Human RNA-binding protein HNRNPD interacts with and regulates the repair of deoxyribouridine in DNA.

Deoxyribouridine (dU) is an abnormal nucleoside in DNA and plays vital roles in multiple biological and physiological processes. Here, we conducted a mass spectrometry-based screen for dU-binding proteins and found that the heterogeneous nuclear ribonucleoprotein D (HNRNPD) could preferentially bind to dU-containing DNA. We also discovered that HNRNPD engages in the 5-Fluorouracil (5FU)-induced DNA damage response and can modulate the repair of dU in DNA in vitro and in human cells. Moreover, using a shuttle vector- and next-generation sequencing-based method, we unveiled the crucial role of HNRNPD in promoting the replicative bypass of dU in human cells. Taken together, these findings suggested that HNRNPD is a novel dU-bearing DNA-binding protein capable of regulating the removal of dU in DNA, and provided new insights into the molecular mechanisms of dU-associated diseases.

RUNX3 inhibits KSHV lytic replication by binding to the viral genome and repressing transcription.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma herpesvirus family, which can cause human malignancies including Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman's diseases. KSHV typically maintains a persistent latent infection within the host. However, after exposure to intracellular or extracellular stimuli, KSHV lytic replication can be reactivated. The reactivation process of KSHV triggers the innate immune response to limit viral replication. Here, we found that the transcriptional regulator RUNX3 is transcriptionally upregulated by the NF-κB signaling pathway in KSHV-infected SLK cells and B cells during KSHV reactivation. Notably, knockdown of RUNX3 significantly promotes viral lytic replication as well as the gene transcription of KSHV. Consistent with this finding, overexpression of RUNX3 impairs viral lytic replication. Mechanistically, RUNX3 binds to the KSHV genome and limits viral replication through transcriptional repression, which is related to its DNA- and ATP-binding ability. However, KSHV has also evolved corresponding strategies to antagonize this inhibition by using the viral protein RTA to target RUNX3 for ubiquitination and proteasomal degradation. Altogether, our study suggests that RUNX3, a novel host-restriction factor of KSHV that represses the transcription of viral genes, may serve as a potential target to restrict KSHV transmission and disease development.IMPORTANCEThe reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latent infection to lytic replication is important for persistent viral infection and tumorigenicity. However, reactivation is a complex event, and the regulatory mechanisms of this process are not fully elucidated. Our study revealed that the host RUNX3 is upregulated by the NF-κB signaling pathway during KSHV reactivation, which can repress the transcription of KSHV genes. At the late stage of lytic replication, KSHV utilizes a mechanism involving RTA to degrade RUNX3, thus evading host inhibition. This finding helps elucidate the regulatory mechanism of the KSHV life cycle and may provide new clues for the development of therapeutic strategies for KSHV-associated diseases.

MetaLnc9 facilitates osteogenesis of human bone marrow mesenchymal stem cells by activating the AKT pathway.

AIM OF THE STUDY: To investigate the role of MetaLnc9 in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs). MATERIALS AND METHODS: We used lentiviruses to knockdown or overexpress MetaLnc9 in hBMSCs. qRT-PCR was employed to determine the mRNA levels of osteogenic-related genes in transfected cells. ALP staining and activity assay, ARS staining and quantification were used to identify the degree of osteogenic differentiation. Ectopic bone formation was conducted to examine the osteogenesis of transfected cells in vivo. AKT pathway activator SC-79 and inhibitor LY294002 were used to validate the relationship between MetaLnc9 and AKT signaling pathway. RESULTS: The expression of MetaLnc9 was significantly upregulated in the osteogenic differentiation of hBMSCs. MetaLnc9 knockdown inhibited the osteogenesis of hBMSCs, whereas overexpression of it promoted the osteogenic differentiation both in vitro and in vivo. Taking a deeper insight, we found that MetaLnc9 enhanced the osteogenic differentiation by activating AKT signaling. The inhibitor of AKT signaling LY294002 could reverse the positive effect on osteogenesis brought by MetaLnc9 overexpression, whereas the activator of AKT signaling SC-79 could reverse the negative effect caused by MetaLnc9 knockdown. CONCLUSION: Our works uncovered a vital role of MetaLnc9 in osteogenesis via regulating the AKT signaling pathway. [Figure: see text].

SIRT1 slows the progression of lupus nephritis by regulating the NLRP3 inflammasome through ROS/TRPM2/Ca2+ channel.

Systemic lupus erythematosus (SLE) is a chronic multisystem inflammatory disease associated with autoantibody formation. Lupus nephritis (LN) is one of the most severe organ manifestations of SLE. The inflammatory response is a key factor in kidney injury, and the NLRP3 inflammasome is frequently associated with the pathogenesis of LN. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD +)-dependent histone deacetylase, is a promising therapeutic target for preventing renal injury. However, the mechanism of SIRT1 in LN remains unclear. Here, we aimed to investigate the mechanism by which SIRT inhibits the NLRP3 inflammasome to slow the progression of LN. We detected the expression of SIRT1 and the infiltration of macrophages in MRL/lpr mice; the results showed that the expression of SIRT1 was decreased, and the symptoms of lupus nephritis were relieved after the use of resveratrol, which upregulated SIRT1. In vitro studies showed that after lipopolysaccharide (LPS) stimulation, SIRT1 expression decreased, and the NLRP3 inflammasome was activated. Upregulation of SIRT1 inhibits NLRP3 inflammasome activation and assembly by interfering with two signalling pathways. First, SIRT1 affects NF-κB expression, transcription, and inflammatory cytokine expression. Second, SIRT1 modulates calcium influx induced by transient receptor potential channel M2 (TRPM2), which could be partly due to the inhibition of reactive oxygen species (ROS) production. Our findings suggest that upregulated SIRT1 inhibits the NLRP3 inflammasome to slow the progression of lupus nephritis by regulating NF-κB and ROS/TRPM2/Ca2+ channels. This study reveals a new anti-inflammatory mechanism of SIRT1, suggesting that SIRT1 may be a potential therapeutic target for the prevention of LN.

S1P/S1PR1 axis promotes macrophage M1 polarization through NLRP3 inflammasome activation in Lupus nephritis.

Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) as well as the leading cause of mortality in patients. Previous studies revealed that S1P level is elevated in plasma samples of SLE patients and murine lupus models. FTY720, targeting S1P receptors, exhibited therapeutic effects in improving the nephritis symptoms of lupus mouse models. However, few studies have discussed the potential relevance of S1P/S1PR to the pathogenesis of LN. Macrophages have been shown to be an important causative agent of renal inflammation, while the pro-inflammatory M1-type promotes kidney injury and inflammation during LN. Importantly, macrophages express various S1P receptors, and how they respond to S1P in the setting of LN remains unclear. Therefore, we examined the level of S1P in the lupus MRL/lpr mice and explored the ensuing interaction of macrophages and S1P. We found that S1P level was elevated in the MRL/lpr mice with a subsequent enhancement of the S1PR1 expression, and blocking S1PR1 by FTY720, the nephritis symptoms of MRL/lpr mice were improved. Mechanistically, we demonstrated that elevated S1P level increase the M1-type macrophage accumulation. And the in-vitro studies proved that S1P/S1PR1 was involved in the promotion of macrophage polarization towards M1 type through activation of NLRP3 inflammasome. These findings confer a novel role to macrophage S1PR1 and provide a new perspective for targeting S1P during LN.

Human Mitochondrial Protein HSPD1 Binds to and Regulates the Repair of Deoxyinosine in DNA.

The generation of deoxyinosine (dI) in DNA is one of the most important sources of genetic mutations, which may lead to cancer and other human diseases. A further understanding of the biological consequences of dI necessitates the identification and functional characterizations of dI-binding proteins. Herein, we employed a mass spectrometry-based proteomics approach to detect the cellular proteins that may sense the presence of dI in DNA. Our results demonstrated that human mitochondrial heat shock protein 60 (HSPD1) can interact with dI-bearing DNA. We further demonstrated the involvement of HSPD1 in the sodium nitrite-induced DNA damage response and in the modulation of dI levels in vitro and in human cells. Together, these findings revealed HSPD1 as a novel dI-binding protein that may play an important role in the mitochondrial DNA damage control in human cells.

KSHV RTA antagonizes SMC5/6 complex-induced viral chromatin compaction by hijacking the ubiquitin-proteasome system.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus with the capacity to establish life-long latent infection. During latent infection, the viral genome persists as a circular episome that associates with cellular histones and exists as a nonintegrated minichromosome in the nucleus of infected cells. Chromatin structure and epigenetic programming are required for the proper control of viral gene expression and stable maintenance of viral DNA. However, there is still limited knowledge regarding how the host regulates the chromatin structure and maintenance of episomal DNA. Here, we found that the cellular protein structural maintenance of chromosome (SMC) complex SMC5/6 recognizes and associates with the KSHV genome to inhibit its replication. The SMC5/6 complex can bind to the KSHV genome and suppress KSHV gene transcription by condensing the viral chromatin and creating a repressive chromatin structure. Correspondingly, KSHV employs an antagonistic strategy by utilizing the viral protein RTA to degrade the SMC5/6 complex and antagonize the inhibitory effect of this complex on viral gene transcription. Interestingly, this antagonistic mechanism of RTA is evolutionarily conserved among γ-herpesviruses. Our work suggests that the SMC5/6 complex is a new host factor that restricts KSHV replication.

Analysis of Rapid Rehabilitation Effect of Children with Severe Viral Encephalitis Based on Continuous Nursing of Omaha System.

Objective: To establish the Omaha System-based intensive care of children with viral encephalitis, compared with the conventional nursing applied in children with severe viral encephalitis for children with clinical symptoms, motor function, the incidence of complications, and the influence of quality of life, to intensive care of children with viral encephalitis way provide certain scientific basis. Methods: 62 cases of severe viral encephalitis diagnosed and treated in our hospital from X month 20XX to X month 20XX were randomly divided into 31 cases of intervention group and 31 cases of control group. The control group received routine nursing, and the intervention group added Omaha system on the basis of the control group. The recovery time of clinical symptoms and signs, FMA score, disability rate, quality of life, and satisfaction of family members were compared between the two groups. Results: The recovery time of clinical signs in the intervention group was shorter than that in the control group. Motor function was improved in both groups, but the improvement effect in intervention group was higher than that in control group. The quality of life in both groups was improved 1-3 months after discharge, but the improvement effect in the intervention group was higher than that in the control group. The incidence of physical dysfunction and behavioral abnormalities was low in the pregroup, and the difference between the two groups was statistically significant (P < 0.05), but the difference between language impairment and intellectual impairment was not statistically significant (P > 0.05). Conclusion: Omaha system nursing can significantly reduce the recovery time of clinical signs, improve FMA score, reduce disability rate, and improve the quality of life and family satisfaction of children with severe viral encephalitis.

Study on Protection of Human Umbilical Vein Endothelial Cells from Amiodarone-Induced Damage by Intermedin through Activation of Wnt/ß-Catenin Signaling Pathway.

Amiodarone (AM) is one of the most effective antiarrhythmic drugs and normally administrated by intravenous infusion which is liable to cause serious phlebitis. The therapeutic drugs for preventing this complication are limited. Intermedin (IMD), a member of calcitonin family, has a broad spectrum of biological effects including anti-inflammatory effects, antioxidant activities, and antiapoptosis. But now, the protective effects of IMD against amiodarone-induced phlebitis and the underlying molecular mechanism are not well understood. In this study, the aim was to investigate the protective efficiency and potential mechanisms of IMD in amiodarone-induced phlebitis. The results of this study revealed that treatment with IMD obviously attenuated apoptosis and exfoliation of vascular endothelial cells and infiltration of inflammatory cells in the rabbit model of phlebitis induced by intravenous infusion of amiodarone compared with control. Further tests in vitro demonstrated that IMD lessened amiodarone-induced endothelial cell apoptosis, improved amiodarone-induced oxidative stress injury, reduced inflammatory reaction, and activated the Wnt/ß-catenin signal pathway which was inhibited by amiodarone. And these effects could be reversed by Wnt/ß-catenin inhibitor IWR-1-endo, and si-RNA knocked down the gene of Wnt pathway. These results suggested that IMD exerted the protective effects against amiodarone-induced endothelial injury via activating the Wnt/ß-catenin pathway. Thus, IMD could be used as a potential agent for the treatment of phlebitis.

Role of sphingosine-1-phosphate mediated signalling in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly prevalent autoimmune disease characterized by the malfunction of the immune system and the persistent presence of an inflammatory environment. Multiple organs can be affected during SLE, leading to heterogeneous manifestations, which eventually result in the death of patients. Due to the lack of understanding regarding the pathogenesis of SLE, the currently available treatments remain suboptimal. Sphingosine-1-phosphate (S1P) is a central bioactive lipid of sphingolipid metabolism, which serves a pivotal role in regulating numerous physiological and pathological processes. As a well-recognized regulator of lymphocyte trafficking, S1P has been shown to be closely associated with autoimmune diseases, including SLE. Importantly, S1P levels have been found to be elevated in patients with SLE. In murine models of lupus, the increased levels of S1P also contribute to disease activity and organ impairment. Moreover, data from several studies also support the hypothesis that S1P receptors and its producer-sphingosine kinases (SPHK) may serve as the potential targets for the treatment of SLE and its co-morbidities. Given the significant success that intervening with S1P signaling has achieved in treating multiple sclerosis, further exploration of its role in SLE is necessary. Therefore, the aim of the present review is to summarize the recent advances in understanding the potential mechanism by which S1P influences SLE, with a primary focus on its role in immune regulation and inflammatory responses.

The p70S6K/PI3K/MAPK feedback loop releases the inhibition effect of high-dose rapamycin on rat mesangial cell proliferation.

Glomerular mesangial cell (MC) proliferation is one of the causative factors of glomerular diseases and one of their prominent pathological features. Rapamycin can inhibit MC proliferation and slow the progression to chronic renal fibrosis. The present study was designed to observe the role of rapamycin in MC proliferation and to explore the mechanism by which rapamycin acts on Akt and MAPK/ERK1/2 pathways in mesangial cells. MTT assay and flow cytometry were used to evaluate the proliferation and the cell cycle phase of glomerular mesangial cells respectively. The mRNA expression level of p70S6K was detected by RT-qPCR. Western blotting was performed to determine p70S6K, PI3K/Akt, and PI3K/MAPK protein expression. We found that rapamycin could reduce mesangial cell proliferation and arrest the cell cycle in the G1 phase, however the inhibition effect of 1000 nmol/L rapamycin was not higher than that in the 100 nmol/L group. The results of western blotting showed that 1000 nmol/L rapamycin more significantly inhibited the phosphorylation of p70S6K than 100 nmol/L, suggesting there should be another signaling pathway that activates the proliferation of MCs. Moreover, our results revealed that 1000 nmol/L rapamycin led to Raf1-MEK1/2-ERK pathway activation through a p70S6K-PI3K-mediated feedback loop in MCs. This study demonstrated that high-dose rapamycin leads to ERK1/2 activation through a p70S6K/PI3K/MAPK feedback loop in rat MCs, thus reducing the inhibitory effect of rapamycin on MC proliferation.

[Osteogenic stimulation of human dental pulp stem cells with gelatin-hydroxyapatite-tricalcium phosphate scaffold].

PURPOSE: To investigate the osteogenic stimulation effect of gelatin-hydroxyapatite-tricalcium phosphate (gelatin-HA-TCP) scaffold on human dental pulp stem cells(hDPSCs). METHODS: Gelatin scaffold and gelatin-HA-TCP scaffold were developed via solution casting/particle washing method, and the physical and mechanical properties of the scaffolds were examined using scanning electron microscopy and a universal testing machine, respectively. The stress intensity(SS) and elastic modulus(E) of the scaffold were measured by vertical electronic universal testing machine. The osteogenic medium was divided into control group (osteogenic medium), experimental group 1 (gelatin scaffold extract + osteogenic medium) and experimental group 2(gelatin-HA-TCP scaffold + osteogenic medium) according to different mediums. The effects of scaffolds on proliferation and alkaline phosphatase(ALP) activity of hDPSCs were evaluated by CCK8 and ALP staining. The expression of osteogenic factors in hDPSCs was detected by alizarin red S staining and reverse transcription-polymerase chain reaction(RT-PCR). Data were processed by SPSS 20.0 software package. RESULTS: Compared with gelatin scaffold, gelatin-HA-TCP scaffold had higher SS and lower E(P<0.05); CD146 and CD105 were positively expressed in hDPSCs, and CD34 and CD45 were negatively expressed in hDPSCs. The number of cells on the 3rd, 5th and 7th day of experimental group 1 was significantly higher than that of the control group (P<0.05); The number of cells on the 1st, 3rd, 5th and 7th day of experimental group 2 was significantly higher than that of the control group (P<0.05); ALP activities on the 4th, 7th and 12th day of experimental group 2 were significantly higher than those of the control group and experimental group 1(P<0.05). Compared with the control group, visual fields were red in both experimental groups, and the unevenly distributed calcified precipitation was also found in both experimental groups. The mRNA expression level of Runt-related transcription factor 2(Runx2) on the 4th day of experimental group 1 and experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of osterix (OSX) on 7th day of experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of bone sialoprotein (BSP) on the 4th day of experimental group 2 was significantly higher than that of the control group and experimental group 1 (P<0.05). CONCLUSIONS: The microscopic characterization of gelatin-HA-TCP scaffold is similar to that of natural bone structure with significant osteoinductive effect on the proliferation and differentiation of hDPSCs, and may be a promising biomaterial for bone tissue engineering.

Effects of ball milling treatment on physicochemical properties and digestibility of Pacific oyster (Crassostrea gigas) protein powder.

The oyster protein was ball milling treated in this work, and the effects on particle size, conformation, physicochemical properties, and in vitro protein digestibility (IVPD) were investigated. After ball milling treatment, the particle size obviously decreased, and the protein powder became denser and more homogeneous. The ball milling treatment could not change the primary structure of oyster protein. However, it could affect the secondary structure and physicochemical properties. The disulfide bond increased from 8.18 to 9.14 µmol/g protein, while the protein surface hydrophobicity index increased from 0.088 to 0.176. The decreasing water-holding capacity from 390% to 226% and the increasing oil-binding capacity from 91.2% to 189.1% were related to the alterations of conformation and physicochemical properties. Ball milling could also improve the IVPD from 54.6% to 82.4%. These results provided theoretical basis for the application of ball milling treatment in the utilization of oyster protein in the food industry.

[Notch activation delayed ageing of human dental pulp cells].

OBJECTIVE: To establish model of dental pulp cells with activated Notch signaling pathway, and investigate the effect of activating Notch signaling pathway on senescence of human dental pulp cells in vitro. METHODS: Human dental pulp cells were isolated, cultured as usual, and used from the 4(th) passage. The cells were divided into the activated group and the negative control group. In the activated group, the way of coating dishes with Jagged1 protein (10 mg/L) was used to activate Notch signaling pathway. The negative control group cells received no treatment. In the 4(th), 8(th), and 10(th) passages, the expression levels of the Notch signaling pathway downstream gene Hes1 were verified by real-time quantitative PCR (RT-qPCR). The cell changes after activating Notch signaling pathway were observed at three levels: (1) The cell morphology changes were observed through invert phase contrast microscope. The cell activity was detected with MTT assay. (2) The alkaline phosphatase (ALP) expression and its activity, and senescence-associated &bgr;-galatosidase (SA-ß-Gal) expression were observed with the kit. (3) The expression changes of senescence related genes were verified using RT-qPCR. The difference between the negative control group and the activated group was analyzed using student's t test. RESULTS: The expression level of the downstream gene Hes1 of Notch signaling pathway increased after coating the dishes with Jagged1 protein, indicating the establishment of the model of dental pulp cells with activated Notch signaling pathway. Compared with the negative control group, the aging cells of the activated group appeared relatively late. In the 8(th) and 10(th) passage, the cell activity increased. In the 10th passage, ALP activity increased, but SA-ß-Gal expression decreased. p16 gene expression decreased in each passage, and p53 gene expression decreased in the 8(th) and 10(th) passages. CONCLUSION: Jagged1 could activate Notch signaling pathway effectively. Through activating Notch signaling pathway, the dental pulp cells showed a trend of senescence delay at different levels, such as cell morphology, metabolic enzyme expressions and related gene expressions.