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.

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.

Using Spin-Coated Silver Nanoparticles/Zinc Oxide Thin Films to Improve the Efficiency of GaInP/(In)GaAs/Ge Solar Cells.

We synthesized a silver nanoparticle/zinc oxide (Ag NP/ZnO) thin film by using spin-coating technology. The treatment solution for Ag NP/ZnO thin film deposition contained zinc acetate (Zn(CH3COO)2), sodium hydroxide (NaOH), and silver nitrate (AgNO3) aqueous solutions. The crystalline characteristics, surface morphology, content of elements, and reflectivity of the Ag NPs/ZnO thin film at various concentrations of the AgNO3 aqueous solution were investigated using X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, and ultraviolet⁻visible⁻near infrared spectrophotometry. The results indicated that the crystalline structure, Ag content, and reflectance of Ag NP/ZnO thin films depended on the AgNO3 concentration. Hybrid antireflection coatings (ARCs) composed of SiNx and Ag NPs/ZnO thin films with various AgNO3 concentrations were deposited on GaInP/(In)GaAs/Ge solar cells. We propose that the optimal ARC consists of SiNx and Ag NP/ZnO thin films prepared using a treatment solution of 0.0008 M AgNO3, 0.007 M Zn(CH3COO)2, and 1 M NaOH, followed by post-annealing at 200 °C. GaInP/(Al)GaAs/Ge solar cells with the optimal hybrid ARC and SiNx ARC exhibit a conversion efficiency of 34.1% and 30.2% with Voc = 2.39 and 2.4 V, Jsc = 16.63 and 15.37 mA/cm², and fill factor = 86.1% and 78.8%.

Combined intranasal nerve growth factor and ventricle neural stem cell grafts prolong survival and improve disease outcome in amyotrophic lateral sclerosis transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a fatal disease that selectively involves motor neurons. Neurotrophic factor supplementation and neural stem cell (NSC) alternative therapy have been used to treat ALS. The two approaches can affect each other in their pathways of action, and there is a possibility for synergism. However, to date, there have been no studies demonstrating the effects of combined therapy in the treatment of ALS. In this study, for the first time, we adopted a method involving the intranasal administration of nerve growth factor combined with lateral ventricle NSC transplantation using G93A-SOD1 transgenic mice as experimental subjects to explore the treatment effect of this combined therapy in ALS. We discover that the combined therapy increase the quantity of TrkA receptors, broaden the migration of exogenous NSCs, further promote active proliferation in neurogenic regions of the brain and enhance the preservation of motor neurons in the spinal cord. Regarding physical activity, the combined therapy improved motor functions, further postponed ALS onset and extended the survival time of the mice.

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.

[Protein expression, purification of PCGF1 and its monoclonal antibody preparation and identification].

AIM: To express the human recombinant PCGF1 protein and prepare monoclonal antibody (mAb) against it. METHODS: The recombinant expression plasmid pET32a-His-PCGF1-128/189 was made and transformed into E.coli (BL21), and then the recombinant fusion protein His-PCGF1-128/189 was expressed and purified. The BALB/c mice were immuned with purified protein His-PCGF1-128/189 as antigen. The mAbs against PCGF1 were prepared by using standard hybridoma technique. The hybridoma cell lines were obtained by ELISA and Western blot screening procedure, the isotype of the mAbs were further identified by immune-double diffusion. Ascites were prepared from one propagated hybridoma cell line and mAbs were purified by using the Kit from Millipore. The valence of mAb was detected by Western blot. RESULTS: The recombinant protein His-PCGF1-128/189 was expressed and purified. Two hybfidmas producing antibodies against PCGF1 were obtained, the isotypes of two mAbs were IgG1, Western blot showed that the antibodies were high sensitive(1:6 000) and high specific for PCGF1. CONCLUSION: The anti-PCGF1 mAb prepared by using recombinant His-PCGF1-128/189 protein as antigen can be used for detecting PCGF1 proteins which are either endogenous or exogenous.

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons.

BACKGROUND: The downstream of tyrosine kinase/docking protein (Dok) adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk) receptor family, which has three members (TrkA, TrkB and TrkC), are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. RESULTS: In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST) precipitation assays and coimmunoprecipitation (Co-IP) experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB) domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3) stimulation. CONCLUSIONS: We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Identification of differentially expressed microRNAs by microarray: a possible role for microRNAs gene in medulloblastomas.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding regulatory RNAs whose aberrant expression may be observed in many malignancies. However, few data are yet available on human primary medulloblastomas. This work aimed to identify that whether miRNAs would be aberrantly expressed in tumor tissues compared with non-tumorous cerebellum tissues from same patients, and to explore a possible role during carcinogenesis. METHODS: A high throughput microRNA microarray was performed in human primary medulloblastoma specimens to investigate differentially expressed miRNAs, and some miRNAs were validated using real-time quantitative RT-PCR method. In addition, the predicted target genes for the most significantly down- or up-regulated miRNAs were analyzed by using a newly modified ensemble algorithm. RESULTS: Nine miRNA species were differentially expressed in medulloblastoma specimens versus normal non-tumorous cerebellum tissues. Of these, 4 were over expressed and 5 were under expressed. The changes ranged from 0.02-fold to 6.61-fold. These findings were confirmed using real-time quantitative RT-PCR for most significant deregulated miRNAs (miR-17, miR-100, miR-106b, and miR-218) which are novel and have not been previously published. Interestingly, most of the predicted target genes for these miRNAs were involved in medulloblastoma carcinogenesis. CONCLUSIONS: MiRNAs are differentially expressed between human medulloblastoma and non-tumorous cerebellum tissue. MiRNAs may play a role in the tumorigenesis of medulloblastoma and maybe serve as potential targets for novel therapeutic strategies in future.

[Expression pattern of polycomb gene Nspc1 at the early developmental stage in zebrafish].

OBJECTIVE: To study the expression pattern of Polycomb gene Nspc1 at the early developmental stage in zebrafish. METHODS: In situ hybridization probe for Nspc1 was designed according to the GenBank information. Collecting zebrafish embryos at different stages including one cell stage, two-cell stage, bud stage, and somites stage, we hybridized them with the prepared probe. Then the hybridization signals at different intervals were observed and photographed at the right time. RESULTS: Nspc1 was expressed globally at the early stage. Its expression specificity began at the somites stage, mainly in the nervous system of the head. CONCLUSION: Nspc1 may play essential roles in the early stage development of zebrafish, especially in the nervous system.

[MiR-9 regulates the expression of CBX7 in human glioma].

OBJECTIVE: To detect the expression of CBX7 in human glioma and investigate the potential regulatory effect of abnormally expressed microRNAs on CBX7 expression. METHODS: Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression pattern of CBX7 in 2 human normal brain tissues, 9 glioma tissues, and 3 glioma cell lines. Miranda algorithm and Ensemble Machine Learning algorithm were combined to predict miRNAs that target human CBX7. The expression of miR-9 in those tissues and cell lines were detected by real-time PCR. After miR-9 overexpression in 293ET and miR-9 knock-down in T98G, luciferase assay and Western blot were used to confirm the effect of miR-9 on CBX7 expression. MTT assay and flow cytometry were applied to detect the effect of miR-9 knock-down on T98G cells. RESULTS: No obvious difference in the CBX7 mRNA level between normal and tumor tissues was observed, while the protein level of CBX7 was abrogated or markedly reduced in glioma tissues and cell lines. Several miRNAs including miR-9 may target CBX7 by bioinformatics prediction. MiR-9 was up-regulated in glioma tissues and cell lines. In 293ET cell, luciferase activity of CBX7-3'UTR reporter was decreased to 24% after miR-9 overexpression. After miR-9 knock-down in T98G cell, the luciferase activity was increased by 1.8 fold and there was no change of CBX7 mRNA, while the protein level of endogenous CBX7 was significantly increased. The number of survival T98G cells increased and cells in G1 phase decreased after miR-9 knock-down. CONCLUSION: In human glioma, CBX7 is down-regulated by the inhibition of miR-9 at posttranscriptional level.

[Role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured rat neurons].

OBJECTIVE: To study the role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured neurons. METHODS: Semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression pattern of Necl1 in the neuronal differentiation cell model in vitro. Western blot was performed to detect the expression pattern of Necl1 in primary cultured rat neurons and in purified synaptosome. Immunofluoresence was used to detect the synapse formation in primary neurons and in 293 cells co-culture and to detect the density of synapses in primary neuron with ectopic expression of Necl1. RESULTS: Necl1 expression increased after retinoic acid (RA) induction in SH-SY5Y and P19 cells. The increase of Necl1 expression was consistent with the days of primary neurons culture in vitro, and Necl1 partly localized in synaptosome. The overexpression of Necl1 in 293 cells induced the synapse formation between cocultured 293 cells and neurons. Ectopic expression of Necl1 in primary neurons increased the density of synapses. CONCLUSION: Necl1 plays an important role in neuronal synapse formation.

[Effect of NECL1 on the proliferation of T98G glioma cell line].

OBJECTIVE: To study the regulation role of tumor suppressor NECL1 on the proliferation of glioma cell line. METHODS: We detected the expression level of NECL1 in human normal brain tissue and glioma cell lines using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. T98G cell line in which NECL1 was silent and whose transfection efficiency was relatively high as target cell was chosen, and the effect of NECL1 on the proliferation of T98G cell line in vitro was detected by using cell growth curve, flow cytometry, and Hoechst staining. RESULTS: NECL1 was abrogated or markedly reduced in 6 glioma cell lines. When NECL1 was overexpressed in T98G cell line, the cell growth rate obviously decreased and the number of apoptotic cells remarkably increased when compared with the control group. CONCLUSION: NECL1 may inhibit the proliferation of T98G cells by inducing its apoptosis.

MiRE: a graphical R package for microRNA-related analysis.

OBJECTIVE: To provide a set of useful analysis tools for the researchers to explore the microRNA data. METHODS: The R language was used for generating the Graphical Users Interface and implementing most functions. Some Practical Extraction and Report Language (Perl) scripts were used for parsing source files. RESULTS: We developed a graphical R package named miRE, which was designated for the analysis of microRNA functions, genomic organization, etc. This package provided effective and convenient tools for molecular biologists to deal with routine analyses in microRNA-related research. With its help, the users would be able to build a desktop-centered microRNA research environment quite easily and effectively. miRE is freely available at http://www. biosino.org/-kanghu/WorkPresentation/miRE/miRE.html. A detailed user manual and tutorials with example code and image are also available. CONCLUSION: miRE is a tool providing an open-source, user-friendly, integrated interface for microRNA-related analysis. With its help, researchers can perform microRNA-related analysis more efficiently.

[Bioluminescent imaging monitoring of a anti-angiogenesis therapeutic gene vasostatin in tumor cell PC3].

OBJECTIVE: To generate a sensitive tool for noninvasive monitoring of a therapeutic gene vasostatin. METHODS: We fused the bioluminescent reporter gene firefly luciferase to the therapeutic transgene vasostatin and ensured that these two proteins would not interrupt each other and kept their own natural character. RESULTS: We therefore examined clones of PC3 cells stably expressing fusion gene and positive controlfluc with bioluminescence. In vivo imaging of PC3-Fluc subcutaneous tumors showed that the mean tumor bioluminescence increased in animals over several weeks. CONCLUSION: Noninvasive monitoring facilitates the detection of gene expression in vivo and in vitro.