Declining Levels of Specialized Synaptic Surface Proteins in nNOS-Expressing Interneurons in Mice Treated Prenatally with Valproic Acid.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorder characterized by impaired social interaction, and repetitive or restricted interests and behaviors. Membrane proteins are a significant part of the proteins in cell and play key functions in synaptic transmission. We have recently shown that neuronal nitric oxide synthase (nNOS) expression was reduced in the basolateral amygdala (BLA) of mice following postnatal valproic acid (VPA) exposure. In the current study, we utilized a label-free proteomics approach to identify and quantify surface protein expression in nNOS-positive interneurons between VPA-treated and control mice. Western blot was used to confirm the expression of selected membrane proteins. Our proteomics data revealed differentially expressed surface proteins in nNOS interneurons, e.g. Narp, AMPA-type glutamate (AMPA) receptor subunit GluA4 and Protein kinase C gamma (PKCγ), which were validated by Western blotting in mice treated with VPA. This work will pave the way for further elucidation of the mechanisms of these differentially membrane proteins in nNOS interneurons-medicated ASD.

GABAB1 receptor knockdown in prefrontal cortex induces behavioral aberrations associated with autism spectrum disorder in mice.

Autism spectrum disorder (ASD) is a set of heterogeneous neurodevelopmental disorders, characterized by social interaction deficit, stereotyped or repetitive behaviors. Apart from these core symptoms, a great number of individuals with ASD exhibit higher levels of anxiety and memory deficits. Previous studies demonstrate pronounced decrease of γ-aminobutyric acid B1 receptor (GABAB1R) protein level of frontal lobe in both ASD patients and animal models. The aim of the present study was to determine the role of GABAB1R in ASD-related behavioral aberrations. Herein, the protein and mRNA levels of GABAB1R in the prefrontal cortex (PFC) of sodium valproic acid (VPA)-induced mouse ASD model were determined by Western blot and qRT-PCR analysis, respectively. Moreover, the behavioral abnormalities in naive mice with GABAB1R knockdown mediated by recombinant adeno-associated virus (rAAV) were assessed in a comprehensive test battery consisted of social interaction, marble burying, self-grooming, open-field, Y-maze and novel object recognition tests. Furthermore, the action potential changes induced by GABAB1R deficiency were examined in neurons within the PFC of mouse. The results show that the mRNA and protein levels of GABAB1R in the PFC of prenatal VPA-induced mouse ASD model were decreased. Concomitantly, naive mice with GABAB1R knockdown exhibited ASD-like behaviors, such as impaired social interaction and communication, elevated stereotypes, anxiety and memory deficits. Patch-clamp recordings also revealed that GABAB1R knockdown provoked enhanced neuronal excitability by increasing action potential discharge frequencies. Overall, these findings support a notion that GABAB1R deficiency might contribute to ASD-like phenotypes, with the pathogenesis most likely resulting from enhanced neuronal excitability. SUBHEADINGS: GABAB1 Knockdown Induces Behavioral Aberrations with ASD.

Functional Evaluation of a Novel GRIN2B Missense Variant Associated with Epilepsy and Intellectual Disability.

Epilepsy, a neurological condition, is widely prevalent among individuals with intellectual disability (ID). It is well established that N-methyl-D-aspartate (NMDA) receptors play an important role in both epilepsy and ID. Autosomal dominant mutations in the GRIN2B gene, which encodes the GluN2B subunit of the NMDA receptor, have been reported to be associated with epilepsy and ID. However, the underlying mechanism of this association is not well-understood. In this study, we identified a novel GRIN2B mutation (c.3272A > C, p.K1091T) in a patient with epilepsy and ID. The proband was a one year and ten months old girl. GRIN2B variant was inherited from her mother. We further investigated the functional consequences of this mutation. Our findings revealed that the p.K1091T mutation created a Casein kinase 2 phosphorylation site. Using recombinant NMDA receptors containing the GluN2B-K1091T along with GluN1 in HEK 293T cells, we observed significant defects in its interactions with postsynaptic density 95. It is accompanied by reduced delivery of the receptors to the cell membrane and a decrease in glutamate affinity. Moreover, primary neurons expressing GluN2B-K1091T also exhibited impaired surface expression of NMDA receptors, a reduction in dendritic spine number and excitatory synaptic transmission. In summary, our study reports a novel GRIN2B mutation and provides functional characteristics of this mutation in vitro, thereby contributing to the understanding of GRIN2B variants in epilepsy and ID.

Ultrasound-Guided Injection of Autologous Platelet-Rich Plasma for Refractory Lateral Epicondylitis of Humerus: Case Series.

Refractory lateral epicondylitis (RLE) is a tendinopathy of the elbow with less effective conservation treatment. Platelet-rich plasma (PRP) is a new treatment option for RLE because of its repair-promoting effect on tissues. Although evidence demonstrates the efficacy of PRP in treating tendinopathies, the therapeutic utility of ultrasound-guided PRP injection for RLE is unknown. Here, we report two cases of RLE treated with PRP. The first patient was a 78-year-old man who received an unknown dose of local glucocorticoid injection at the local community clinic in June 2016. His pain recurred after exertion. The second patient was a 54-year-old woman who received a glucocorticoid injection (0.5 mL of compound betamethasone and 1.5 mL of 0.9% normal saline) in October 2020. Her pain could not be relieved. A physician diagnosed patients with RLE based on their medical history, symptoms, and clinical signs. The doctor injected PRP (the first patient in November 2020, the second in March 2021) under ultrasound guidance into the patient's attachment point of the extensor tendon at the lateral humeral epicondyle. The doctor evaluated the effectiveness of the treatment by ultrasonography, visual analogue scale, and the patient-rated tennis elbow evaluation. After four weeks of treatment, the pain was relieved, and functions continued to improve in the following three months. Moreover, the ultrasonography showed that the damaged tendons were repaired. Together, we demonstrate that ultrasound-guided PRP injection could considerably relieve pain, improve elbow joint functions in patients with RLE, and provide visible evidence that PRP repairs tendon damage.

2-Oxo-2,3-dihydro-1H-imidazo[1,2-a]pyridinium iodide.

In the title compound, C(7)H(7)N(2)O(+)·I(-), the carbonyl C and O atoms of the cation and the iodide ion are situated on mirror planes. The mean plane of the imidazo[1,2-d]pyridinium cation is perpendicular to the mirror plane as a consequence of the disorder of the cation over two opposite orientations of equal occupancy. In the crystal, N-H⋯I interactions are present.

Identification and functional characterization of Lys-trimethylation of lactate dehydrogenase A.

Background: Trimethylation of histones has been extensively studied, where histone methyltransferases catalyze the transfer of methyl groups from S-adenosyl methionine. Thus far, there have been no researches on the trimethylation of non-histone proteins. The precise mechanisms by which trimethylation affects cell progress and the related protein functions remain unclear. Purpose: The objective of this study was to identify the Lys-trimethylated proteins in kidney-derived cells and tissues, as well as to better understand the mechanisms underlying Lys-trimethylation-mediated cell metabolism. Methods: The levels of Lys-trimethylation in kidney-derived cells and tissues were assayed by Western blotting. Additionally, high-resolution mass spectrometry was used to analyze kidney-derived cells and tissues, and the eukaryotic expression vectors that led to the mutations of lysine were constructed and transfected into HEK293T cells. The LDHA activity of HEK293T cells was detected under conditions of Lys-trimethylation inhibition, and the proliferation of HEK293T cells was measured using EdU and Western blotting analyses. Results: The different proteins in kidney-derived cells and tissues showed different levels of Lys-trimethylation. In particular, lactate dehydrogenase A (LDHA) was Lys-trimethylated on lysine (K5). Inhibition of the Lys-trimethylation in LDHA increased the LDH activity of HEK293T cells and upregulated their proliferation. Conclusion: We suggested that LDHA affects the metabolism and proliferation of cells via a Lys-trimethylation-mediated mechanism; Lys-trimethylation might be a potential target for therapeutic research or used as a prognostic and treatment biomarker of several diseases.

Decreased Number and Expression of nNOS-Positive Interneurons in Basolateral Amygdala in Two Mouse Models of Autism.

The basolateral amygdala (BLA) controls socio-emotional behaviors and is involved in the etiology of autism. We have recently shown that virtually every neuronal nitric oxide synthase (nNOS) positive cell is a GABAergic inhibitory interneuron in the mouse BLA. Here, stereology was used to quantify the number of nNOS-expressing interneurons in valproic acid (VPA)-exposed C57BL/6J (B6) and BTBR T+Itpr3tf/J (BTBR) mice models of autism. Additionally, the protein and mRNA levels of nNOS in the BLA were quantitatively assessed by western blot and qRT-PCR analysis, respectively. Our results showed the decreased number of nNOS interneurons in the BLA of animal models relative to autism. Consistently, nNOS was significantly reduced in the VPA-exposed and BTBR mice at both protein and mRNA levels. Together, these preliminary findings suggest that down-regulation of nNOS may be an attractive target for the pharmacological intervention in autism.

Gastrodin Rescues Autistic-Like Phenotypes in Valproic Acid-Induced Animal Model.

Autism spectrum disorder (ASD) is an immensely challenging developmental disorder characterized by impaired social interaction, restricted/repetitive behavior, and anxiety. GABAergic dysfunction has been postulated to underlie these autistic symptoms. Gastrodin is widely used clinically in the treatment of neurological disorders and showed to modulate GABAergic signaling in the animal brain. The present study aimed to determine whether treatment with gastrodin can rescue valproic acid (VPA) induced autistic-like phenotypes, and to determine its possible mechanism of action. Our results showed that administration of gastrodin effectively alleviated the autistic-associated behavioral abnormalities as reflected by an increase in social interaction and decrement in repetitive/stereotyped behavior and anxiety in mice as compared to those in untreated animals. Remarkably, the amelioration in autistic-like phenotypes was accompanied by the restoration of inhibitory synaptic transmission, α5 GABAA receptor, and type 1 GABA transporter (GAT1) expression in the basolateral amygdala (BLA) of VPA-treated mice. These findings indicate that gastrodin may alleviate the autistic symptoms caused by VPA through regulating GABAergic synaptic transmission, suggesting that gastrodin may be a potential therapeutic target in autism.

Label-free quantitative proteomics unravels the importance of RNA processing in glioma malignancy.

Glioma, one of the most common cancers in human, is classified to different grades according to the degrees of malignancy. Glioblastoma (GBM) is known to be the most malignant (Grade IV) whereas low-grade astrocytoma (LGA, Grade II) is relatively benign. The mechanism underlying the pathogenesis and progression of glioma malignancy remains unclear. Here we report a quantitative proteomic study to elucidate the differences between GBM and LGA using liquid chromatography and tandem mass spectrometry followed by label-free quantification. A total of 136 proteins were differentially expressed in GBM for at least five folds in comparison with LGA. Ontological analysis revealed a close correlation between GBM-associated proteins and RNA processing. Interaction network analysis indicated that the GBM-associated proteins in the RNA processing were linked to crucial signaling transduction modulators including epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 1 (STAT1), and mitogen-activated protein kinase 1 (MAPK1), which were further connected to the proteins important for neuronal structural integrity, development and functions. Upregulation of 40S ribosomal protein S5 (RPS5), Ferritin Heavy chain (FTH1) and STAT1, and downregulation of tenascin R (TNR) were validated as representatives by immune assays. In summary, we revealed a panel of GBM-associated proteins and the important modulators centered at the RNA-processing network in glioma malignancy that may become novel biomarkers and help elucidate the underlying mechanism.

Yersinia pestis acetyltransferase-mediated dual acetylation at the serine and lysine residues enhances the auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.

Lysine acetylation is known as a post translational modification (PTM) by histone acetyltransferases (HAT) that modifies histones and non-histone proteins to regulate gene expression. Serine acetylation, however, is reported in mammalian hosts by serine acetyltransferase of Yersinia pestis (YopJ) during infection. The protein target and cellular function of bacterial YopJ in mammalian systems are not fully addressed. Here we report dual acetylation at the serine and lysine residues by transiently expressed serine acetyltransferase YopJ mimicking Y. pestis infection in HeLa cells. Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- (YopJ/non-YopJ 1.3-fold, p = 0.02) and 8 Lys- (YopJ/non-YopJ 3.5-fold, p = 0.00003) acetylation sites. Specifically, YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252. YopJ-mediated Ser- and Lys-acetylation of MARCH8 is further confirmed by Western blotting using the specific antibodies against MARCH8 Sac71 and pan-acetyl lysine. Functional study demonstrates that YopJ-mediated Ser- and Lys-acetylation affects the auto-ubiquitination of MARCH8. The mutant C172A of YopJ previously shown to abolish the acetyltransferase activity also reduces Ser- and Lys-acetylation and diminishes the auto-ubiquitination of MARCH8. In support, MARCH8 is indeed acetylated at serine and lysine in vitro by purified YopJ but the activity is reduced by the C172A mutant in YopJ. Our study provides evidence that bacterial serine acetyltransferase YopJ mediates Ser- and Lys-acetylation and affects auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.

H2A/K pseudogene mutation may promote cell proliferation.

Little attention has been paid to the histone H2A/K pseudogene. Results from our laboratory showed that 7 of 10 kidney cancer patients carried a mutant H2A/K pseudogene; therefore, we were interested in determining the relationship between mutant H2A/K and cell proliferation. We used shotgun and label-free proteomics methods to study whether mutant H2A/K lncRNAs affected cell proliferation. Quantitative proteomic analysis indicated that the expression of mutant H2A/K lncRNAs resulted in the upregulation of many oncogenes, which promoted cell proliferation. Further interaction analyses revealed that a proliferating cell nuclear antigen (PCNA)-protein interaction network, with PCNA in the center, contributes to cell proliferation in cells expressing the mutant H2A/K lncRNAs. Western blotting confirmed the critical upregulation of PCNA by mutant H2A/K lncRNA expression. Finally, the promotion of cell proliferation by mutant H2A/K lncRNAs (C290T, C228A and A45G) was confirmed using cell proliferation assays. Although we did not determine the exact mechanism by which the oncogenes were upregulated by the mutant H2A/K lncRNAs, we confirmed that the mutant H2A/K lncRNAs promoted cell proliferation by upregulating PCNA and other oncogenes. The hypothesis that cell proliferation is promoted by the mutant H2A/K lncRNAs was supported by the protein expression and cell proliferation assay results. Therefore, mutant H2A/K lncRNAs may be a new factor in renal carcinogenesis.

Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics.

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.

Myelin protein zero and its antibody in serum as biomarkers of n-hexane-induced peripheral neuropathy and neurotoxicity effects.

BACKGROUND: Chronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy. METHODS: We compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody. RESULTS: P0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls. CONCLUSIONS: P0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.