibitor MK-2206 completely abolished antiglucotoxicity effect of FLZ. Furthermore, FLZ treatment increased cytosolic ATP/ADP ratio. Taken together, our results suggest that FLZ could be a potential therapeutic agent to treat the hyperglycemia-induced ß-cell failure.

Roles of the lateral fenestration residues of the P2X4 receptor that contribute to the channel function and the deactivation effect of ivermectin.

P2X receptors are cation-permeable ion channels gated by extracellular adenosine triphosphate (ATP). Available crystallographic data suggest that ATP-binding ectodomain is connected to the transmembrane pore domain by three structurally conserved linker regions, which additionally frame the lateral fenestrations through which permeating ions enter the channel pore. The role of these linker regions in relaying the conformational change evoked by ATP binding of the ectodomain to the pore-forming transmembrane domain has not been investigated systematically. Using P2X4R as our model, we employed alanine and serine replacement mutagenesis to determine how the side chain structure of these linker regions influences gating. The mutants Y54A/S, F198A/S, and W259A/S all trafficked normally to the plasma membrane of transfected HEK293 cells but were poorly responsive to ATP. Nevertheless, the function of the F198A/S mutants could be recovered by pretreatment with the known positive allosteric modulator of P2X4R, ivermectin (IVM), although the IVM sensitivity of this mutant was significantly impaired relative to wild type. The functional mutants Y195A/S, F200A/S, and F330A/S exhibited ATP sensitivities identical to wild type, consistent with these side chains playing no role in ATP binding. However, Y195A/S, F200A/S, and F330A/S all displayed markedly changed sensitivity to the specific effects of IVM on current deactivation, suggesting that these positions influence allosteric modulation of gating. Taken together, our data indicate that conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain meaningfully contribute to signal transduction and channel gating in P2X receptors.

Vitamin E isomer δ-tocopherol enhances the efficiency of neural stem cell differentiation via L-type calcium channel.

The effects of the vitamin E isomer δ-tocopherol on neural stem cell (NSC) differentiation have not been investigated until now. Here we investigated the effects of δ-tocopherol on NSC neural differentiation, maturation and its possible mechanisms. Neonatal rat NSCs were grown in suspended neurosphere cultures, and were identified by their expression of nestin protein and their capacity for self-renewal. Treatment with a low concentration of δ-tocopherol induced a significant increase in the percentage of ß-III-tubulin-positive cells. δ-Tocopherol also stimulated morphological maturation of neurons in culture. We further observed that δ-tocopherol stimulation increased the expression of voltage-dependent Ca(2+) channels. Moreover, a L-type specific Ca(2+) channel blocker verapamil reduced the percentage of differentiated neurons after δ-tocopherol treatment, and blocked the effects of δ-tocopherol on NSC differentiation into neurons. Together, our study demonstrates that δ-tocopherol may act through elevation of L-type calcium channel activity to increase neuronal differentiation.

Involvement of the left-flipper-to-dorsal-fin interface of the zebrafish P2X4 receptor in ATP binding and structural rearrangement.

P2X receptors are trimeric ATP-activated non-selective cation channels. The ATP binding pocket is positioned between two neighboring subunits. Accompanying ligand binding, subunit-subunit contacts are most likely involved in receptor function and drive a conformational change to open the ion permeation pathway. In this way, we sought to determine the function of side chains of the zebrafish P2X4 receptor ectodomain left-flipper-to-dorsal-fin interface residues in ligand binding. By combining site-directed mutagenesis and electrophysiology methods, we showed that cysteine substitutions of I212, S215, Y216 and L217 resulted in decreased sensitivity to ATP. In addition, the ATP induced current at L217C was completely inhibited by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)), indicating a role for this residue in ATP action. Deletion of residues 285-293 from the zebrafish P2X4 receptor abolished channel function. However, insertion of the same sequence frame into a homologous position of the rat P2X6 receptor did not rescue channel function, suggesting that these residues are necessary but not sufficient for achieving the correct ATP-induced conformation.

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique.

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.