DiDBiT-TMT: A Novel Method to Quantify Changes in the Proteomic Landscape Induced by Neural Plasticity.

Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.

Cubic Na0.5Bi0.5TiO3 nanoperovskite significantly expands the application of sensitive immunosensor for the detection of carcinoembryonic antigen.

Nanosized sodium bismuth perovskite titanate (NBT) was synthesized and first used as the electrochemical immune sensing platform for the sensitive detection of carcinoembryonic antigen (CEA). Gold nanoparticles (Au NPs) grew on the surface of NBT through forming Au-N bond to obtain Au@NBT, and a label-free electrochemical immunosensor was proposed using Au@NBT as an immunosensing recognizer towards CEA. The well-ordered crystal structure of NBT was not changed at all after the modification of Au NPs outside, but significantly improved the conductivity, catalytic activity, and biocompatibility of the Au@NBT-modified electrode. The unique cubic crystal nanostructure of NBT offered a large active area for both Au NP modification and the subsequent immobilization of biomolecules over the electrode surface, triggering the effective generation of promising properties of the proposed Au@NBT-based electrochemical immunosensor. As expected, favorable detection performances were achieved using this immunosensor towards CEA detection, where a good linear relationship between the current response and CEA concentration was obtained in the concentration range 10 fg mL-1 to 100 ng mL-1 with a low detection limit (LOD) of 13.17 fg mL-1. Also, the significantly enhanced selectivity, and stability guaranteed the promising electrochemical properties of this immunosensor. Furthermore, the analysis of real serum samples verified the high feasibility of this new method in clinical CEA detection. This work opens a new window for the application of nanoperovskite in the early detection of CEA.

Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function.SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.

Quantitative BONCAT Allows Identification of Newly Synthesized Proteins after Optic Nerve Injury.

Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here, we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine, a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in more than 1000 proteins at 1 or 5 d after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.SIGNIFICANCE STATEMENT Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.

Temporal Quantitative Profiling of Newly Synthesized Proteins during Aß Accumulation.

Accumulation of aggregated amyloid beta (Aß) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of Aß deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice. This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post Aß deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to Aß accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

Multi-biomarker is an early-stage predictor for progression of Coronavirus disease 2019 (COVID-19) infection.

Coronavirus disease 2019 (COVID-19) has spread widely in the communities in many countries. Although most of the mild patients could be cured by their body's ability to self-heal, many patients quickly progressed to severe disease and had to undergo treatment in the intensive care unit (ICU). Thus, it is very important to effectively predict which patients with mild disease are more likely to progress to severe disease. A total of 72 patients hospitalized with COVID-19 in Shandong Provincial Public Health Clinical Center and 1141 patients included in the published papers were enrolled in this study. We determined that the combination of interleukin-6 (IL-6), Neutrophil (NEUT), and Natural Killer (NK) cells had the highest prediction accuracy (with 75% sensitivity and 95% specificity) for progression of COVID-19 infection. A binomial regression equation that accounted for a multiple risk score for the combination of IL-6, NEUT, and NK was also established. The multiple risk score is a good indicator for early stratification of mild patients into risk categories, which is very important for adjusting the treatment plan and preventing death.

Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy.

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced natural killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

Containing misinformation spreading in temporal social networks.

Many researchers from a variety of fields, including computer science, network science, and mathematics, have focused on how to contain the outbreaks of Internet misinformation that threaten social systems and undermine societal health. Most research on this topic treats the connections among individuals as static, but these connections change in time, and thus social networks are also temporal networks. Currently, there is no theoretical approach to the problem of containing misinformation outbreaks in temporal networks. We thus propose a misinformation spreading model for temporal networks and describe it using a new theoretical approach. We propose a heuristic-containing (HC) strategy based on optimizing the final outbreak size that outperforms simplified strategies such as those that are random-containing and targeted-containing. We verify the effectiveness of our HC strategy on both artificial and real-world networks by performing extensive numerical simulations and theoretical analyses. We find that the HC strategy dramatically increases the outbreak threshold and decreases the final outbreak threshold.

Quantitative temporal analysis of protein dynamics in cardiac remodeling.

Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly-synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle. This analysis revealed a complex combination of adaptive and maladaptive alterations at acute and prolonged time points including the identification of proteins not previously associated with CR. We also combined the PALM dataset with our published protein turnover rate dataset to identify putative biochemical mechanisms underlying CR. The novel integration of analyzing NSPs together with their protein turnover rates demonstrated that alterations in specific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulation of protein synthesis and degradation.

Proteomics and pulse azidohomoalanine labeling of newly synthesized proteins: what are the potential applications?

INTRODUCTION: Measuring the immediate changes in cells that arise from changing environmental conditions is crucial to understanding the underlying mechanisms involved. These changes can be measured with metabolic stable isotope fully labeled proteomes, but requires looking for changes in the midst of a large background. In addition, labeling efficiency can be an issue in primary and fully differentiated cells. Area covered: Azidohomoalanine (AHA), an analog of methionine, can be accepted by cellular translational machinery and incorporated into newly synthesized proteins (NSPs). AHA-NSPs can be coupled to biotin via CuAAC-mediated click-chemistry and enriched using avidin-based affinity purification. Thus, AHA-containing proteins or peptides can be enriched and efficiently separated from the whole proteome. In this review, we describe the development of mass spectrometry (MS) based AHA strategies and discuss their potential to measure proteins involved in immune response, secretome, gut microbiome, and proteostasis as well as their potential for clinical uses. Expert commentary: AHA strategies have been used to identify synthesis activity and to compare two biological conditions in various biological model organisms. In combination with instrument development, improved sample preparation and fractionation strategies, MS-based AHA strategies have the potential for broad application, and the methods should translate into clinical use.

HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification.

Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.

The alteration of H4-K16ac and H3-K27met influences the differentiation of neural stem cells.

The neural stem cell therapy provides a promising future for patients with central nerve system damage, thus an insight into its differentiation mechanism is urgently needed. Herein, we aimed to identify various histone modifications and reveal their impact on the differentiation of neural stem cells (NSCs) toward neurons. Firstly, we labeled primary NSCs using the stable isotope labeling with amino acids in cell culture (SILAC) technique. Then we induced these NSCs to differentiate by all-trans retinoic acid (atRA) or SB216763. Next, we identified the alteration of histone modifications in early-differentiated NSCs by mass spectrometry and verified them by Western blot. Interestingly, these modification alterations and phenotype changes were found similar in NSCs induced by the two different drugs. More interestingly, during the differentiation process H3-K27met was significantly up-regulated while H4-K16ac was not altered at the global level but down-regulated in some low-abundance combinatorial codes. We inhibited the methyltransferase of H3-K27 and deacetylase of H4-K16 simultaneously and found the differentiation procedure was obviously delayed. The function of H4-K16ac and H3-K27met in NSCs differentiation would be useful to reveal the differentiation mechanism and valuable for further neural stem cell therapy.

Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice.

Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.

Activated cyclin-dependent kinase 5 promotes microglial phagocytosis of fibrillar ß-amyloid by up-regulating lipoprotein lipase expression.

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar ß-amyloid (Aß) by activated microglia is essential for Aß clearance in Alzheimer disease. However, the mechanism underlying Aß clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aß. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aß treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aß stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aß treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aß, but did not affect degradation of internalized Aß. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aß phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aß internalization. Furthermore, Aß plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aß. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aß phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.

Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.

Neurons dynamically regulate their proteome in response to sensory input, a key process underlying experience-dependent plasticity. We characterized the visual experience-dependent nascent proteome within a brief, defined time window after stimulation using an optimized metabolic labeling approach. Visual experience induced cell type-specific and age-dependent alterations in the nascent proteome, including proteostasis-related processes. We identified Emerin as the top activity-induced candidate plasticity protein and demonstrated that its rapid activity-induced synthesis is transcription-independent. In contrast to its nuclear localization and function in myocytes, activity-induced neuronal Emerin is abundant in the endoplasmic reticulum and broadly inhibits protein synthesis, including translation regulators and synaptic proteins. Downregulating Emerin shifted the dendritic spine population from predominantly mushroom morphology to filopodia and decreased network connectivity. In mice, decreased Emerin reduced visual response magnitude and impaired visual information processing. Our findings support an experience-dependent feed-forward role for Emerin in temporally gating neuronal plasticity by negatively regulating translation.

Electronic Structure Modification of MnO2 Nanosheet Arrays with Enhanced Water Oxidation Activity and Stability by Nitrogen Plasma.

The strategic design of catalysts for the oxygen evolution reaction (OER) is crucial in tackling the substantial energy demands associated with hydrogen production in electrolytic water splitting. Despite extensive research on birnessite (δ-MnO2) manganese oxides to enhance catalytic activity by modulating Mn3+ species, the ongoing challenge is to simultaneously stabilize Mn3+ while improving overall activity. Herein, oxygen (O) vacancies and nitrogen (N) doping have been simultaneously introduced into the MnO2 through a simple nitrogen plasma approach, resulting in efficient OER performance. The optimized N-MnO2v electrocatalyst exhibits outstanding OER activity in alkaline electrolyte, reducing the overpotential by nearly 160 mV compared to pure pristine MnO2 (from 476 to 312 mV) at 10 mA cm-2, and a small Tafel slope of 89 mV dec-1. Moreover, it demonstrates excellent durability over a 122 h stability test. The introduction of O vacancies and incorporation of N not only fine-tune the electronic structure of MnO2, increasing the Mn3+ content to enhance overall activity, but also play a crucial role in stabilizing Mn3+, thereby leading to exceptional stability over time. Subsequently, density functional theory calculations validate the optimized electronic structure of MnO2 achieved through the two engineering methods, effectively lowering the intermediate adsorption free energy barrier. Our synergistic approach, utilizing nitrogen plasma treatment, opens a pathway to concurrently enhance the activity and stability of OER electrocatalysts, applicable not only to Mn-based but also to other transition metal oxides.

Nanomaterials promote the fast development of electrochemical MiRNA biosensors.

Cancer has become the leading cause of death worldwide. In recent years, molecular diagnosis has demonstrated great potential in the prediction and diagnosis of cancer. MicroRNAs (miRNAs) are short oligonucleotides that regulate gene expression and cell function and are considered ideal biomarkers for cancer detection, diagnosis, and patient prognosis. Therefore, the specific and sensitive detection of ultra-low quantities of miRNA is of great significance. MiRNA biosensors based on electrochemical technology have advantages of high sensitivity, low cost and fast response. Nanomaterials show great potential in miRNA electrochemical detection and promote the rapid development of electrochemical miRNA biosensors. Some methods and signal amplification strategies for miRNA detection in recent years are reviewed herein, followed by a discussion of the latest progress in electrochemical miRNA detection based on different types of nanomaterial. Future perspectives and challenges are also proposed for further exploration of nanomaterials to bring breakthroughs in electrochemical miRNA detection.

Characterization of an aldo-keto reductase from Thermotoga maritima with high thermostability and a broad substrate spectrum.

A novel aldo-keto reductase gene, Tm1743, from Thermotoga maritima was overexpressed in Escherichia coli. The enzyme displayed the highest activity at 90 °C and at pH 9. It retained 63 % of its activity after 15 h at 85 °C. The enzyme also could tolerate (up to 10 % v/v) acetonitrile, ethanol and 2-propanol with slightly increased activities. Methanol, DMSO and acetone decreased activity slightly. Furthermore, Tm1743 exhibited broad substrate specificity towards various keto esters, ketones and aldehydes, with relative activities ranging from 2 to 460 % compared to the control. Its optimum substrate, 2,2,2-trifluoroacetophenone, was asymmetrically reduced in a coupled NADPH-regeneration system with an enantioselectivity of 99.8 % and a conversion of 98 %.

Development of a whole-cell biocatalyst with NADPH regeneration system for biosulfoxidation.

A formate dehydrogenase gene (fdh) originated from Candida boidinii was co-expressed in E. coli BL21 (DE3) with the cyclohexanone monooxygenase gene (chmo) cloned from Acinetobacter calcoaceticus NCIMB 9871. The co-expression system was then used as a whole-cell biocatalyst to synthesize chiral phenyl methyl sulfoxide (PMSO) from thioanisole (PMS) and the reaction conditions were investigated. When the initial concentration of PMS was 20 mM, the specific productivity of PMSO in this system was 2.07 µmol g(-1) cw min(-1) (cw: wet cell weight) and the ee value for the R-sulfoxide was 99 %. In contrast, when chmo was the only gene expressed in E. coli, the specific productivity of PMSO was 0.053 µmol g(-1) cw min(-1) with no exact enantioselectivity. Further determination of NADPH concentration in the whole-cell catalysts suggested that co-expression of fdh with chmo significantly improved NADPH supply. Thus, this whole-cell biocatalyst system is highly advantageous for the synthesis of optically pure R-sulfoxide.

Proteomic screen reveals diverse protein transport between connected neurons in the visual system.

Intercellular transfer of toxic proteins between neurons is thought to contribute to neurodegenerative disease, but whether direct interneuronal protein transfer occurs in the healthy brain is not clear. To assess the prevalence and identity of transferred proteins and the cellular specificity of transfer, we biotinylated retinal ganglion cell proteins in vivo and examined biotinylated proteins transported through the rodent visual circuit using microscopy, biochemistry, and mass spectrometry. Electron microscopy demonstrated preferential transfer of biotinylated proteins from retinogeniculate inputs to excitatory lateral geniculate nucleus (LGN) neurons compared with GABAergic neurons. An unbiased mass spectrometry-based screen identified ∼200 transneuronally transported proteins (TNTPs) isolated from the visual cortex. The majority of TNTPs are present in neuronal exosomes, and virally expressed TNTPs, including tau and ß-synuclein, were detected in isolated exosomes and postsynaptic neurons. Our data demonstrate transfer of diverse endogenous proteins between neurons in the healthy intact brain and suggest that TNTP transport may be mediated by exosomes.