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Objective To investigate the analgesic effect of transcranial direct current stimulation (tDCS) on chronic pain in a rat model of monosodium iodoacetate (MIA)-induced osteoarthritis (OA) and its mechanism. Methods Healthy male Sprague-Dawley rats were randomly divided into sham-operation group, MIA group, tDCS group, and sham-tDCS group, with 9 rats in each group. The rats in the sham-operation group were given intra-articular injection of normal saline into the left knee joint, and those in the MIA group, the tDCS group, and the sham-tDCS group were given MIA injection at the same site to establish a model of chronic pain in OA. On day 21 after MIA injection, the rats in the tDCS group and the sham-tDCS group were treated with real or sham tDCS, respectively, for 8 d. The pain behavioral test was performed for the rats in each group on day 1, 2, and 7 after treatment; the periaqueductal gray (PAG) of rats were collected on day 2 after treatment and Western blot was used to measure the expression of brain-derived neurotrophic factor (BDNF). Results The pain behavioral test showed that compared with the sham-tDCS group, the tDCS group had a significantly longer thermal paw withdrawal latency at each time point after treatment (F=11.25-21.48,P

Objective·To prepare a high-purity cyanobacterial serine protease inhibitor, screen its target proteases, and detect its inhibitory activity.Methods·A novel serine protease inhibitor from Arthrospira platensis was identified in the Expanded Human Oral Microbiome Database (eHOMD) by amino acid sequence alignment and named as arthropin. The fusion expression vector pSUMO3-arthropin was constructed and transferred into Escherichia coli (E. coli) BL21(DE3) system for fusion protein expression. The recombinant arthropin was purified by a four-step chromatographic purification approach of nickel affinity chromatography, enzymatic digestion, reverse nickel affinity chromatography, and anion exchange chromatography. In addition, the recombinant arthropin was co-incubated with 14 serine proteases such as activated factor Ⅸ (FⅨa), FⅩa, FⅪa, activated protein C (APC) and kallikrein 1 (KLK1), respectively, and then analyzed by SDS-PAGE. The inhibitory rate of arthropin on KLK1 was assayed with kinetic methods. The crystallization conditions of the recombinant arthropin were screened preliminarily, and the suitable crystals were picked for X-ray diffraction to collect the data. Finally, a sub-stable structure model of arthropin was predicted with AlphaFlod Colab.Results·SDS-PAGE showed that the fused arthropin was successfully expressed in the E. coli BL21(DE3) system, and following purification, the high-purity recombinant arthropin, the relative molecular mass of which was similar to the theoretical value (45 800), was obtained. The co-incubation analysis of recombinant arthropin with 14 serine proteases revealed that arthropin was able to form stable covalent complexes with 9 proteases, including FⅩa, APC, FⅨa, FⅪa, trypsin, cathepsin G, KLK1, KLK7 and thrombin. Arthropin inhibited KLK1 with a second-order association rate constant of 1.7×103 L/(mol·s). Moreover, the recombinant arthropin crystalised under the condition of 25% PEG MME 550, 0.1 mol/L MES (pH 6.5) and 0.01 mol/L ZnCl2 , and the crystals preliminarily diffracted to a resolution of 10 Å (1 Å=1×10-10 m). The analysis of the structure predicted by AlphaFlod Colab revealed that arthropin had the classical structural features of the inhibitory serpin.Conclusion·Arthropin, a serpin from Arthrospira platensis, was successfully obtained with high purity and a broad-spectrum of serine protease inhibition, but at a low inhibitory rate.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.