Long-Acting Heterodimeric Paclitaxel-Idebenone Prodrug-Based Nanomedicine Promotes Functional Recovery after Spinal Cord Injury.

After spinal cord injury (SCI), successive systemic administration of microtubule-stabilizing agents has been shown to promote axon regeneration. However, this approach is limited by poor drug bioavailability, especially given the rapid restoration of the blood-spinal cord barrier. There is a pressing need for long-acting formulations of microtubule-stabilizing agents in treating SCI. Here, we conjugated the antioxidant idebenone with microtubule-stabilizing paclitaxel to create a heterodimeric paclitaxel-idebenone prodrug via an acid-activatable, self-immolative ketal linker and then fabricated it into chondroitin sulfate proteoglycan-binding nanomedicine, enabling drug retention within the spinal cord for at least 2 weeks and notable enhancement in hindlimb motor function and axon regeneration after a single intraspinal administration. Additional investigations uncovered that idebenone can suppress the activation of microglia and neuronal ferroptosis, thereby amplifying the therapeutic effect of paclitaxel. This prodrug-based nanomedicine simultaneously accomplishes neuroprotection and axon regeneration, offering a promising therapeutic strategy for SCI.

Evaluation of the clinical application value of cytokine expression profiles in the differential diagnosis of prostate cancer.

BACKGROUND: The significance of tumor-secreted cytokines in tumor development has gained substantial attention. Nevertheless, the precise role of tumor-related inflammatory cytokines in prostate cancer (PCa) remains ambiguous. OBJECTIVES: To gain deeper insights into the inflammatory response in the process of PCa. METHODS: A total of 233 cases were collected, including 80 cases of prostate hyperplasia as disease control, 65 cases of postoperative prostate cancer and 36 cases of prostate cancer as PCa group. Additionally, 52 patients undergoing physical examinations during the same period were collected as the healthy control. The levels of 12 inflammatory cytokines in peripheral blood samples were analyzed using flow cytometric bead array technology. The levels of total prostate-specific antigen (TPSA) and free prostate-specific antigen (FPSA) in peripheral blood samples were analyzed using electrochemiluminescence technology. RESULTS: Our findings revealed significant increases in serum IL-8 levels in PCa group compared to the healthy control group. Additionally, IL-6, IL-10, IFN-γ and IL-12p70 levels were markedly elevated in the PCa group compared to the disease control group (all p < 0.05). Conversely, the level of IL-4, TNF-α, IL-1ß, IL-17A and IFN-α were lower in the PCa group compared to those in control group. Following surgery, the concentration of IL-6 decreased; whereas, the concentrations of IL-4, TNF-α, IL-17A, IL-1ß, IL-12p70, and IFN-α increased, demonstrating significant differences (p < 0.05). The differential upregulation of IL-6 or downregulation of IL-17A in peripheral blood exhibited diagnostic efficacy in PCa patients. Moreover, we observed a significant increase in IL-17A levels, accompanied by decreased of IL-2, IL-4, IL-10, TNF-a, IFN-γ, IL-1ß, and IL-12P70 in patients with distant metastasis. CONCLUSION: The peripheral blood cytokines are closely associated with the occurrence and development of prostate cancer, especially the serum levels of IL-6 and IL-17A may be useful as potential predictors of PCa diagnosis.

Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood-spinal cord barrier breakdown during acute spinal cord injury in rats.

BACKGROUND: Nafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action. METHODS: Time to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood-spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence. RESULTS: The optimal administration time window of nafamostat was 2-12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2-12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site. CONCLUSIONS: Our study provides preclinical information of nafamostat about the administration time window of 2-12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.

Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli.

Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system.IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

Iron-sulphur cluster biogenesis factor LYRM4 is a novel prognostic biomarker associated with immune infiltrates in hepatocellular carcinoma.

BACKGROUND: LYRM4 is necessary to maintain the stability and activity of the human cysteine desulfurase complex NFS1-LYRM4-ACP. The existing experimental results indicate that cancer cells rely on the high expression of NFS1. However, the role of LYRM4 in liver hepatocellular carcinoma (LIHC) remains unclear. METHODS: In this study, we combined bioinformatics analysis and clinical specimens to evaluate the mRNA, protein expression, and gene regulatory network of LYRM4 in LIHC. Furthermore, we detected the activity of several classical iron-sulphur proteins in LIHC cell lines through UV-vis spectrophotometry. RESULTS: The mRNA and protein levels of LYRM4 were upregulated in LIHC. Subsequent analysis revealed that the LYRM4 mRNA expression was related to various clinical stratifications, prognosis, and survival of LIHC patients. In addition, the mRNA expression of LYRM4 was significantly associated with ALT, tumour thrombus, and encapsulation of HBV-related LIHC patients. IHC results confirmed that LYRM4 was highly expressed in LIHC tissues and showed that the expression of LYRM4 protein in LIHC was significantly correlated with age and serum low-density lipoprotein (LDL) and triglyceride (TG) content. In particular, the mRNA expression of key iron- sulphur proteins POLD1 and PRIM2 was significantly overexpressed and correlated with poor prognosis in LIHC patients. Compared with hepatocytes, the activities of mitochondrial complex I and aconitate hydratase (ACO2) in LIHC cell lines were significantly increased. These results indicated that the iron-sulphur cluster (ISC) biosynthesis was significantly elevated in LIHC, leading to ISC-dependent metabolic reprogramming. Changes in the activity of ISC-dependent proteins may also occur in paracancerous tissues. Further analysis of the biological interaction and gene regulation networks of LYRM4 suggested that these genes were mainly involved in the citric acid cycle and oxidative phosphorylation. Finally, LYRM4 expression in LIHC was significantly positively correlated with the infiltrating levels of six immune cell types, and both factors were strongly associated with prognosis. CONCLUSION: LYRM4 could be a novel prognostic biomarker and molecular target for LIHC therapy. In particular, the potential regulatory networks of LYRM4 overexpression in LIHC provide a scientific basis for future research on the role of the ISC assembly mechanism and LYRM4-mediated sulphur transfer routes in carcinogenesis.

Biodetection and bioremediation of copper ions in environmental water samples using a temperature-controlled, dual-functional Escherichia coli cell.

Although a variety of whole-cell biosensors and biosorbents have been developed for detection and removal of heavy metal contaminants, few whole cells can be applied to both monitoring and remediation of copper pollution in water. In this study, a modified plasmid was constructed by incorporating a copper-sensing element and a copper-adsorbing element into a temperature-inducible plasmid, pBV220. This plasmid was subsequently transformed into an engineered Escherichia coli strain lacking copA and cueO. This dual-functional E. coli cell selectively responded to copper ions with a linear detection range of 0.01-25 µM at 37 °C and could express surface-displayed CueR when treated at 42 °C without any costly chemical inducers. The display of CueR on the cell surface specifically enhanced its copper adsorption capacity and rapidly removed copper ions from aqueous solutions. In addition, the CueR surface-displayed cells could be regenerated by adsorption-desorption cycles via pH regulation. Moreover, by simply using two different temperatures, the detection or adsorption of copper using this dual-functional whole cell was achieved without any cross-interference. Most importantly, it provided highly sensitive, accurate quantification, and effective removal of copper in real environmental water samples. Thus, this E. coli cell can be used for large-scale detection and remediation of copper pollutants.

EGFR phosphorylates FAM129B to promote Ras activation.

Ras GTPase-activating proteins (GAPs) are important regulators for Ras activation, which is instrumental in tumor development. However, the mechanism underlying this regulation remains elusive. We demonstrate here that activated EGFR phosphorylates the Y593 residue of the protein known as family with sequence similarity 129, member B (FAM129B), which is overexpressed in many types of human cancer. FAM129B phosphorylation increased the interaction between FAM129B and Ras, resulting in reduced binding of p120-RasGAP to Ras. FAM129B phosphorylation promoted Ras activation, increasing ERK1/2- and PKM2-dependent ß-catenin transactivation and leading to the enhanced glycolytic gene expression and the Warburg effect; promoting tumor cell proliferation and invasion; and supporting brain tumorigenesis. Our studies unearthed a novel and important mechanism underlying EGFR-mediated Ras activation in tumor development.

Hematopoietic stem cell transplantation without in vivo T-cell depletion for pediatric aplastic anemia: A single-center experience.

For young patients, HLA-MRD HSCT is the first-line treatment of SAA. However, due to China's birth control policy, few patients could find suitable sibling donors and HLA-MUD. More and more transplantation centers have used Haplo-D as the donor source for young adult and pediatric patients. However, studies with larger amount of pediatric patients are rare. We retrospectively analyzed the data of children with AA who were treated with allogeneic HSCT and compared the therapeutic efficacy of Haplo-HSCT and MRD/MUD group. A total of 62 patients were enrolled. Implantation was successfully performed in 58 patients. There was no significant difference in the time for reconstruction of hematopoietic function between patients in the two groups. Thirty-two had grade I-IV aGVHD with incidence of 51.61%. The incidence of aGVHD was 79.41% for patients in the Haplo-HSCT, significantly higher than that of 17.86% for patients in the MRD/MUD group (P < .01). However, the incidence of cGVHD was not significantly different between patients in the two groups (26.47% vs 10.71%, P = .09), the incidence of CMV infection was 28.57% and 52.94% for patients in the MRD/MUD and Haplo group, respectively, showing no significant difference (P = .053). The incidence of EBV infection was 47.06% for patients in the Haplo group and 28.57% for patients in the MRD/MUD group, showing no significant difference (P = .11). However, the 3- and 5-year cumulative OS and FFS rates showed statistically significant difference in the two groups, P = .012 and .045, respectively. Compared to Haplo-HSCT, MRD/MUD is more economic. In this study, we achieved good Haplo transplantation results. The incidences of cGVHD and CMV/EBV were not significantly different between Haplo group and MRD/MUD group. Although OS and FFS of the Haplo group were not as good as those of the MRD/MUD group, it is still acceptable as an alternative treatment under emergency.

Polygonatum odoratum Polysaccharides Modulate Gut Microbiota and Mitigate Experimentally Induced Obesity in Rats.

Increasing evidence suggests that the gut microbiota plays vital roles in metabolic diseases. Polygonatum odoratum extract alleviates hyperglycemia and hyperlipidemia, but the underlying mechanism remains unclear. This study investigated the effects of P. odoratum polysaccharides (POPs) on high-fat diet (HFD)-induced obesity in rats and whether these effects were related to modulation of gut microbiota. POP treatment attenuated weight gain, fat accumulation, epididymal adipocyte size, liver triglycerides, and total liver cholesterol content in HFD-fed rats. POP administration also increased short-chain fatty acids (SCFAs), including isobutyric acid, butyric acid, and valeric acid. POP upregulated the expression of genes involved in adipocyte differentiation (Pparg, Cebpa, Cebpb) and lipolysis (Ppara, Atgl), and downregulated those related to lipid synthesis (Srebpf1, Fabp4, Fas), with corresponding changes in PPARγ and FABP4 protein expression. Finally, POP enhanced species richness and improved the gut microbiota community structure, reducing the relative abundances of Clostridium, Enterococcus, Coprobacillus, Lactococcus, and Sutterella. Principal coordinates analysis (PCoA) revealed a clear separation between HFD-fed rats and all other treatment groups. Correlation analysis identified negative and positive associations between obesity phenotypes and 28 POP-influenced operational taxonomic units (OTUs), including putative SCFA-producing bacteria. Our data suggest that POP supplementation may attenuate features of obesity in HFD-fed rats in association with the modulation of gut microbiota.

Artesunate induces ROS-dependent apoptosis via a Bax-mediated intrinsic pathway in Huh-7 and Hep3B cells.

Artesunate (ARS), an artemisinin derivative, has been demonstrated to possess antitumor activity in various human tumor cells. This study aims to investigate the molecular mechanism by which ARS induces apoptosis in human hepatocellular carcinoma cells (Huh-7 and Hep3B cells). ARS effectively induced externalization of phosphatidylserine (PS), depolarization of mitochondrial membrane, release of cytochrome c from mitochondria, and activation of caspase-9 and 3, characteristics of the intrinsic apoptosis. Pretreatment with antioxidant N-Acetyle-Cysteine (NAC) completely blocked ARS-induced reactive oxygen species (ROS) generation and apoptosis in the two cell lines. ARS increased cellular iron ions level in Huh-7/Hep3B cells, but decreased cellular iron ions level in HepG2 cells. Pifithrin-alpha (PFT), an inhibitor of p53, significantly enhanced ARS-induced cytotoxicity in HepG2 cells, and the forced expression of wild-type p53 significantly enhanced ARS-induced cytotoxicity in Hep3B cells. In addition, ARS induced translocation and activation of the proapoptotic Bax, and silencing Bax remarkably inhibited ARS-induced apoptosis and ΔΨm collapse in Huh-7 and Hep3B cells, demonstrating the key role of Bax in ARS-induced apoptosis. Collectively, our data demonstrate that ARS induces ROS-dependent apoptosis via a Bax-mediated intrinsic pathway in Huh-7 and Hep3B cells.

Artesunate induces apoptosis via a ROS-independent and Bax-mediated intrinsic pathway in HepG2 cells.

This study aims to explore the detail molecular mechanism by which artesunate (ARS), an artemisinin derivative, induces apoptosis in HepG2 cells. ARS induced a loss of mitochondrial transmemberane potential (ΔΨm), phosphatidylserine (PS) externalization, as well as activations of Bax/Bak and caspases indicative of apoptosis induction. Silencing Bax but not Bak significantly inhibited ARS-induced apoptosis, demonstrating the key role of the Bax-mediated intrinsic pathway. Although ARS increased intracellular reactive oxygen species (ROS), ARS-induced apoptosis was neither prevented by pretreatment with ROS scavengers nor potentiated by pretreatment with l-buthionine-sulfoximine (BSO) that enhanced the ARS-induced intracellular ROS generation, demonstrating that ROS was not involved in ARS-induced apoptosis. In addition, ARS did not induce Bid translocation to mitochondria, and the cytotoxicity of ARS was not prevented by silencing Bim, Puma or Mcl-1, but was significantly enhanced by HA14-1 pretreatment, demonstrating that Bcl-2/-xl instead of Bid and Bim as well as Puma may be the upstream factor to regulate the Bax-mediated intrinsic pathway. Collectively, our data demonstrate that ARS induces ROS-independent apoptosis via the Bax-mediated intrinsic pathway in HepG2 cells.

Copper binding in IscA inhibits iron-sulphur cluster assembly in Escherichia coli.

Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.

Lysinuric protein intolerance with novel mutations in solute carrier family 7A member 7 in a Chinese family.

Introduction: Lysinuric protein intolerance (LPI) is a rare genetic disorder caused by mutations in the solute carrier family 7A member 7 (SLC7A7) gene. Case presentation: We presented two siblings with LPI, carrying novel mutations of c.776delT (p.L259Rfs*18) and c.155G>T (p.G52V) in SLC7A7. The younger sibling, preferring protein-rich foods, showed severe symptoms, including alveolar proteinosis, macrophage activation syndrome, severe diarrhea, and disturbance of consciousness with involuntary movements. In contrast, the elder sibling only had mild symptoms, likely due to aversion to protein-rich food since toddler age. Conclusion: LPI is a congenital genetic metabolic disease with multi-system involvement. Initiating appropriate protein-restricted diet therapy as soon as possible could help prevent the progression of LPI.

Celastrol inhibits oligodendrocyte and neuron ferroptosis to promote spinal cord injury recovery.

BACKGROUND: Spinal cord injury (SCI) is a traumatic injury to the central nervous system and can cause lipid peroxidation in the spinal cord. Ferroptosis, an iron-dependent programmed cell death, plays a key role in the pathophysiology progression of SCI. Celastrol, a widely used antioxidant drug, has potential therapeutic value for nervous system. PURPOSE: To investigate whether celastrol can be a reliable candidate for ferroptosis inhibitor and the molecular mechanism of celastrol in repairing SCI by inhibiting ferroptosis. METHODS: First, a rat SCI model was constructed, and the recovery of motor function was observed after treatment with celastrol. The regulatory effect of celastrol on ferroptosis pathway Nrf2-xCT-GPX4 was detected by Western blot and immunofluorescence. Finally, the ferroptosis model of neurons and oligodendrocytes was constructed in vitro to further verify the mechanism of inhibiting ferroptosis by celastrol. RESULTS: Our results demonstrated that celastrol promoted the recovery of spinal cord tissue and motor function in SCI rats. Further in vitro and in vivo studies showed that celastrol significantly inhibited ferroptosis in neurons and oligodendrocytes and reduced the accumulation of ROS. Finally, we found that celastrol could inhibit ferroptosis by up-regulating the Nrf2-xCT-GPX4 axis to repair SCI. CONCLUSION: Celastrol effectively inhibits ferroptosis after SCI by upregulating the Nrf2-xCT-GPX4 axis, reducing the production of lipid ROS, protecting the survival of neurons and oligodendrocytes, and improving the functional recovery.

PPIH gene regulation system and its prognostic significance in hepatocellular carcinoma: a comprehensive analysis.

BACKGROUND: Peptidyl-prolyl isomerase H (PPIH) is a member of the cyclophilin protein family, which functions as a molecular chaperone and is involved in the splicing of pre-mRNA. According to reports, the malignant progression of HCC related to hepatitis B virus (HBV) is tightly associated with RNA-binding proteins. Nevertheless, there is no research on PPIH expression or its function in the occurrence and progression of HCC. RESULTS: We are the first to reveal that the mRNA and protein levels of Ppih are substantially overexpressed in HCC, as the outcomes show. A significant correlation existed between enriched expression of Ppih within HCC and more advanced, poorly differentiated, and TP53-mutated tumors. CONCLUSION: These findings, which suggest that Ppih may serve as a predictive biomarker for people with HCC, serve as a starting point for further investigation into the function of Ppih in the progression of carcinogenesis. METHODS: Accordingly, we utilized clinical samples and bioinformatics analysis to assess Ppih's mRNA, protein expression, and gene regulatory system in HCC. Additionally, Wilcoxon signed-rank testing and logistic regression were utilized to inspect the association between clinicopathological factors and Ppih. Clinical pathological traits linked to overall survival (OS) among HCC patients were examined via TCGA data via Cox regression and the Kaplan-Meier approach. Additionally, via TCGA data collection, gene set enrichment assessment was also conducted.

The discovery of the new mechanism: Celastrol improves spinal cord injury by increasing cAMP through VIP-ADCYAP1R1-GNAS pathway.

Spinal cord injury (SCI) is a debilitating condition that results in significant impairment of motor function and sensation. Despite the ongoing efforts to develop effective treatments, there are currently very limited options available for patients with SCI. Celastrol, a natural anti-inflammatory compound extracted from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory and anti-apoptotic properties. In this study, we aimed to explore the therapeutic potential of celastrol for SCI and elucidate the underlying molecular mechanisms involved. We found that local tissue often experiences a significant decrease in cAMP content and occurrs apoptosis after SCI. However, the treatment of celastrol could promote the production of cAMP by up-regulating the VIP-ADCYAP1R1-GNAS pathway. This could effectively inhibit the phosphorylation of JNK and prevent apoptosis, ultimately improving the exercise ability after SCI. Together, our results reveal celastrol may be a promising therapeutic agent for the treatment of SCI.

Roles of conserved active site residues in the IscS cysteine desulfurase reaction.

Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury.

Maintaining the integrity of the blood-spinal cord barrier is critical for the recovery of spinal cord injury. Ferroptosis contributes to the pathogenesis of spinal cord injury. We hypothesized that ferroptosis is involved in disruption of the blood-spinal cord barrier. In this study, we administered the ferroptosis inhibitor liproxstatin-1 intraperitoneally after contusive spinal cord injury in rats. Liproxstatin-1 improved locomotor recovery and somatosensory evoked potential electrophysiological performance after spinal cord injury. Liproxstatin-1 maintained blood-spinal cord barrier integrity by upregulation of the expression of tight junction protein. Liproxstatin-1 inhibited ferroptosis of endothelial cell after spinal cord injury, as shown by the immunofluorescence of an endothelial cell marker (rat endothelium cell antigen-1, RECA-1) and ferroptosis markers Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Liproxstatin-1 reduced brain endothelial cell ferroptosis in vitro by upregulating glutathione peroxidase 4 and downregulating Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Furthermore, inflammatory cell recruitment and astrogliosis were mitigated after liproxstatin-1 treatment. In summary, liproxstatin-1 improved spinal cord injury recovery by inhibiting ferroptosis in endothelial cells and maintaining blood-spinal cord barrier integrity.

Peripheral ulcerative keratitis, nodular episcleritis, and pulmonary nodules as the initial signs of rheumatic arthritis: A Case Report.

Background: Rheumatoid vasculitis (RV) is a rare but potentially devastating complication of rheumatoid arthritis (RA). It typically occurs in patients with extra-articular manifestations. Here we reported a case of PUK with nodular episcleritis and pulmonary nodules that occurred in the same patient without joint involvement. Case presentation: A 43-year-old Chinese woman, exhibited a partial crescent-shaped marginal corneal ulcer in the right eye at admission and the ulcer developed rapidly into nearly 360-degree ulcers in both eyes within one week. Nodular episcleritis was observed in the right eye. Conjunctival biopsy revealed vasculitis. Her rheumatoid factor (RF) and anti-cyclic citrullinated protein antibody were positive, while anti-neutrophilic cytoplasmic antibody (c-ANCA) and anti-protease 3 were negative. Pulmonary nodules were found, without joint involvement. The ocular condition did not relieve under the topical and systemic use of corticosteroids, or under other immunosuppressive agents until the infliximab therapy. PUK recurrence was observed after the discontinuation of infliximab. Conclusions: Rapidly deteriorated PUK with nodular episcleritis and pulmonary nodules occurred in the same patient is a special case of RA without joint involvement. This case reinforces the concept that RV may be the initial sign of RA. Infliximab can be used to prevent further progress of RA-related PUK in some refractory cases.

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments. These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment. In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.