Disease-linked mutations in Munc18-1 deplete synaptic Doc2.

Heterozygous de novo mutations in the neuronal protein Munc18-1/STXBP1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia and tremor, summarized as STXBP1 encephalopathies. Although haploinsufficiency is the prevailing disease mechanism, it remains unclear how the reduction in Munc18-1 levels causes synaptic dysfunction in disease as well as how haploinsufficiency alone can account for the significant heterogeneity among patients in terms of the presence, onset and severity of different symptoms. Using biochemical and cell biological readouts on mouse brains, cultured mouse neurons and heterologous cells, we found that the synaptic Munc18-1 interactors Doc2A and Doc2B are unstable in the absence of Munc18-1 and aggregate in the presence of disease-causing Munc18-1 mutants. In haploinsufficiency-mimicking heterozygous knockout neurons, we found a reduction in Doc2A/B levels that is further aggravated by the presence of the disease-causing Munc18-1 mutation G544D as well as an impairment in Doc2A/B synaptic targeting in both genotypes. We also demonstrated that overexpression of Doc2A/B partially rescues synaptic dysfunction in heterozygous knockout neurons but not heterozygous knockout neurons expressing G544D Munc18-1. Our data demonstrate that STXBP1 encephalopathies are not only characterized by the dysfunction of Munc18-1 but also by the dysfunction of the Munc18-1 binding partners Doc2A and Doc2B, and that this dysfunction is exacerbated by the presence of a Munc18-1 missense mutant. These findings may offer a novel explanation for the significant heterogeneity in symptoms observed among STXBP1 encephalopathy patients.

LncRNA-miRNA interactions prediction based on meta-path similarity and Gaussian kernel similarity.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are two typical types of non-coding RNAs that interact and play important regulatory roles in many animal organisms. Exploring the unknown interactions between lncRNAs and miRNAs contributes to a better understanding of their functional involvement. Currently, studying the interactions between lncRNAs and miRNAs heavily relies on laborious biological experiments. Therefore, it is necessary to design a computational method for predicting lncRNA-miRNA interactions. In this work, we propose a method called MPGK-LMI, which utilizes a graph attention network (GAT) to predict lncRNA-miRNA interactions in animals. First, we construct a meta-path similarity matrix based on known lncRNA-miRNA interaction information. Then, we use GAT to aggregate the constructed meta-path similarity matrix and the computed Gaussian kernel similarity matrix to update the feature matrix with neighbourhood information. Finally, a scoring module is used for prediction. By comparing with three state-of-the-art algorithms, MPGK-LMI achieves the best results in terms of performance, with AUC value of 0.9077, AUPR of 0.9327, ACC of 0.9080, F1-score of 0.9143 and precision of 0.8739. These results validate the effectiveness and reliability of MPGK-LMI. Additionally, we conduct detailed case studies to demonstrate the effectiveness and feasibility of our approach in practical applications. Through these empirical results, we gain deeper insights into the functional roles and mechanisms of lncRNA-miRNA interactions, providing significant breakthroughs and advancements in this field of research. In summary, our method not only outperforms others in terms of performance but also establishes its practicality and reliability in biological research through real-case analysis, offering strong support and guidance for future studies and applications.

A Novel Role for DOC2B in Ameliorating Palmitate-Induced Glucose Uptake Dysfunction in Skeletal Muscle Cells via a Mechanism Involving ß-AR Agonism and Cofilin.

Diet-related lipotoxic stress is a significant driver of skeletal muscle insulin resistance (IR) and type 2 diabetes (T2D) onset. ß2-adrenergic receptor (ß-AR) agonism promotes insulin sensitivity in vivo under lipotoxic stress conditions. Here, we established an in vitro paradigm of lipotoxic stress using palmitate (Palm) in rat skeletal muscle cells to determine if ß-AR agonism could cooperate with double C-2-like domain beta (DOC2B) enrichment to promote skeletal muscle insulin sensitivity under Palm-stress conditions. Previously, human T2D skeletal muscles were shown to be deficient for DOC2B, and DOC2B enrichment resisted IR in vivo. Our Palm-stress paradigm induced IR and ß-AR resistance, reduced DOC2B protein levels, triggered cytoskeletal cofilin phosphorylation, and reduced GLUT4 translocation to the plasma membrane (PM). By enhancing DOC2B levels in rat skeletal muscle, we showed that the deleterious effects of palmitate exposure upon cofilin, insulin, and ß-AR-stimulated GLUT4 trafficking to the PM and glucose uptake were preventable. In conclusion, we revealed a useful in vitro paradigm of Palm-induced stress to test for factors that can prevent/reverse skeletal muscle dysfunctions related to obesity/pre-T2D. Discerning strategies to enrich DOC2B and promote ß-AR agonism can resist skeletal muscle IR and halt progression to T2D.

DOC2B is a negative regulator of Wnt/ß-catenin signaling pathway in cervical cancer.

DOC2B is a ubiquitously expressed isoform of the double C-2 protein family that requires Ca2+ for most of its physiological functions. Initial findings have indicated that DOC2B participates in exocytosis, vesicular transport, insulin secretion and regulation, glucose homeostasis, and neurotransmitter release. Aberrant expression of DOC2B has been reported in diabetes, leukemia, and cervical cancer (CC). Our earlier studies have demonstrated the inhibitory effects of DOC2B on CC cell proliferation, migration, invasion, and EMT and suggested the possible role of DOC2B in Wnt signaling inhibition. However, the association between DOC2B downregulation and Wnt/ß-catenin signaling activation and the underlying molecular mechanism remain elusive. Herein, we found that DOC2B inhibited Wnt/ß-catenin pathway by enhancing the expression of the components of the CTNNB1 destruction complex and by fostering proteasomal degradation of CTNNB1. The translocation of CTNNB1 to the nucleus and its interaction with TCF/LEF family transcription factors was perturbed in the presence of DOC2B in a GSK3ß independent manner. Further, we have identified DKK1 as one of the upregulated genes in the presence of DOC2B. DKK1 inhibition in DOC2B expressing cells by WAY262611 reactivated Wnt/ß-catenin signaling, relieved DOC2B induced senescence, and alleviated the inhibitory effects of DOC2B on the aforementioned malignant behaviors. We have provided evidence for DOC2B-DKK1-senescence-Wnt/ß-catenin-EMT signaling crosstalk to have tumor growth regulatory functions in CC. Thus, targeting DOC2B-DKK1-senescence-Wnt/ß-catenin-EMT signaling crosstalk via activation of DOC2B may offer a novel approach to restraint malignant behaviors in CC.

Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence.

Senescence induction and epithelial-mesenchymal transition (EMT) events are the opposite sides of the spectrum of cancer phenotypes. The key molecules involved in these processes may get influenced or altered by genetic and epigenetic changes during tumor progression. Double C2-like domain beta (DOC2B), an intracellular vesicle trafficking protein of the double C2 protein family, plays a critical role in exocytosis, neurotransmitter release, and intracellular vesicle trafficking. DOC2B is repressed by DNA promoter hypermethylation and functions as a tumor growth regulator in cervical cancer. To date, the molecular mechanisms of DOC2B in cervical cancer progression and metastasis is elusive. Herein, the biological functions and molecular mechanisms regulated by DOC2B and its impact on senescence and EMT are described. DOC2B inhibition promotes proliferation, growth, and migration by relieving G0/G1-S arrest, actin remodeling, and anoikis resistance in Cal27 cells. It enhanced tumor growth and liver metastasis in nude mice with the concomitant increase in metastasis-associated CD55 and CD61 expression. Inhibition of EMT and promotion of senescence by DOC2B is a calcium-dependent process and accompanied by calcium-mediated interaction between DOC2B and CDH1. In addition, we have identified several EMT and senescence regulators as targets of DOC2B. We show that DOC2B may act as a metastatic suppressor by inhibiting EMT through induction of senescence via DOC2B-calcium-EMT-senescence axis.

Ca2+ sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons.

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.

Down-Regulation of Double C2 Domain Alpha Promotes the Formation of Hyperplastic Nerve Fibers in Aganglionic Segments of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is a common developmental anomaly of the gastrointestinal tract in children. The most significant characteristics of aganglionic segments in HSCR are hyperplastic extrinsic nerve fibers and the absence of endogenous ganglion plexus. Double C2 domain alpha (DOC2A) is mainly located in the nucleus and is involved in Ca2+-dependent neurotransmitter release. The loss function of DOC2A influences postsynaptic protein synthesis, dendrite morphology, postsynaptic receptor density and synaptic plasticity. It is still unknown why hyperplastic extrinsic nerve fibers grow into aganglionic segments in HSCR. We detected the expression of DOC2A in HSCR aganglionic segment colons and established three DOC2A-knockdown models in the Neuro-2a cell line, neural spheres and zebrafish separately. First, we detected the protein and mRNA expression of DOC2A and found that DOC2A was negatively correlated with AChE+ grades. Second, in the Neuro-2a cell lines, we found that the amount of neurite outgrowth and mean area per cell were significantly increased, which suggested that the inhibition of DOC2A promotes nerve fiber formation and the neuron's polarity. In the neural spheres, we found that the DOC2A knockdown was manifested by a more obvious connection of nerve fibers in neural spheres. Then, we knocked down Doc2a in zebrafish and found that the down-regulation of Doc2a accelerates the formation of hyperplastic nerve fibers in aganglionic segments in zebrafish. Finally, we detected the expression of MUNC13-2 (UNC13B), which was obviously up-regulated in Grade3/4 (lower DOC2A expression) compared with Grade1/2 (higher DOC2A expression) in the circular muscle layer and longitudinal muscle layer. The expression of UNC13B was up-regulated with the knocking down of DOC2A, and there were protein interactions between DOC2A and UNC13B. The down-regulation of DOC2A may be an important factor leading to hyperplastic nerve fibers in aganglionic segments of HSCR. UNC13B seems to be a downstream molecule to DOC2A, which may participate in the spasm of aganglionic segments of HSCR patient colons.

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

Doc2-mediated superpriming supports synaptic augmentation.

Various forms of synaptic plasticity underlie aspects of learning and memory. Synaptic augmentation is a form of short-term plasticity characterized by synaptic enhancement that persists for seconds following specific patterns of stimulation. The mechanisms underlying this form of plasticity are unclear but are thought to involve residual presynaptic Ca2+ Here, we report that augmentation was reduced in cultured mouse hippocampal neurons lacking the Ca2+ sensor, Doc2; other forms of short-term enhancement were unaffected. Doc2 binds Ca2+ and munc13 and translocates to the plasma membrane to drive augmentation. The underlying mechanism was not associated with changes in readily releasable pool size or Ca2+ dynamics, but rather resulted from superpriming a subset of synaptic vesicles. Hence, Doc2 forms part of the Ca2+-sensing apparatus for synaptic augmentation via a mechanism that is molecularly distinct from other forms of short-term plasticity.

Multi-Criteria Recommendation Systems to Foster Online Grocery.

With the exponential increase in information, it has become imperative to design mechanisms that allow users to access what matters to them as quickly as possible. The recommendation system (RS) with information technology development is the solution, it is an intelligent system. Various types of data can be collected on items of interest to users and presented as recommendations. RS also play a very important role in e-commerce. The purpose of recommending a product is to designate the most appropriate designation for a specific product. The major challenge when recommending products is insufficient information about the products and the categories to which they belong. In this paper, we transform the product data using two methods of document representation: bag-of-words (BOW) and the neural network-based document combination known as vector-based (Doc2Vec). We propose three-criteria recommendation systems (product, package and health) for each document representation method to foster online grocery shopping, which depends on product characteristics such as composition, packaging, nutrition table, allergen, and so forth. For our evaluation, we conducted a user and expert survey. Finally, we compared the performance of these three criteria for each document representation method, discovering that the neural network-based (Doc2Vec) performs better and completely alters the results.

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee ß-cell Function and Protection.

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic ß-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to ß-cells improves whole-body glucose homeostasis, enhances ß-cell function, and surprisingly, protection of ß-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of ß-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.

Automatic Detection of Pornographic and Gambling Websites Based on Visual and Textual Content Using a Decision Mechanism.

Pornographic and gambling websites become increasingly stubborn via disguising, misleading, blocking, and bypassing, which hinder the construction of a safe and healthy network environment. However, most traditional approaches conduct the detection process through a single aspect of these sites, which would fail to handle the more intricate and challenging situations. To alleviate this problem, this study proposed an automatic detection system for porn and gambling websites based on visual and textual content using a decision mechanism (PG-VTDM). This system can be applied to the intelligent wireless router at home or school to realize the identification, blocking, and warning of ill-suited websites. First, Doc2Vec was employed to learn the textual features that can be used to represent the textual content in the hypertext markup language (HTML) source code of the websites. In addition, the traditional bag-of-visual-words (BoVW) was improved by introducing local spatial relationships of feature points for better representing the visual features of the website screenshot. Then, based on these two types of features, a text classifier and an image classifier were both trained. In the decision mechanism, a data fusion algorithm based on logistic regression (LR) was designed to obtain the final prediction result by measuring the contribution of the two classification results to the final category prediction. The efficiency of this proposed approach was substantiated via comparison experiments using gambling and porn website datasets crawled from the Internet. The proposed approach outperformed the approach based on a single feature and some state-of-the-art approaches, with accuracy, precision, and F-measure all over 99%.

Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane.

Double C2 domain protein B (DOC2B) is a high-affinity Ca2+ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)-bisphosphate [PI(4, 5)P2 ] and target SNAREs (t-SNAREs). Here, we show that PI(4, 5)P2 hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t-SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P2 in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P2 -binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P2 are required for the DOC2B-PM association and revealing multiple PI(4, 5)P2 -C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP-independent and occurs in a diffusion-like manner. Our data suggest that the Ca2+ -triggered translocation of DOC2B is diffusion-driven and aimed at PI(4, 5)P2 -containing membranes.

DOC2B promotes insulin sensitivity in mice via a novel KLC1-dependent mechanism in skeletal muscle.

AIMS/HYPOTHESIS: Skeletal muscle accounts for >80% of insulin-stimulated glucose uptake; dysfunction of this process underlies insulin resistance and type 2 diabetes. Insulin sensitivity is impaired in mice deficient in the double C2 domain ß (DOC2B) protein, while whole-body overexpression of DOC2B enhances insulin sensitivity. Whether insulin sensitivity in the skeletal muscle is affected directly by DOC2B or is secondary to an effect on other tissues is unknown; the underlying molecular mechanisms also remain unclear. METHODS: Human skeletal muscle samples from non-diabetic or type 2 diabetic donors were evaluated for loss of DOC2B during diabetes development. For in vivo analysis, new doxycycline-inducible skeletal-muscle-specific Doc2b-overexpressing mice fed standard or high-fat diets were evaluated for insulin and glucose tolerance, and insulin-stimulated GLUT4 accumulation at the plasma membrane (PM). For in vitro analyses, a DOC2B-overexpressing L6-GLUT4-myc myoblast/myotube culture system was coupled with an insulin resistance paradigm. Biochemical and molecular biology methods such as site-directed mutagenesis, co-immunoprecipitation and mass spectrometry were used to identify the molecular mechanisms linking insulin stimulation to DOC2B. RESULTS: We identified loss of DOC2B (55% reduction in RNA and 40% reduction in protein) in the skeletal muscle of human donors with type 2 diabetes. Furthermore, inducible enrichment of DOC2B in skeletal muscle of transgenic mice enhanced whole-body glucose tolerance (AUC decreased by 25% for female mice) and peripheral insulin sensitivity (area over the curve increased by 20% and 26% for female and male mice, respectively) in vivo, underpinned by enhanced insulin-stimulated GLUT4 accumulation at the PM. Moreover, DOC2B enrichment in skeletal muscle protected mice from high-fat-diet-induced peripheral insulin resistance, despite the persistence of obesity. In L6-GLUT4-myc myoblasts, DOC2B enrichment was sufficient to preserve normal insulin-stimulated GLUT4 accumulation at the PM in cells exposed to diabetogenic stimuli. We further identified that DOC2B is phosphorylated on insulin stimulation, enhancing its interaction with a microtubule motor protein, kinesin light chain 1 (KLC1). Mutation of Y301 in DOC2B blocked the insulin-stimulated phosphorylation of DOC2B and interaction with KLC1, and it blunted the ability of DOC2B to enhance insulin-stimulated GLUT4 accumulation at the PM. CONCLUSIONS/INTERPRETATION: These results suggest that DOC2B collaborates with KLC1 to regulate insulin-stimulated GLUT4 accumulation at the PM and regulates insulin sensitivity. Our observation provides a basis for pursuing DOC2B as a novel drug target in the muscle to prevent/treat type 2 diabetes.

Open-Ended Technological Innovation.

We detect ongoing innovation in empirical data about human technological innovations. Ongoing technological innovation is a form of open-ended evolution, but it occurs in a nonbiological, cultural population that consists of actual technological innovations that exist in the real world. The change over time of this population of innovations seems to be quite open-ended. We take patented inventions as a proxy for technological innovations and mine public patent records for evidence of the ongoing emergence of technological innovations, and we compare two ways to detect it. One way detects the first instances of predefined patent pigeonholes, specifically the technology classes listed in the United States Patent Classification (USPC). The second way embeds patents in a high-dimensional semantic space and detects the emergence of new patent clusters. After analyzing hundreds of years of patent records, both methods detect the emergence of new kinds of technologies, but clusters are much better at detecting innovations that are unanticipated and undetected by USPC pigeonholes. Our clustering methods generalize to detect unanticipated innovations in other evolving populations that generate ongoing streams of digital data.

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

Loss of Doc2-Dependent Spontaneous Neurotransmission Augments Glutamatergic Synaptic Strength.

Action potential-evoked vesicle fusion comprises the majority of neurotransmission within chemical synapses, but action potential-independent spontaneous neurotransmission also contributes to the collection of signals sent to the postsynaptic cell. Previous work has implicated spontaneous neurotransmission in homeostatic synaptic scaling, but few studies have selectively manipulated spontaneous neurotransmission without substantial changes in evoked neurotransmission to study this function in detail. Here we used a quadruple knockdown strategy to reduce levels of proteins within the soluble calcium-binding double C2 domain (Doc2)-like protein family to selectively reduce spontaneous neurotransmission in cultured mouse and rat neurons. Activity-evoked responses appear normal while both excitatory and inhibitory spontaneous events exhibit reduced frequency. Excitatory miniature postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), increase in amplitude after quadruple knockdown. This increase in synaptic efficacy correlates with reduced phosphorylation levels of eukaryotic elongation factor 2 and also requires the presence of elongation factor 2 kinase. Together, these data suggest that spontaneous neurotransmission independently contributes to the regulation of synaptic efficacy, and action potential-evoked and spontaneous neurotransmission can be segregated at least partially on a molecular level.SIGNIFICANCE STATEMENT Action potential-evoked and spontaneous neurotransmission have been observed in nervous system circuits as long as methods have existed to measure them. Despite being well studied, controversy still remains about whether these forms of neurotransmission are regulated independently on a molecular level or whether they are simply a continuum of neurotransmission modes. In this study, members of the Doc2 family of presynaptic proteins were eliminated, which caused a reduction in spontaneous neurotransmission, whereas action potential-evoked neurotransmission remained relatively normal. This protein loss also caused an increase in synaptic strength, suggesting that spontaneous neurotransmission is able to communicate independently with the postsynaptic neuron and trigger downstream signaling cascades that regulate the synaptic state.

DNA promoter methylation-dependent transcription of the double C2-like domain ß (DOC2B) gene regulates tumor growth in human cervical cancer.

Double C2-like domain ß (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Plasmodium falciparum double C2 domain protein, PfDOC2, binds to calcium when associated with membranes.

The pathogenesis of malaria is strongly correlated with secretion of the micronemes, the apical organelles which contain the adhesins required for invasion of Plasmodium falciparum into human erythrocytes. A critical event in P. falciparum erythrocyte invasion is the production of calcium transients. After entering the cell, Ca(2+) binds to soluble Ca(2+)-binding proteins, such as the double C2 domains (DOC2). Recently, deletion of a P. falciparum DOC2 protein, PfDOC2, was shown to cause impairment in microneme secretion. However, PfDOC2 remains poorly characterized. Here, we report that PfDOC2 is expressed throughout the erythrocytic cycle and demonstrate that it is associated with membrane fractions and binds to calcium when it is part of these membranous structures. In summary, we show that PfDOC2 is a calcium lipid-binding protein of the protein kinase C type of DOC2 proteins.

DeepPPThermo: A Deep Learning Framework for Predicting Protein Thermostability Combining Protein-Level and Amino Acid-Level Features.

Using wet experimental methods to discover new thermophilic proteins or improve protein thermostability is time-consuming and expensive. Machine learning methods have shown powerful performance in the study of protein thermostability in recent years. However, how to make full use of multiview sequence information to predict thermostability effectively is still a challenge. In this study, we proposed a deep learning-based classifier named DeepPPThermo that fuses features of classical sequence features and deep learning representation features for classifying thermophilic and mesophilic proteins. In this model, deep neural network (DNN) and bi-long short-term memory (Bi-LSTM) are used to mine hidden features. Furthermore, local attention and global attention mechanisms give different importance to multiview features. The fused features are fed to a fully connected network classifier to distinguish thermophilic and mesophilic proteins. Our model is comprehensively compared with advanced machine learning algorithms and deep learning algorithms, proving that our model performs better. We further compare the effects of removing different features on the classification results, demonstrating the importance of each feature and the robustness of the model. Our DeepPPThermo model can be further used to explore protein diversity, identify new thermophilic proteins, and guide directed mutations of mesophilic proteins.