Research trends in chemogenetics for neuroscience in recent 14 years: A bibliometric study in CiteSpace.

BACKGROUND: Chemogenetics has been widely adopted in Neuroscience. Neuroscience has become a hot research topic for scientists. Therefore, the purpose of this study is to explore the current status and trends in the global application of chemogenetics in neuroscience over the last 14 years via CiteSpace. METHODS: Publications related to chemogenetics in neuroscience were retrieved from the Science Citation Index-Extended Web of Science from 2008 to 2021. We used CiteSpace to analyze publications, citations, cited journals, countries, institutions, authors, cited authors, cited references, and keywords. RESULTS: A total of 947 records were retrieved from 2008 to 2021 on February 21, 2022. The number and rate of publications and citations increased significantly. Journal of Neuroscience was the most cited journal, and BRAIN RES BULL ranked first in the centrality of cited journals. The United States of America (USA) had the highest number of publications among the countries. Takashi Minamoto was the most prolific author and Armbruster BN ranked the first among authors cited. The first article in the frequency ranking of the references cited was published by Roth BL. The keyword of "nucleus accumben (NAc)" had the highest frequency. The top 3 keywords with the strongest citation bursts include "transgenic mice," "cancer," and "blood-brain barrier." CONCLUSION: The period 2008 to 2021 has seen a marked increase in research on chemogenetics in neuroscience. The application of chemogenetics is indispensable for research in the field of neuroscience. This bibliometrics study provides the current situation and trend in chemogenetic methods in neuroscience in recent 14 years, which may help researchers to identify the hot topics and frontiers for future studies in this field.

Proteomic Analysis of the Amygdala Reveals Dynamic Changes in Glutamate Transporter-1 During Progression of Complete Freund's Adjuvant-Induced Pain Aversion.

Pain sufferer usually show an aversion to the environment associated with pain, identified as pain aversion. The amygdala, an almond-shaped limbic structure in the medial temporal lobe, exerts a critical effect on emotion and pain formation. However, studies on inflammatory pain-induced aversion are still relatively limited, and the available evidence is not enough to clarify its inherent mechanisms. Proteomics is a high-throughput, comprehensive, and objective study method that compares the similarities and differences of protein expression under different conditions to screen potential targets. The current study aimed to identify potential pivotal proteins in the amygdala of rats after complete Freund's adjuvant (CFA)-induced pain aversion via proteomics analysis. Immunohistochemistry was performed to confirm the expression of glutamate transporter-1 (GLT-1) in the amygdala during different periods of pain aversion. Thirteen proteins were found to be different between the day 2 and day 15 groups. Among the 13 differentially expressed proteins, Q8R64 denotes GLT-1, which utilises synaptic glutamate to remain optimal extracellular glutamic levels, thereby preventing accumulation in the synaptic cleft and consequent excitotoxicity. The variation in GLT-1 expression was correlated with the variation tendency of pain aversion, which implies a potential link between the modulation of pain aversion and the excitability of glutamatergic neurons. This study demonstrated that exposure to inflammatory pain results in aversion induced from pain, leading to extensive biological changes in the amygdala.

[Central mechanism of acupuncture intervention in anxiety and depression under fMRI].

Despite the significant efficacy of acupuncture in alleviating anxiety and depression, the mechanism remains unclear. In recent years, functional magnetic resonance imaging (fMRI) technology has provided a visual method for deciphering the mechanism of acupuncture in treating anxiety and depression. This paper summarized the clinical studies about the imaging changes of anxiety and depression during the treatment with acupuncture under fMRI. The available studies demonstrated that acupuncture may act on functional nuclei and brain regions such as hippocampus, amygdala, cingulate gyrus, frontal lobe, and temporal lobe. The paper can lay a foundation for the further study of the central mechanism of acupuncture in the treatment of anxiety and depression.

Electro-Acupuncture for Bladder Pain Syndrome: A Protocol of a Randomized Controlled Trial and Study for Central Mechanism.

Objective: The efficacy of conventional treatments for treating bladder pain syndrome (BPS) remains unsatisfactory. Electro-acupuncture (EA) is one of the complementary treatments with great analgesic effect and minimal side effect, but evidence of the efficacy of EA on BPS is limited. Thus, this study aims to investigate the efficacy and safety of EA for treating BPS and study on central mechanism of patients with BPS. Methods/Design: The study is a randomized controlled and assessor-blinded design trial. A total of 84 participants will be randomly assigned to medication group (n=21), EA group (n=42) and sham electro-acupuncture (SA) group (n=21) in a 1:2:1 allocation ratio. This trial will include baseline period, 4-week treatment period and 4-week follow-up period. Participants in medication group will undergo treatment of amitriptyline for a period of 4 weeks. Participants in EA and SA groups will receive a 30 min EA or SA treatment for a total of 12 sessions over 4 weeks. The primary outcome is the Visual Analog Scale (VAS). The secondary outcomes include the O'Leary-Sant questionnaire, 24-hour voiding diary, Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD) and functional magnetic resonance imaging (fMRI). The VAS will be collected at baseline, week 2, week 4, and week 8 after randomization. The O'Leary-Sant questionnaire, HAMA and HAMD will be assessed at baseline, week 4 and week 8 after randomization. The 24-hour voiding diary will be assessed every single day. The fMRI data will be collected at baseline and week 4. Discussion: The results will provide evidence on the efficacy and safety of EA in the management of BPS and investigate the central mechanism of EA in treating patients with BPS. Trial Registration: ClinicalTrials.gov identifier: NCT05279963. Registered on 15 March 2022.

Detection and quantitation of succinimide in intact protein via hydrazine trapping and chemical derivatization.

The formation of aspartyl succinimide is a common post-translational modification of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV-Vis, liquid chromatography mass spectrometry (LC-MS), and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages are imparted by fluorescence labeling including the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, for example, without optimization, 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions, and related processes relevant to protein pharmaceuticals.

In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS.

Characterization of N-glycans by liquid chromatography-positive electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) using a microfluidic chip packed with porous graphitized carbon (PGC) represents a rapidly developing area in oligosaccharide analysis. Positive ion ESI-MS generates B/Y-type glycosidic fragment ions under collisional-induced dissociation (CID). Although these ions facilitate glycan sequencing, they provide little information on linkage and positional isomers. Isomer identification in these cases is by retention on the PGC stationary phase where the specific structural isomers can, in principle, be separated. In this paper, we broaden the applicability of the PGC microfluidic chip/MS platform by implementing fluoride-mediated negative ESI-MS. Ammonium fluoride, added to the mobile phase, aids in the formation of pseudomolecular oligosaccharide anions due to the ability of fluoride to abstract a proton from the glycan structure. The negative charge results in the generation of C-type glycosidic fragments, highly informative A-type cross-ring fragment ions, and additional gas-phase ion reaction products (e.g., D- and E-type ions), which, when combined, lead to in-depth oligosaccharide characterization, including linkage and positional isomers. Due to the separation of anomers by the PGC phase, comparison of oligosaccharides with an intact reducing terminus to their experimentally prepared corresponding alditols was performed, revealing a more sensitive MS and, especially, MS/MS analysis from the glycans with a free reducing end. Fluoride also ensured recovery of charged oligosaccharides from the PGC stationary phase. Application to the characterization of N-glycans released from polyclonal human and murine serum IgG is presented to demonstrate the effectiveness of the chip/negative ESI approach.

Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein L-isoaspartate O-methyltransferase-deficient mice.

The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD.

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

Protein isoaspartate methyltransferase-mediated 18O-labeling of isoaspartic acid for mass spectrometry analysis.

Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with (18)O via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in (18)O-water to regenerate isoAsp. The specific replacement of (16)O with (18)O at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the ß-delta sleep-inducing peptide (DSIP) are demonstrated.

Analysis of isoaspartic Acid by selective proteolysis with Asp-N and electron transfer dissociation mass spectrometry.

A ubiquitous yet underappreciated protein post-translational modification, isoaspartic acid (isoAsp, isoD, or beta-Asp), generated via the deamidation of asparagine or isomerization of aspartic acid in proteins, plays a diverse and crucial role in aging, as well as autoimmune, cancer, neurodegeneration, and other diseases. In addition, formation of isoAsp is a major concern in protein pharmaceuticals, as it may lead to aggregation or activity loss. The scope and significance of isoAsp have, up to now, not been fully explored, as an unbiased screening of isoAsp at low abundance remains challenging. This difficulty is due to the subtle difference in the physicochemical properties between isoAsp and Asp, e.g., identical mass. In contrast, endoprotease Asp-N (EC 3.4.24.33) selectively cleaves aspartyl peptides but not the isoaspartyl counterparts. As a consequence, isoaspartyl peptides can be differentiated from those containing Asp and also enriched by Asp-N digestion. Subsequently, the existence and site of isoaspartate can be confirmed by electron transfer dissociation (ETD) mass spectrometry. As little as 0.5% of isoAsp was detected in synthetic beta-amyloid and cytochrome c peptides, even though both were initially assumed to be free of isoAsp. Taken together, our approach should expedite the unbiased discovery of isoAsp.