Quantitative Determination of Four Potential Genotoxic Impurities in the Active Pharmaceutical Ingredients in TSD-1 Using UPLC-MS/MS.

A novel method of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed for the identification and quantification of four potential genotoxic impurities (PGIs) in the active pharmaceutical ingredients of TSD-1, a novel P2Y12 receptor antagonist. Four PGIs were named, 4-nitrobenzenesulfonic acid, methyl 4-nitrobenzenesulfonate, ethyl 4-nitrobenzenesulfonate, and isopropyl 4-nitrobenzenesulfonate. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying four PGIs at 15.0 ppm in samples of 0.5 mg/mL concentration. This validated approach presented very low limits (0.1512−0.3897 ng/mL), excellent linearity (coefficients > 0.9900), and a satisfactory recovery range (94.9−115.5%). The method was sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity, and robustness and, thus, has high practicality in the pharmaceutical quality control of TSD-1.

Size Effects of Au/Ni-Coated Polymer Particles on the Electrical Performance of Anisotropic Conductive Adhesive Films under Flexible Mechanical Conditions.

In soft electronics, anisotropic conductive adhesive films (ACFs) are the trending interconnecting approach due to their substantial softness and superior bondability to flexible substrates. However, low bonding pressure (≤1 MPa) and fine-pitch interconnections of ACFs become challenging while being extended in advanced device developments such as wafer-level packaging and three-dimensional multi-layer integrated circuit board assembly. To overcome these difficulties, we studied two types of ACFs with distinct conductive filler sizes (ACF-1: ~20 µm and ACF-2: ~5 µm). We demonstrated a low-pressure thermo-compression bonding technique and investigated the size effect of conductive particles on ACF's mechanical properties in a customized testing device, which consists of flexible printing circuits and Flex on Flex assemblies. A consistency of low interconnection resistance (<1 Ω) after mechanical stress (cycling bending test up to 600 cycles) verifies the assembly's outstanding electrical reliability and mechanical stability and thus validates the great effectiveness of the ACF bonding technique. Additionally, in numerical studies using the finite element method, we developed a generic model to disclose the size effect of Au/Ni-coated polymer fillers in ACF on device reliability under mechanical stress. For the first time, we confirmed that ACFs with smaller filler particles are more prone to coating fracture, leading to deteriorated electrical interconnections, and are more likely to peel off from substrate electrode pads resulting in electrical faults. This study provides guides for ACF design and manufacturing and would facilitate the advancement of soft wearable electronic devices.

A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice.

The axotomy-inducible enzyme Nna1 defines a subfamily of M14 metallocarboxypeptidases, and its mutation underlies the Purkinje cell degeneration (pcd) mouse. However, the relationship among its catalytic activity, substrate specificities, and the critical processes of neurodegeneration/axon regeneration is incompletely understood. Here we used a transgenic rescue strategy targeting expression of modified forms of Nna1 to Purkinje cells in pcd mice to determine structure-activity relationships for neuronal survival and in parallel characterized the enzymatic properties of purified recombinant Nna1. The Nna1 subfamily uniquely shares conserved substrate-determining residues with aspartoacylase that, when mutated, cause Canavan disease. Homologous mutations (D1007E and R1078E) inactivate Nna1 in vivo, as does mutation of its catalytic glutamate (E1094A), which implies that metabolism of acidic substrates is essential for neuronal survival. Consistent with reports that Nna1 is a tubulin glutamylase, recombinant Nna1-but not the catalytic mutants-removes glutamate from tubulin. Recombinant Nna1 metabolizes synthetic substrates with 2 or more C-terminal glutamate (but not aspartate) residues (V(max) for 3 glutamates is ∼7-fold higher than 2 glutamates although K(M) is similar). Catalysis is not ATP/GTP dependent, and mutating the ATP/GTP binding site of Nna1 has no effect in vivo. Nna1 is a monomeric enzyme essential for neuronal survival through hydrolysis of polyglutamate-containing substrates.

Hsa_circ_0008673 Promotes Breast Cancer Progression by MiR-578/GINS4 Axis.

BACKGROUND: Circular RNAs (circRNAs) play a crucial role in breast cancer (BC) development. This study aimed to explore the new potential mechanism of hsa_circ_0008673 in BC. MATERIALS AND METHODS: Hsa_circ_0008673, microRNA-578 (miR-578) and recombinant human GINS complex subunit 4 (GINS4) abundances were measured via quantitative real-time PCR or western blot. Cell proliferation, metastasis, angiogenesis and apoptosis were assessed via EdU assay, transwell assay, tube formation assay, and flow cytometry. The interactions among hsa_circ_0008673, miR-578 and GINS4 were tested via dual-luciferase reporter analysis and RNA pull-down assay. Animal studies were performed to assess the effect of hsa_circ_0008673 on BC tumor growth. RESULTS: Hsa_circ_0008673 level was increased in BC tissues and cells. Hsa_circ_0008673 silencing repressed BC cell growth, metastasis and angiogenesis, as well as hampered BC tumor growth. Hsa_circ_0008673 acted as miR-578 sponge, and miR-578 targeted GINS4. Furthermore, hsa_circ_0008673 modulated GINS4 expression through sponging miR-578. Additionally, miR-578 inhibitor or GINS4 overexpression could reverse the inhibitory effect of hsa_circ_0008673 silencing on BC cell progression. CONCLUSION: Hsa_circ_0008673 might promote BC progression via modulating miR-578/GINS4 pathway.

Learning-Based Rate Control for Video-Based Point Cloud Compression.

Due to limited transmission resources and storage capacity, efficient rate control is important in Video-based Point Cloud Compression (V-PCC). In this paper, we propose a learning-based rate control method to improve the rate-distortion (RD) performance of V-PCC. A low-latency synchronous rate control structure is designed to reduce the overhead of pre-coding. The basic unit (BU) parameters are predicted accurately based on our proposed CNN-LSTM neural network, instead of the online updating approach, which can be inaccurate due to low consistency between adjacent 2D frames in V-PCC. When determining the quantization parameters for the BU, a patch-based clipping method is proposed to avoid unnecessary clipping. This approach is able to improve the RD performance and subjective dynamic point cloud quality. Experiments show that our proposed rate control method outperforms present approaches.

Low-Complexity Error Resilient HEVC Video Coding: A Deep Learning Approach.

Intra/inter switching-based error resilient video coding effectively enhances the robustness of video streaming when transmitting over error-prone networks. But it has a high computation complexity, due to the detailed end-to-end distortion prediction and brute-force search for rate-distortion optimization. In this article, a Low Complexity Mode Switching based Error Resilient Encoding (LC-MSERE) method is proposed to reduce the complexity of the encoder through a deep learning approach. By designing and training multi-scale information fusion-based convolutional neural networks (CNN), intra and inter mode coding unit (CU) partitions can be predicted by the networks rapidly and accurately, instead of using brute-force search and a large number of end-to-end distortion estimations. In the intra CU partition prediction, we propose a spatial multi-scale information fusion based CNN (SMIF-Intra). In this network a shortcut convolution architecture is designed to learn the multi-scale and multi-grained image information, which is correlated with the CU partition. In the inter CU partition, we propose a spatial-temporal multi-scale information fusion-based CNN (STMIF-Inter), in which a two-stream convolution architecture is designed to learn the spatial-temporal image texture and the distortion propagation among frames. With information from the image, and coding and transmission parameters, the networks are able to accurately predict CU partitions for both intra and inter coding tree units (CTUs). Experiments show that our approach significantly reduces computation time for error resilient video encoding with acceptable quality decrement.

The Purkinje cell degeneration (pcd) mouse: an unexpected molecular link between neuronal degeneration and regeneration.

The spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), was first identified through its ataxic behavior. Since its discovery in the 1970s, the strain has undergone extensive investigation, although another quarter century elapsed until the mutant gene (agtpbp1 a.k.a. Nna1) underlying the pcd phenotype was identified. As Nna1 was initially discovered as a gene induced in motor neurons following axotomy the finding that its loss leads to selective neuronal degeneration points to a novel and unexpected common molecular mechanism contributing to the apparently opposing processes of degeneration and regeneration. The elucidation of this mechanism may of course have significant implications for an array of neurological disorders. Here we will first review the principle features of the pcd phenotype and then discuss the functional implications of more recent findings emanating from the characterization of Nna1, the protein that is lost in pcd. We also provide new data on the genetic dissection of the cell death pathways operative in pcd(3J) mice, proving that granule cell death and Purkinje cell death in these mice have distinct molecular bases. We also provide new information on the structure of mouse Nna1 as well as Nna1 protein levels in pcd(3J) mice.

Identification of candidate Purkinje cell-specific markers by gene expression profiling in wild-type and pcd(3J) mice.

The identification of mRNAs that have restricted expression patterns in the brain represents powerful tools with which to characterize and manipulate the nervous system. Here, we describe a strategy using microarray technology (Affymetrix Mouse Genome 430 2.0 Arrays) to identify mRNA transcripts that are candidate markers of cerebellar Purkinje neurons. Initially, gene expression profiles were compared between cerebella of 4-month-old Purkinje cell degeneration (pcd(3J)) mice, in which most Purkinje cells had already degenerated and wild-type littermates with a normal complement of Purkinje neurons. Of 14,563 probe sets expressed in wild-type cerebellum, 797 showed a significant (p<0.0001) reduction in pcd(3J) mice. These probes could represent transcripts with varying levels of specificity for Purkinje cells as well as transcripts in other cell types that decline as a secondary consequence of Purkinje cell loss. Ranking of the probe signals revealed that well-known Purkinje cell-specific transcripts such as calbindin and L7/pcp2 clustered in a group that was <33% of wild-type levels. Therefore, to identify potentially new Purkinje cell-specific transcripts that cluster with the known markers, more stringent selection criteria were applied (<33% of wild-type signal and p<0.0001). With these criteria, 55 independent transcripts were identified of which 33 were annotated genes and 22 were ESTs and RIKEN cDNAs. A literature search revealed that 25 of the 33 annotated genes were expressed in Purkinje cells, with no data being available on the other 8. Thus, the additional 8 annotated and 22 un-annotated genes are clustered with many genes expressed in Purkinje cells making them candidate markers. To confirm the microarray data, eight representative annotated genes were selected including five reported to be in Purkinje neurons and three for which no data was available. Semi-quantitative RT-PCR demonstrated reduced expression of all eight transcripts in cerebella from pcd(3J) mice. The promoters of genes expressed selectively in subsets of neurons can be used to direct heterologous gene expression in transgenic mice and the more restricted the expression pattern the greater their utility. Therefore, microarray analysis was used to assess expression levels of all 55 transcripts in cerebral cortex, striatum, substantia nigra and ventral tegmental area. This permitted the identification of a set of genes whose promoters might have utility for selectively targeting gene expression to cerebellar Purkinje cells.

The carboxypeptidase-like substrate-binding site in Nna1 is essential for the rescue of the Purkinje cell degeneration (pcd) phenotype.

The Purkinje cell degeneration (pcd) phenotype is characterized by adult onset neurodegeneration resulting from mutations in Nna1, a gene encoding an intracellular protein with a putative metallocarboxypeptidase domain. As Nna1 is also induced in axotomized motor neurons, the elucidation of its function can shed light on previously unsuspected mechanisms common to degenerative and regenerative responses. Structural modeling revealed that Nna1 and three related gene products constitute a new subfamily of metallocarboxypeptidases with a distinctive substrate-binding site. To test whether the metallocarboxypeptidase domain is functionally essential, transgenic mice were generated that expressed Nna1 or a substrate-binding site mutant of Nna1 selectively in Purkinje cells using the L7/pcp2 promoter. When bred onto a homozygous pcd(3J) background, wild type but not mutant Nna1 rescued ataxic behavior and Purkinje cell loss. Therefore, loss of Nna1 in Purkinje cells leads directly to their degeneration and Nna1's carboxypeptidase domain is essential for survival of these neurons.