Proteomic Analysis of the Sphincter in a Neurogenic Bladder Caused by T10 Spinal Cord Injury.

OBJECTIVE: This study aimed to conduct proteomic analysis of the sphincter in a neurogenic bladder caused by T10 spinal cord injury. The differentially expressed proteins (DEPs) of the sphincters (internal urethral sphincter) in the neurogenic bladders (NBs) of rats after complete transection of the T10 spinal cord segment were screened using tandem mass tag (TMT)-based quantitative labeling, and their biological information was analyzed. METHODS: Twelve adult Sprague Dawley rats out of 40 were randomly assigned to the blank group (n = 12), while the remaining 28 were placed in the T10 spinal cord injury model via modified Hassan Shaker spinal cord transection; 12 of these rats were then randomly selected as the model group. The rats in both groups underwent urodynamics detection and hematoxylin and eosin (H&E) staining. The proteins expressed in the bladder sphincter were detected using TMT-based quantitative proteomics. DEPs were defined as proteins with fold change >1.5 or <1/1.5, p < 0.05, and unique peptide ≥2. The DEPs were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using KOBAS 3.0., and gene ontology functional annotation analysis was performed using the Cytoscape 3.7.1. BiNGO plug-in. The protein-protein interaction network was then constructed using the interactive gene-retrieval tool STRING and Cytoscape software. RESULTS: The leak-point pressure and maximum cystometric volume in the model group were significantly higher than those in the blank group (p < 0.01), and H&E staining showed continuous interruption of the bladder sphincter fibers in the model group. A total of 250 DEPs were screened in the bladder sphincter, 83 of which were up-regulated and 167 of which were down-regulated. KEGG analysis of the DEPs was used to screen 15 pathways, including metabolic pathways, extracellular matrix (ECM)-receptor interaction, adhesion spots, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, the cytochalasin signaling pathway, and the advanced glycation end-products (AGE)/receptor for AGEs (RAGE) signaling pathway in diabetic complications and vascular smooth muscle contraction. CONCLUSIONS: It is of great significance to explore the pathological mechanism of non-inhibitory contraction of the bladder sphincter caused by spinal cord injury above the T10 segment from the perspective of ECM-receptor interaction, focal adhesion-activated PI3K/Akt signaling pathway, and cell relaxation signaling pathways. Synaptic vesicle glycoprotein (Sv2A) involved in the release of neurotransmitters from synaptic vesicles, arrestin ß2 inhibitory proteins involved in α-adrenergic receptors and G-protein-coupled receptor internalization, and calmodulin and calmodulin binding protein involved in calcium-sensitive signaling pathways may be potential targets for developing new ways to treat bladder sphincter overactivity caused by T10 spinal cord injury.

Metabolic Engineering Strategies Based on Secondary Messengers (p)ppGpp and C-di-GMP To Increase Erythromycin Yield in Saccharopolyspora erythraea.

Secondary messengers (such as (p)ppGpp and c-di-GMP) were proved to play important roles in antibiotic biosynthesis in actinobacteria. In this study, we found that transcription levels of erythromycin-biosynthetic ( ery) genes were upregulated in nutrient limitation, which depended on (p)ppGpp in Saccharopolyspora erythraea. Further study demonstrated that the expression of ery genes and intracellular concentrations of (p)ppGpp showed synchronization during culture process. The erythromycin yield was significantly improved (about 200%) by increasing intracellular concentration of (p)ppGpp through introduction of C-terminally truncated (p)ppGpp synthetase RelA (1.43 kb of the N-terminal segment) from Streptomyces coelicolor into S. erythraea strain NRRL2338 (named as WT/pIB-P BAD- relA1-489). As the intracellular concentration of (p)ppGpp in an industrial erythromycin-high-producing strain E3 was greatly higher (about 10- to 100-fold) than WT strain, the applications of the above-described strategy did not work in E3 strain. Further research revealed that low concentration of 2-oxoglutarate in E3 strain exerted a "nitrogen-rich" pseudosignal, leading to the downregulation of nitrogen metabolism genes, which limited the use of nitrogen sources and thus the high intracellular (p)ppGpp concentration. Furthermore, the secondary messenger, c-di-GMP, was proved to be able to activate ery genes transcription by enhancing binding of BldD to promoters of ery genes. Overexpressing the diguanylate cyclase CdgB from S. coelicolor in S. erythraea increased the intracellular c-di-GMP concentration, and improved erythromycin production. These findings demonstrated that increasing the concentration of intracellular secondary messengers can activate ery genes transcription, and provided new strategies for designing metabolic engineering based on secondary messengers to improve antibiotics yield in actinobacteria.

Stiff person syndrome with elevated titers of antibodies against cardiolipin and ß2 glycoprotein 1: a case report and literature review.

We reported a Stiff person syndrome (SPS) patient with elevated autoantibodies against cardiolipin and ß2 glycoprotein 1 but without glutamic acid decarboxylase (GAD) antibodies. A 40-year male was admitted due to limited mouth opening for 1 week. His blood routine, biochemical, infectious diseases, tumor markers, radiographic examinations were all normal. At day 3 (D3) after admission, he developed paroxysmal systemic muscle rigidity. At D6, the on-duty physician occasionally gave oral clonazepam, which effectively relieved the symptom. At D13, the titers of cardiolipin and ß2 glycoprotein 1 autoantibodies elevated but the remaining autoantibodies were all in normal ranges. After clonazepam treatment for 1 week, the symptoms were basically relieved, and the titers of these two antibodies returned to normal range with the relief of symptoms. During the 3 years of follow-up, the symptoms did not present again, and the titers of both antibodies were stable in the normal ranges. He had no tumor and other immune system diseases. In summary, we reported a SPS case with elevated cardiolipin and ß2 glycoprotein 1 autoantibodies. The patient was highly responsive to clonazepam therapy, and had favorable outcome in the 3 years follow-up. Our report is helpful for better understand the heterogeneous feature of SPS.