Integration of temporal & spatial properties of dynamic functional connectivity based on two-directional two-dimensional principal component analysis for disease analysis.

Dynamic functional connectivity, derived from resting-state functional magnetic resonance imaging (rs-fMRI), has emerged as a crucial instrument for investigating and supporting the diagnosis of neurological disorders. However, prevalent features of dynamic functional connectivity predominantly capture either temporal or spatial properties, such as mean and global efficiency, neglecting the significant information embedded in the fusion of spatial and temporal attributes. In addition, dynamic functional connectivity suffers from the problem of temporal mismatch, i.e., the functional connectivity of different subjects at the same time point cannot be matched. To address these problems, this article introduces a novel feature extraction framework grounded in two-directional two-dimensional principal component analysis. This framework is designed to extract features that integrate both spatial and temporal properties of dynamic functional connectivity. Additionally, we propose to use Fourier transform to extract temporal-invariance properties contained in dynamic functional connectivity. Experimental findings underscore the superior performance of features extracted by this framework in classification experiments compared to features capturing individual properties.

Multi-classifier fusion based on belief-value for the diagnosis of autism spectrum disorder.

Introduction: Autism Spectrum Disorder (ASD) has a significant impact on the health of patients, and early diagnosis and treatment are essential to improve their quality of life. Machine learning methods, including multi-classifier fusion, have been widely used for disease diagnosis and prediction with remarkable results. However, current multi-classifier fusion methods lack the ability to measure the belief level of different samples and effectively fuse them jointly. Methods: To address these issues, a multi-classifier fusion classification framework based on belief-value for ASD diagnosis is proposed in this paper. The belief-value measures the belief level of different samples based on distance information (the output distance of the classifier) and local density information (the weight of the nearest neighbor samples on the test samples), which is more representative than using a single type of information. Then, the complementary relationships between belief-values are captured via a multilayer perceptron (MLP) network for effective fusion of belief-values. Results: The experimental results demonstrate that the proposed classification framework achieves better performance than a single classifier and confirm that the fusion method used can effectively fuse complementary relationships to achieve accurate diagnosis. Discussion: Furthermore, the effectiveness of our method has only been validated in the diagnosis of ASD. For future work, we plan to extend this method to the diagnosis of other neuropsychiatric disorders.

Transcriptome Profiles of IncRNA and mRNA Highlight the Role of Ferroptosis in Chronic Neuropathic Pain With Memory Impairment.

Background: Chronic neuropathic pain is commonly associated with memory loss, which increases the risk of dementia, lowers life quality and spending. On the other hand, the molecular processes are unknown, and effective therapies have yet to be discovered. Long non-coding RNAs (lncRNAs) are emerging potential therapeutic targets for chronic pain, but their role in chronic pain-induced memory impairment is unknown. Methods: We established a CCI-induced memory impairment rat model. To investigate and validate the gene expression alterations in the hippocampus of CCI-induced memory impairment, we used RNA-Seq, bioinformatics analysis, qRT-PCR, western blot, immunostaining, Nissl staining, and Diaminobenzidine-enhanced Perls' stain. Results: CCI rats displayed long-term memory deficits in the Y maze and novel objective recognition tests, and chronic mechanical and thermal pain hypersensitivity in the hind paws. We found a total of 179 differentially expressed mRNAs (DEmRNAs) (81 downregulated and 98 upregulated) and 191 differentially expressed long noncoding RNAs (DElncRNAs) (87 downregulated and 105 upregulated) between the hippocampus CA1 of CCI-induced memory impairment model and the sham control, using RNA-Seq expression profiles. The most enriched pathways involving oxidation and iron metabolism were explored using a route and function pathway analysis of DEmRNAs and DElncRNAs. We also discovered that ATF3 was considerably overexpressed in the hippocampal CA1 area, and gene markers of ferroptosis, such as GPX4, SLC7A11, SLC1A5, and PTGS2, were dysregulated in the CCI-induced memory impairment paradigm. Furthermore, in the hippocampus CA1 of CCI-induced memory impairment, lipid peroxidation and iron overload were considerably enhanced. Fer-1 treatment reversed ferroptosis damage of CCI with memory impairment model. Finally, in CCI-induced memory impairment, a competing RNA network analysis of DElncRNAs and DEmRNAs was performed to investigate the putative regulatory link of DElncRNAs on DEmRNAs via miRNA sponging. Conclusion: Using RNA-Seq, we created a genome-wide profile of the whole hippocampus of a rat model of CCI-induced memory impairment. In the hippocampus, pathways and function analyses revealed numerous intriguing genes and pathways involved in ferroptosis and memory impairment in response to chronic pain stress. As a result, our research may aid in the identification of potential and effective treatments for CCI-induced memory impairment.

Genetic code degeneracy is established by the decoding center of the ribosome.

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson-Crick base pair at the second position of the anticodon-codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

Nanocasting of fibrous morphology on a substrate for long-term propagation of human induced pluripotent stem cells.

Human-induced pluripotent stem cells (hiPSCs) can be self-renewed for many generations on nanofibrous substrates. Herein, a casting method is developed to replicate the nanofibrous morphology into a thin layer of polymethylsiloxane (PDMS). The template is obtained by electrospinning and chemical crosslinking of gelatin nanofibers on a glass slide. The replicas of the template are surface-functionalized by gelatin and used for propagation of hiPSCs over tenth generations. The performance of the propagated hiPSCs is checked by immunofluorescence imaging, flowcytometry, and RT-PCR, confirming the practicability of this method. The results are also compared to those obtained using electrospun nanofiber substrates. Inherently, the PDMS replica is of low stiffness and can be reproduced easily. Compared to other patterning techniques, casting is more flexible and cost-effective, suggesting that this method might find applications in cell-based assays that rely on stringent consideration of both substrate stiffness and surface morphology.

Activation of neurotrophin signalling with light­inducible receptor tyrosine kinases.

Optogenetics combined with protein engineering based on natural light­sensitive dimerizing proteins has evolved as a powerful strategy to study cellular functions. The present study focused on tropomyosin kinase receptors (Trks) that have been engineered to be light­sensitive. Trk belongs to the superfamily of receptor tyrosine kinases (RTKs), which are single­pass transmembrane receptors that are activated by natural ligands and serve crucial roles in cellular growth, differentiation, metabolism and motility. However, functional variations exist among receptors fused with light­sensitive proteins. The present study proposed a signal transduction model for light­induced receptor activation. This model is based on analysis of previous light­induced Trk receptors reported to date and comparisons to the activation mechanism of natural receptors. In this model, quantitative differences on the dimerization induced from either top­to­bottom or bottom­to­up may lead to the varying amplitude of intracellular signals. We hypothesize that the top­to­bottom propagation is more favourable for activation and yields better results compared with the bottom­to­top direction. The careful delineation of the dimerization mechanisms fine­tuning activation will guide future design for an optimum cellular output with the precision of light.

Lysine-Specific Histone Demethylase 1 Promotes Oncogenesis of the Esophageal Squamous Cell Carcinoma by Upregulating DUSP4.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer (EC) and has a poor prognosis due to its aggressive nature. Accordingly, it is necessary to find novel prognostic biomarkers and therapeutic targets for ESCC. Lysine-specific histone demethylase 1 (LSD1) plays a core role in the regulation of ESCC oncogenesis. However, the detailed mechanism of LSD1-regulated ESCC growth has not been elucidated. This study aims to explore molecular mechanism underlying the LSD1-regulated ESCC's oncogenesis. After LSD1 silencing, we detected differentially expressed genes (DEGs) in human ESCC cell line, TE-1, by transcriptome sequencing. Subsequently, we investigated expression pattern of the selected molecules in the ESCC tissues and cell lines by qRT-PCR and Western blotting. Furthermore, we explored the roles of selected molecules in ESCC using gene silencing and overexpression assays. Transcriptome sequencing showed that the expression of dual specificity phosphatase 4 (DUSP4) in TE-1 was significantly attenuated after the LSD1 silencing. In addition, the DUSP4 mRNA expression level was significantly higher in the ESCC tissues, especially in those derived from patients with invasion or metastasis. Moreover, the DUSP4 expression was positively associated with the LSD1 expression in the ESCC tissues. DUSP4 overexpression promoted proliferation, invasion, and migration of the ESCC cells, while DUSP4 silencing had an opposite effect. DUSP4 overexpression also enhanced tumorigenicity of the ESCC cells in vivo, while DUSP4 silencing inhibited tumor growth. Importantly, inhibition of cell proliferation, invasion, and migration by the LSD1 inhibitor (ZY0511) was reversed by DUSP4 overexpression. Conclusively, we found that LSD1 promotes ESCC's oncogenesis by upregulating DUSP4, the potential therapeutic and diagnostic target in ESCC.

GluN2A and GluN2B NMDA receptors use distinct allosteric routes.

Allostery represents a fundamental mechanism of biological regulation that involves long-range communication between distant protein sites. It also provides a powerful framework for novel therapeutics. NMDA receptors (NMDARs), glutamate-gated ionotropic receptors that play central roles in synapse maturation and plasticity, are prototypical allosteric machines harboring large extracellular N-terminal domains (NTDs) that provide allosteric control of key receptor properties with impact on cognition and behavior. It is commonly thought that GluN2A and GluN2B receptors, the two predominant NMDAR subtypes in the adult brain, share similar allosteric transitions. Here, combining functional and structural interrogation, we reveal that GluN2A and GluN2B receptors utilize different long-distance allosteric mechanisms involving distinct subunit-subunit interfaces and molecular rearrangements. NMDARs have thus evolved multiple levels of subunit-specific allosteric control over their transmembrane ion channel pore. Our results uncover an unsuspected diversity in NMDAR molecular mechanisms with important implications for receptor physiology and precision drug development.

Optoproteomics elucidates the interactome of L-type amino acid transporter 3 (LAT3).

Membrane protein interactions are crucial for diverse biological processes. We report the application of genetic code expansion in combination with photo-crosslinking chemistry, as we termed "optoproteomics", to identify proteins interacting with the human L-type membrane amino acid transporter 3 (LAT3, also known as SLC43A1). The site-specifically incorporated photo-cross-linker p-azido-L-phenylalanine (AzF), which reacts with proteins in their proximity, enabled the capture of weak and transient partners of LAT3 in living cells. We identify 11 unique interacting proteins which are light-sensitive and 19 unique proteins that are site-specific, validating the approach and providing insights into the LAT3 protein-protein interaction network currently unavailable.

Nanodefensin-encased hydrogel with dual bactericidal and pro-regenerative functions for advanced wound therapy.

Background: Host defense peptides (HDPs) have emerged as a novel therapeutic paradigm for wound management; however, their clinical applications remain a challenge owing to their poor pharmacological properties and lack of suitable pharmaceutical formulations. Nanodefensin (ND), a nanoengineered human α-defensin 5 (HD5), has shown improved pharmacological properties relative to the parent compound. In this study, we engineered a nanodefensin-encased hydrogel (NDEFgel), investigated the effects of NDEFgel on wound healing, and elucidated underlying mechanisms. Method: ND was chemically synthesized and tested functions by in vitro antimicrobial and scratch assays and western blotting. Different NDEFgels were evaluated by in vitro characterizations including degradation, drug release and antimicrobial activity. In full-thickness excisional murine models, the optimal NDEFgel was directly applied onto wound sites, and the efficacy was assessed. Moreover, the underlying mechanisms of pro-regenerative effect developed by NDEFgel were also explored. Results: Apart from bactericidal effects, ND modulated fibroblast behaviors by promoting migration and differentiation. Among the tested hydrogels, the Pluronic F127 (Plu) hydrogel represented the most desirable carrier for ND delivery owing to its favorable controlled release and compatibility with ND. Local treatment of NDEFgel on the wound bed resulted in accelerated wound regeneration and attenuated bacterial burden. We further demonstrated that NDEFgel therapy significantly upregulated genes related to collagen deposition and fibroblasts, and increased the expression of myofibroblasts and Rac1. We therefore found that Rac1 is a critical factor in the ND-induced modulation of fibroblast behaviors in vitro through a Rac1-dependent cytoskeletal rearrangement. Conclusion: Our results indicate that NDEFgel may be a promising dual-action therapeutic option for advanced wound management in the future.

Photosensitive tyrosine analogues unravel site-dependent phosphorylation in TrkA initiated MAPK/ERK signaling.

Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; thus, TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. However, mechanistic insights into TrKA signaling are lacking due to lack of site-dependent phosphorylation control. Here we engineer two light-sensitive tyrosine analogues, namely p-azido-L-phenylalanine (AzF) and the caged-tyrosine (ONB), through amber codon suppression to optically manipulate the phosphorylation state of individual intracellular tyrosines in TrkA. We identify TrkA-AzF and ONB mutants, which can activate the ERK pathway in the absence of NGF ligand binding through light control. Our results not only reveal how TrkA site-dependent phosphorylation controls the defined signaling process, but also extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy.

D Amino Acids Highlight the Catalytic Power of the Ribosome.

The possible mechanism(s) by which ribosomes make peptide bonds during protein synthesis have been explored for decades. Yet, there is no agreement on how the catalytic site, the peptidyl transferase center (PTC), promotes this reaction. Here, we discuss the results of recent investigations of translation with D amino acids that provide fresh insights into that longstanding question.

A deafness-associated mitochondrial DNA mutation altered the tRNASer(UCN) metabolism and mitochondrial function.

Mutations in mitochondrial DNA (mtDNA) have been associated with deafness and their pathophysiology remains poorly understood. In this study, we investigated the pathogenic mechanism of deafness-associated 7505A > G variant in the mitochondrial tRNASer(UCN). The m.7505A > G variant affected the highly conserved adenine at position 11 (A11), disrupted the highly conserved A11-U24 base-pairing of DHU stem of tRNASer(UCN) and introduced a tertiary base pairing (G11-C56) with the C56 in the TΨC loop. We therefore hypothesized that the m.7505A > G variant altered both structure and function of tRNASer(UCN). We demonstrated that the m.7505A > G variant perturbed the conformation and stability of tRNASer(UCN), as indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wild type molecule. Using the cybrids constructed by transferring mitochondria from the Chinese family into mitochondrial DNA (mtDNA)-less cells, we demonstrated the m.7505A > G variant led to significantly decreased steady-state levels of tRNASer(UCN) in the mutant cybrids, as compared with those of control cybrids. The aberrant tRNASer(UCN) metabolism resulted in the variable decreases in mtDNA-encoded polypeptides in the mutant cybrids. Furthermore, we demonstrated that the m.7505A > G variant decreased the activities of mitochondrial respiratory complexes I, III and IV, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the mutant cybrids. These results demonstrated that the m.7505A > G variant affected both structure and function of tRNASer(UCN) and consequently altered mitochondrial function. Our findings highlighted critical insights into the pathophysiology of maternally inherited deafness, which is manifested by the aberrant tRNA metabolism.

Control of Protein Activity and Gene Expression by Cyclofen-OH Uncaging.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

Genetic Code Expansion and Optoproteomics.

Nature has invented photoreceptor proteins that are involved in sensing and response to light in living organisms. Genetic code expansion (GCE) technology has provided new tools to transform light insensitive proteins into novel photoreceptor proteins. It is achieved by the site-specific incorporation of unnatural amino acids (Uaas) that carry light sensitive moieties serving as "pigments" that react to light via photo-decaging, cross-linking, or isomerization. Over the last two decades, various proteins including ion channels, GPCRs, transporters, and kinases have been successfully rendered light responsive owing to the functionalities of Uaas. Very recently, Cas9 protein has been engineered to enable light activation of genomic editing by CRISPR. Those novel proteins have not only led to discoveries of dynamic protein conformational changes with implications in diseases, but also facilitated the screening of ligand-protein and protein-protein interactions of pharmacological significance. This review covers the genetic editing principles for genetic code expansion and design concepts that guide the engineering of light-sensitive proteins. The applications have brought up a new concept of "optoproteomics" that, in contrast to "optogenetics," aims to combine optical methods and site-specific proteomics for investigating and intervening in biological functions.

Design of Light-Sensitive NMDARs by Genetically Encoded Photo-Cross-Linkers.

Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application. Immunoblotting analysis has been used to confirm inter-subunit crosslinking.

Optocontrol of glutamate receptor activity by single side-chain photoisomerization.

Engineering light-sensitivity into proteins has wide ranging applications in molecular studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology.

Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene.

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross-linkers.

Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.