A Novel Rabbit Anti-Myoglobin Monoclonal Antibody's Potential Application in Rhabdomyolysis Associated Acute Kidney Injury.

Myoglobin (Mb) is the main constituent of vertebrate skeletal muscle and myocardium and plays an essential role in oxygen binding, storage, transport, and earliest disease diagnosis. This study focuses on preparing the novel recombinant rabbit anti-Mb monoclonal antibody and applying it to a diagnosis of Mb deposition in rhabdomyolysis-associated acute kidney injury (RM-AKI). The full-length coding sequence of rat Mb was cloned and expressed, and the high-quality and titer rabbit anti-Mb polyclonal antibodies were produced by the immunogen His-Mb fusion protein. A new hybridoma cell was obtained by hybridoma screening technology. With the help of DNA sequencing and a molecular clonal, anti-Mb monoclonal antibody heavy and light chains expression plasmid was constructed. Finally, the recombinant rabbit anti-Mb monoclonal antibody with extraordinarily high affinity (KD = 1.21 pM) was obtained. Meanwhile, it had broad species reactivity (mouse, rat, human, and horse) and good tissue specificity (skeletal muscle and myocardium). It also had a very good performance in western blotting, immunohistochemistry, and immunofluorescence assay to detect the Mb level in the kidney, myocardium, and skeletal muscle of RM-AKI. This study will be significantly helpful for Mb-associated disease diagnosis, and pathogenesis exploration, and further may act as a neutralizing antibody for disease treatment.

Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells.

Repetitive low-level blast (rLLB) exposure is a potential risk factor for the health of soldiers or workers who are exposed to it as an occupational characteristic. Alveolar macrophages (AMs) are susceptible to external blast waves and produce pro-inflammatory or anti-inflammatory effects. However, the effect of rLLB exposure on AMs is still unclear. Here, we generated rLLB waves through a miniature manual Reddy-tube and explored their effects on MH-S cell morphology, phenotype transformation, oxidative stress status, and apoptosis by immunofluorescence, real-time quantitative PCR (qPCR), western blotting (WB) and flow cytometry. Ipatasertib (GDC-0068) or PDTC was used to verify the role of the Akt/NF-κB signaling pathway in these processes. Results showed that rLLB treatment could cause morphological irregularities and cytoskeletal disorders in MH-S cells and promote their polarization to the M1 phenotype by increasing iNOS, CD86 and IL-6 expression. The molecular mechanism is through the Akt/NF-κB signaling pathway. Moreover, we found reactive oxygen species (ROS) burst, Ca2+ accumulation, mitochondrial membrane potential reduction, and early apoptosis of MH-S cells. Taken together, our findings suggest rLLB exposure may cause M1 polarization and early apoptosis of AMs. Fortunately, it is blocked by specific inhibitors GDC-0068 or PDTC. This study provides a new treatment strategy for preventing and alleviating health damage in the occupational population caused by rLLB exposure.

Pathway network of pyroptosis and its potential inhibitors in acute kidney injury.

Acute kidney injury (AKI) is a worldwide problem, and there is no effective drug to eliminate AKI. The death of renal cells is an important pathological basis of intrinsic AKI. At present, targeted therapy for TEC death is a research hotspot in AKI therapy. There are many ways of cell death involved in the occurrence and development of AKI, such as apoptosis, necrosis, ferroptosis, and pyroptosis. This article mainly focuses on the role of pyroptosis in AKI. The assembly and activation of NLRP3 inflammasome is a key event in the occurrence of pyroptosis, which is affected by many factors, such as the activation of the NF-κB signaling pathway, mitochondrial instability and excessive endoplasmic reticulum (ER) stress. The activation of NLRP3 inflammasome can trigger its downstream inflammatory cytokines, which will lead to pyroptosis and eventually induce AKI. In this paper, we reviewed the possible mechanism of pyroptosis in AKI and the potential effective inhibitors of various key targets in this process. It may provide potential therapeutic targets for novel intrinsic AKI therapies based on pyroptosis, so as to develop better therapeutic strategies.

Emerging medical therapies in crush syndrome - progress report from basic sciences and potential future avenues.

Crush injury is a disease that is commonly found in victims of earthquakes, debris flows, mine disasters, explosions, terrorist attacks, local wars, and other accidents. The complications that arise due to the crush injury inflicted on victims give rise to crush syndrome (CS). If not treated in time, the mortality rate of CS is very high. The most important measure that can be taken to reduce mortality in such situations is to immediately start treatment. However, the traditional treatment methods such as fluid resuscitation, diuresis, and hemodialysis are not feasible enough to be carried out at the disaster scene. So there is a need for developing new treatments that are efficient and convenient. Because it is difficult to diagnose in the disaster area and reach the treatment equipment and treat on time. It has become a new research needs to be directed into identifying new medical treatment targets and methods using the etiology and pathophysiological mechanisms of CS. In recent years, a large number of new anti-oxidant and anti-inflammatory drug therapies have been shown to be highly efficacious in CS rat/mouse models. Some of them are expected to become specific drugs for the emergency treatment of a large number of patients who may develop CS in the aftermath of earthquakes, wars, and other disasters in the future. Hence, we have reviewed the latest research on the medical therapy of CS as a source for anyone wishing to pursue research in this direction.

Damage-Associated Molecular Patterns and Their Signaling Pathways in Primary Blast Lung Injury: New Research Progress and Future Directions.

Primary blast lung injury (PBLI) is a common cause of casualties in wars, terrorist attacks, and explosions. It can exist in the absence of any other outward signs of trauma, and further develop into acute lung injury (ALI) or a more severe acute respiratory distress syndrome (ARDS). The pathogenesis of PBLI at the cellular and molecular level has not been clear. Damage-associated molecular pattern (DAMP) is a general term for endogenous danger signals released by the body after injury, including intracellular protein molecules (HMGB1, histones, s100s, heat shock proteins, eCIRP, etc.), secretory protein factors (IL-1ß, IL-6, IL-10, TNF-α, VEGF, complements, etc.), purines and pyrimidines and their derived degradation products (nucleic acids, ATP, ADP, UDPG, uric acid, etc.), and extracellular matrix components (hyaluronic acid, fibronectin, heparin sulfate, biglycan, etc.). DAMPs can be detected by multiple receptors including pattern recognition receptors (PRRs). The study of DAMPs and their related signaling pathways, such as the mtDNA-triggered cGAS-YAP pathway, contributes to revealing the molecular mechanism of PBLI, and provides new therapeutic targets for controlling inflammatory diseases and alleviating their symptoms. In this review, we focus on the recent progress of research on DAMPs and their signaling pathways, as well as the potential therapeutic targets and future research directions in PBLI.

HIV and syphilis and sexual risk behaviours among men who have sex with men attending university in China: a systematic review and meta-analysis.

Background This study was conducted to summarise the HIV epidemic, sexual behaviours and HIV testing among men who have sex with men (MSM) attending university in China. METHODS: Five databases were searched for student MSM information in English and Chinese language publications. Meta-analyses were performed to calculate the pooled prevalence of HIV and syphilis, pooled mean age at first anal intercourse (AFAI) and the rate of other HIV-related behaviours among MSM attending university in China. Univariate meta-regression and subgroup analysis were conducted to explore potential sources of heterogeneity. Publication bias was measured using Egger's test. RESULTS: Thirty-three articles representing 31 studies were included in the analysis. The pooled HIV prevalence was 4.1% (95% CI 3.1-5.0%). The estimated AFAI was 18.7 years, but 37.5% of students had their first anal intercourse before 18 years of age. Most (88.2%) had their first sexual intercourse with a male partner. Of the MSM attending university, 4.2% of MSM engaged in commercial sex (either selling or buying sex), 10.3% had ever engaged in group sex, 13.1% had had sex with a female partner in the past month and 10.1% had ever used drugs. Most (77.7%) sought sex partners via geosocial networking gay apps or the Internet, and 42.9% had ever tested for HIV. There was a tendency for an increase in lifetime HIV testing rate from 32% in 2005-07 to 53% in 2014-16. CONCLUSIONS: This review found high HIV prevalence, early AFAI and a high prevalence of sexual risk behaviours among MSM attending university in China. Interventions aimed at increasing HIV testing and reducing sexual risk behaviours are urgently needed among this young population.

Study on the allocation efficiency of medical and health resources in Hainan Province: Based on the super-efficiency SBM-Malmquist model.

The equity and efficiency of medical and health resource allocation is the key point of health reform in all countries. Poor allocation efficiency of health resources will seriously affect the sustainable and high-quality development of health causes. Hainan Province is the only free trade port with Chinese characteristics in China, which means that Hainan has ushered in a brand-new development under the policy of free trade port. This study aims to adopt policies to improve the efficiency of medical and health resource allocation in Hainan Province and provide references for other regions. In this study, the Super-efficiency SBM and Malmquist models were used to analyze the static and dynamic efficiency of medical and health resource allocation in Hainan Province during 2016-2020. The results showed that, statically, the average efficiency of comprehensive allocation of health resources in Hainan Province from 2016 to 2020 was 0.975, showing poor overall performance and significant regional differences. Dynamically, the average index of allocation efficiency of medical and health resources was 0.934, showing a negative growth trend. The technical efficiency and scale efficiency of health resource allocation efficiency showed positive growth, while the technical progress and pure technical efficiency showed negative growth. It shows that it is influenced by both scale efficiency and technological progress, among which technological progress is the key factor. Therefore, some policy suggestions are put forward to further optimize the allocation of medical and health resources and improve utilization efficiency.

Rosmarinic acid plus deferasirox inhibits ferroptosis to alleviate crush syndrome-related AKI via Nrf2/Keap1 pathway.

BACKGROUND: Myoglobin (Mb) induced death of renal tubular epithelial cells (RTECs) is a major pathological factor in crush syndrome-related acute kidney injury (CS-AKI). It is unclear whether ferroptosis is involved and could be a target for treatment. PURPOSE: This study aimed to evaluate the potential therapeutic effects of combining the natural small molecule rosemarinic acid (RA) and the iron chelator deferasirox (Dfe) on CS-AKI through inhibition of ferroptosis. METHODS: Sequencing data were downloaded from the GEO database, and differential expression analysis was performed using the R software limma package. The CS-AKI mouse model was constructed by squeezing the bilateral thighs of mice for 16 h with 1.5 kg weight. TCMK1 and NRK-52E cells were induced with 200 µM Mb and then treated with RA combined with Dfe (Dfe + RA, both were 10 µM). Functional and pathological changes in mouse kidney were evaluated by glomerular filtration rate (GFR) and HE pathology. Immunofluorescence assay was used to detect Mb levels in kidney tissues. The expression levels of ACSL4, GPX4, Keap1, and Nrf2 were analyzed by WB. RESULTS: We found that AKI mice in the GSE44925 cohort highly expressed the ferroptosis markers ACSL4 and PTGS2. CS-AKI mice showed a rapid decrease in GFR, up-regulation of ACSL4 expression in kidney tissue, and down-regulation of GPX4 expression, indicating activation of the ferroptosis pathway. Mb was found to deposit in renal tubules, and it has been proven to cause ferroptosis in TCMK1 and NRK-52E cells in vitro. We found that Dfe had a strong iron ion scavenging effect and inhibited ACSL4 expression. RA could disrupt the interaction between Keap1 andNrf2, stabilize Nrf2, and promote its nuclear translocation, thereby exerting antioxidant effects. The combination of Dfe and RA effectively reversed Mb induced ferroptosis in RTECs. CONCLUSION: In conclusion, we found that RA combined with Dfe attenuated CS-AKI by inhibiting Mb-induced ferroptosis in RTECs via activating the Nrf2/Keap1 pathway.

Nspc1 regulates the key pluripotent Oct4-Nanog-Sox2 axis in P19 embryonal carcinoma cells via directly activating Oct4.

Nspc1 is an identified transcription repressor. However, transiently up-regulated or down-regulated Nspc1 in P19 embryonal carcinoma cells affects expression levels of Oct4, Sox2 and Nanog in a positive correlation. Luciferase activity assays verified that Nspc1 regulates the Oct4 promoter in a dose dependent manner. ChIP assay shows that Nspc1 activates Oct4 by directly binding to the (-1021 to -784) region of Oct4 promoter. Dominant negative analysis indicated the activation is dependent on the retinoid acid response element (RARE). We demonstrated Nspc1 has a positive role in maintaining the pluripotency of P19 cells by directly regulating Oct4.

Association analysis of USF1 gene polymorphisms and total unstable carotid plaque area in atherosclerotic stroke patients.

Polymorphisms of the upstream stimulatory factor 1 (USF1) have been associated with carotid artery intima-media thickness and coronary atherosclerotic lesions. Unstable carotid plaque is an atherosclerotic change of vascular morphology that has been correlated with cerebrovascular ischemic symptoms. Associations of three single nucleotide polymorphisms of the USF1 gene with total unstable carotid plaque area (CPA) were investigated in Chinese atherosclerotic stroke patients. We recruited 668 atherosclerotic stroke patients and 602 controls. Total unstable CPA values were measured by ultrasound. Genotypes were analyzed using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) or mismatched PCR-RFLP. A significant difference in total unstable CPA was found for rs2516838 and rs2516839 genotypes (P = 0.039 and 0.046, respectively) in atherosclerotic stroke patients with unstable carotid plaque. Furthermore, in multiple logistic regression analysis adjusted by age, sex, BMI, hypertension, smoking status, glucose, total cholesterol, triglycerides, high-density lipoprotein-cholesterols, low-density lipoprotein-cholesterols and high-sensitivity C-reactive protein, significant associations were seen between the total unstable CPA values and genotypes of the rs2516838 or the rs2516839 in these patients. The rare allele C of rs2516838 or rare allele A of rs2516839 could predict relative low total unstable CPA values. The rs2516838 and rs2516839 polymorphisms of USF1 influence total unstable CPA in atherosclerotic stroke patients, which might be new markers to predict the risk of recurrence for this disease.

Ferroptosis in acute kidney injury following crush syndrome: A novel target for treatment.

BACKGROUND: Crush syndrome (CS) is a kind of traumatic and ischemic injury that seriously threatens life after prolonged compression. It is characterized by systemic inflammatory reaction, myoglobinuria, hyperkalemia and acute kidney injury (AKI). Especially AKI, it is the leading cause of death from CS. There are various cell death forms in AKI, among which ferroptosis is a typical form of cell death. However, the role of ferroptosis has not been fully revealed in CS-AKI. AIM OF REVIEW: This review aimed to summarize the evidence of ferroptosis in CS-AKI and its related molecular mechanism, discuss the therapeutic significance of ferroptosis in CS-AKI, and open up new ideas for the treatment of CS-AKI. KEY SCIENTIFIC CONCEPTS OF REVIEW: One of the main pathological manifestations of CS-AKI is renal tubular epithelial cell dysfunction and cell death, which has been attributed to massive deposition of myoglobin. Large amounts of myoglobin released from damaged muscle deposited in the renal tubules, impeding the normal renal tubules function and directly damaging the tubules with oxidative stress and elevated iron levels. Lipid peroxidation damage and iron overload are the distinguishing features of ferroptosis. Moreover, high levels of pro-inflammatory cytokines and damage-associated molecule pattern molecules (HMGB1, double-strand DNA, and macrophage extracellular trap) in renal tissue have been shown to promote ferroptosis. However, how ferroptosis occurs in CS-AKI and whether it can be a therapeutic target remains unclear. In our current work, we systematically reviewed the occurrence and underlying mechanism of ferroptosis in CS-AKI.

Biotherapy of experimental acute kidney injury: emerging novel therapeutic strategies.

Acute kidney injury (AKI) is a complex and heterogeneous disease with high incidence and mortality, posing a serious threat to human life and health. Usually, in clinical practice, AKI is caused by crush injury, nephrotoxin exposure, ischemia-reperfusion injury, or sepsis. Therefore, most AKI models for pharmacological experimentation are based on this. The current research promises to develop new biological therapies, including antibody therapy, non-antibody protein therapy, cell therapy, and RNA therapy, that could help mitigate the development of AKI. These approaches can promote renal repair and improve systemic hemodynamics after renal injury by reducing oxidative stress, inflammatory response, organelles damage, and cell death, or activating cytoprotective mechanisms. However, no candidate drugs for AKI prevention or treatment have been successfully translated from bench to bedside. This article summarizes the latest progress in AKI biotherapy, focusing on potential clinical targets and novel treatment strategies that merit further investigation in future pre-clinical and clinical studies.

Delayed step-by-step decompression with DSF alleviates skeletal muscle crush injury by inhibiting NLRP3/CASP-1/GSDMD pathway.

Crush injury (CI) is a common disease in earthquake and traffic accidents. It refers to long-term compression that induces ischemia and hypoxia injury of skeletal muscle rich parts, leading to rupture of muscle cells and release of contents into the blood circulation. Crush syndrome (CS) is the systemic manifestation of severe, traumatic muscle injury. CI rescue faces a dilemma. Ischemic reperfusion due to decompression is a double-edged sword for the injured. Death often occurs when the injured are glad to be rescued. Programmed cell death (PCD) predominates in muscle CI or ischemia-reperfusion injury. However, the function and mechanism of pyroptosis and apoptosis in the pathogenesis of skeletal muscle injury in CI remain elusive. Here, we identified that pyroptosis and apoptosis occur independently of each other and are regulated differently in the injured mice's skeletal muscle of CI. While in vitro model, we found that glucose-deprived ischemic myoblast cells could occur pyroptosis. However, the cell damage degree was reduced if the oxygen was further deprived. Then, we confirmed that delayed step-by-step decompression of CI mice could significantly reduce skeletal muscle injury by substantially inhibiting NLRP3/Casp-1/GSDMD pyroptosis pathway but not altering the Casp-3/PARP apoptosis pathway. Moreover, pyroptotic inhibitor DSF therapy alone, or the combination of delayed step-by-step decompression and pyroptotic inhibitor therapy, significantly alleviated muscle injury of CI mice. The new physical stress relief and drug intervention method proposed in this study put forward new ideas and directions for rescuing patients with CI, even CS-associated acute kidney injury (CS-AKI).

Review on visualization technology in simulation training system for major natural disasters.

Major natural disasters have occurred frequently in the last few years, resulting in increased loss of life and economic damage. Most emergency responders do not have first-hand experience with major natural disasters, and thus, there is an urgent need for pre-disaster training. Due to the scenes unreality of traditional emergency drills, the failure to appeal to the target audience and the novel coronavirus pandemic, people are forced to maintain safe social distancing. Therefore, it is difficult to carry out transregional or transnational emergency drills in many countries under the lockdown. There is an increasing demand for simulation training systems that use virtual reality, augmented reality, and mixed reality visualization technologies to simulate major natural disasters. The simulation training system related to natural disasters provides a new way for popular emergency avoidance science education and emergency rescue personnel to master work responsibilities and improve emergency response capabilities. However, to our knowledge, there is no overview of the simulation training system for major natural disasters. Hence, this paper uncovers the visualization techniques commonly used in simulation training systems, and compares, analyses and summarizes the architecture and functions of the existing simulation training systems for different emergency phases of common natural disasters. In addition, the limitations of the existing simulation training system in practical applications and future development directions are discussed to provide reference for relevant researchers to better understand the modern simulation training system.

ß1-Blocker improves survival and ventricular remodelling in rats with lethal crush injury.

BACKGROUND: Crush injury/crush syndrome (CI/CS) is the second most common cause of death during earthquakes. Most studies of CI/CS have mainly focused on kidney injury after decompression. Few studies have focused on myocardial injury caused by crush injury and its potential mechanisms. METHODS: We first verified cardiomyocyte injury during compression in rats with a crush injury. The survival rate, electrocardiographic results, histological results, catecholamine changes and cardiac ß1-AR expression were evaluated. Next, we explored the effects of pretreatment with a selective ß1-blocker (bisoprolol) with or without fluid resuscitation on rats with a crush injury. In addition to evaluating the survival rates, biochemical and histological analyses and echocardiographic measurements were also performed. RESULTS: Reduced heart rates, elevated ST segments, and tall-peaked T waves were observed in the rats with a crush injury. The changes in the myocardial enzymes and pathological results demonstrated that myocardial damage occurred during compression in rats with a crush injury. The levels of the catecholamine norepinephrine in both the serum and myocardial tissue were elevated during compression. Pretreatment with a selective ß1-blocker combined with fluid resuscitation significantly improved the survival rates of the rats with lethal crush injury. The myocardial enzymes and pathological results showed that the combined therapy decreased myocardial damage. The echocardiography measurements showed that the rats that received the combined therapy exhibited decreased left ventricular mass (LVM), left ventricular volume at end-systole (LVVs) and left ventricular internal diameter (LVID) compared with the rats with a crush injury. CONCLUSIONS: Our findings demonstrated the presence of myocardial injury in the early stage of compression in rats with a crush injury. Pretreatment with a ß1-blocker (bisoprolol) with fluid resuscitation significantly reduced mortality, decreased myocardial tissue damage, and improved ventricular remodelling in rats with a lethal crush injury.

Myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI.

Crush syndrome (CS) is a life-threatening illness in traffic accidents and earthquakes. Crush syndrome-induced acute kidney injury (CS-AKI) is considered to be mainly due to myoglobin (Mb) circulation and deposition after skeletal muscle ruptures and releases. Macrophages are the primary immune cells that fight foreign substances and play critical roles in regulating the body's natural immune response. However, what effect does myoglobin have on macrophages and the mechanisms involved in the CS-AKI remain unclear. This study aims to look into how myoglobin affects macrophages of the CS-AKI model. C57BL/6 mice were used to construct the CS-AKI model by digital crush platform. Biochemical analysis and renal histology confirmed the successful establishment of the CS-AKI mouse model. Ferrous myoglobin was used to treat Raw264.7 macrophages to mimic the CS-AKI cell model in vitro. The macrophage polarization toward M1 type and activation of RIG-I as myoglobin sensor were verified by real-time quantitative PCR (qPCR), Western blotting (WB), and immunofluorescence (IF). Macrophage pyroptosis was observed under light microscopy. The interaction between RIG-I and caspase1 was subsequently explored by co-immunoprecipitation (Co-IP) and IF. Small interfering RNA (siRIG-I) and pyroptosis inhibitor dimethyl fumarate (DMF) were used to verify the role of macrophage polarization and pyroptosis in CS-AKI. In the kidney tissue of CS-AKI mice, macrophage infiltration and M1 type were found. We also detected that in the cell model of CS-AKI in vitro, ferrous myoglobin treatment promoted macrophages polarization to M1. Meanwhile, we observed pyroptosis, and myoglobin activated the RIG-I/Caspase1/GSDMD signaling pathway. In addition, pyroptosis inhibitor DMF not only alleviated kidney injury of CS-AKI mice but also inhibited macrophage polarization to M1 phenotype and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway. Our research found that myoglobin promotes macrophage polarization to M1 type and pyroptosis via the RIG-I/Caspase1/GSDMD signaling pathway in CS-AKI.

Major signaling pathways and key mediators of macrophages in acute kidney injury (Review).

Acute kidney injury (AKI) has become a global public health problem with high morbidity and mortality rates, as well as high healthcare costs. Immune cells, particularly macrophages, which regulate tissue development, destroy pathogens, control homeostasis and repair wounds, play crucial and complex roles in AKI. In various types of AKI, numerous rapidly recruited monocytes and tissue­resident macrophages act in a coordinated manner. Thus, elucidating the phenotypic and functional characteristics of macrophages in AKI is essential for identifying potential therapeutic targets. Macrophage­sensing mediators and macrophage­derived mediators participate in the major macrophage­related signaling pathways in AKI, which regulate macrophage polarization and determine disease progression. In conclusion, macrophages change their roles and regulatory mechanisms during the occurrence and development of AKI. The aim of the present review was to contribute to an improved understanding of AKI and to the identification of novel therapeutic targets for this condition.

RIG-I, a novel DAMPs sensor for myoglobin activates NF-κB/caspase-3 signaling in CS-AKI model.

BACKGROUND: Acute kidney injury (AKI) is the main life-threatening complication of crush syndrome (CS), and myoglobin is accepted as the main pathogenic factor. The pattern recognition receptor retinoicacid-inducible gene I (RIG-I) has been reported to exert anti-viral effects function in the innate immune response. However, it is not clear whether RIG-I plays a role in CS-AKI. The present research was carried out to explore the role of RIG-I in CS-AKI. METHODS: Sprague-Dawley rats were randomly divided into two groups: the sham and CS groups (n = 12). After administration of anesthesia, the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions. The rats in both groups were denied access to food and water. Rats were sacrificed at 12 h or 36 h after pressure was relieved. The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination. In addition, RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI. Furthermore, NRK-52E cells were treated with 200 µmol/L ferrous myoglobin to mimic CS-AKI at the cellular level. The cells and cell supernatant samples were collected at 6 h or 24 h. Small interfering RNAs (siRNA) was used to knock down RIG-I expression. The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative Real-time PCR (qPCR), Western blotting analysis, and immunohistochemistry (IHC) staining. Tumor necrosis factor-α (TNF-α) was detected by ELISA. Co-Immunoprecipitation (Co-IP) assays were used to detect the interaction between RIG-I and myoglobin. RESULTS: RNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway. qPCR, Western blotting, and IHC assays showed that RIG-I, nuclear factor kappa-B (NF-κB) P65, p-P65, and the apoptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group (P < 0.05). However, the levels of interferon regulatory factor 3 (IRF3), p-IRF3 and the antiviral factor interferon-beta (IFN-ß) showed no significant changes between the sham and CS groups. Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group. Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis. CONCLUSION: RIG-I is a novel damage-associated molecular patterns (DAMPs) sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI. In the development of CS-AKI, specific intervention in the RIG-I pathway might be a potential therapeutic strategy for CS-AKI.

MicroRNA-128 inhibits glioma cells proliferation by targeting transcription factor E2F3a.

MicroRNAs are approximately 21nt single-stranded RNAs and function as regulators of gene expression. Previous studies have shown that microRNAs play crucial roles in tumorigenesis by targeting the mRNAs of oncogenes or tumor suppressors. Here we show that brain-enriched miR-128 is down-regulated in glioma tissues and cell lines when compared to normal brain tissues. Overexpression of miR-128 in glioma cells inhibited cell proliferation. A bioinformatics search revealed a conserved target site within the 3'untranslated region (UTR) of E2F3a, a transcription factor that regulates cell cycle progression. The protein levels of E2F3a in gliomas and normal brain tissues were negatively correlated to the expression levels of miR-128 in these tissues. Overexpression of miR-128 suppressed a luciferase-reporter containing the E2F3a-3'UTR and reduced the level of E2F3a protein in T98G cells. Moreover, knocking down of E2F3a had similar effect as overexpression of miR-128, and overexpression of E2F3a can partly rescue the proliferation inhibition caused by miR-128. Taken together, our study demonstrates that miR-128 can inhibit proliferation of glioma cells through one of its targets, E2F3a.

Human D-Tyr-tRNA(Tyr) deacylase contributes to the resistance of the cell to D-amino acids.

DTD (D-Tyr-tRNA(Tyr) deacylase) is known to be able to deacylate D-aminoacyl-tRNAs into free D-amino acids and tRNAs and therefore contributes to cellular resistance against D-amino acids in Escherichia coli and yeast. We have found that h-DTD (human DTD) is enriched in the nuclear envelope region of mammalian cells. Treatment of HeLa cells with D-Tyr resulted in nuclear accumulation of tRNA(Tyr). D-Tyr treatment and h-DTD silencing caused tRNA(Tyr) downregulation. Furthermore, inhibition of protein synthesis by D-Tyr treatment and h-DTD silencing were also observed. D-Tyr, D-Asp and D-Ser treatment inhibited mammalian cell viability in a dose-dependent manner; overexpression of h-DTD decreased the inhibition rate, while h-DTD-silenced cells became more sensitive to the D-amino acid treatment. Our results suggest that h-DTD may play an important role in cellular resistance against D-amino acids by deacylating D-aminoacyl tRNAs at the nuclear pore. We have also found that m-DTD (mouse DTD) is specifically enriched in central nervous system neurons, its nuclear envelope localization indicates that D-aminoacyl-tRNA editing may be vital for the survival of neurons under high concentration of D-amino acids.