Effect and significance of viral proteins regulating activity of retroelement long interspersed element 1 / 中国生物制品学杂志

@#Abstract：Long interspersed element⁃1（LINE⁃1）is the only known active and autonomously transposable retroelement in human cells，which is related to autoimmune diseases and plays important roles in activating and regulating the antiviral innate immunity of cells，especially the level of interferon（IFN）. This paper reviews the mechanisms of several non ⁃ structural proteins from human immunodeficiency virus（HIV），hepatitis B virus（HBV）and other viruses participating in the regulation of LINE ⁃ 1 activity. These mechanisms not only ensure the normal expression of viral genome，but also participate in the cellular innate immunity regulation，the inhibition of which may provide new strategies to develop treatments of diseases caused by viruses.

LncRNA-NEF improves immune imbalance and PD-1/PD-1L-mediated Treg-Th17 cell balance in postmenopausal osteoporosis mice by miR-21 adsorption / 中华内分泌外科杂志

Objective:To investigate the regulatory mechanism of long non-coding RNA (lncRNA) NEF on T cell immune function in postmenopausal osteoporosis (PMOP) mice.Methods:Female Balb/c mice were used to construct OVX model ( n=46) and sham control group ( n=16) . Bone marrow mesenchymal stem cells (BM-SCs) from these two groups of mice were cultured. NEF recombinant expression vector (pIRSE2-NEF) was constructed and transfected into BMSCs. RT-qPCR was used to detect NEF and miR-21 levels in BMSCs cells in sham group, OVX group, and pIRSE2-NEF group. Luciferase gene report experiment was used to verify the binding effect of NEF and miR-21. The remaining 40 OVX mice were divided into 4 groups, including OVX group ( n=10) , pIRSE2-NEF injection group (pIRSE2-NEF group, n=10) , pIRSE2-NEF combined with PD-1 inhibitor group (pIRSE2-NEF+ PD-L1-IN-1 group, n=10) , and pIRSE2-NEF combined with miR-21 mimic (mimic) group (pIRSE2-NEF+ mimic group, n=10) . The remaining 10 mice in sham group were used as the control group. ELI-SA was used to detect the levels of IFN-γ, IL-2, IL-4, IL-13 and PD-1/PD-1L in peripheral blood. Flow cytometry was used to detect the shift of serum Treg-Th17 cell subsets. Results:Compared with the Sham group (1.01±0.04, 1.00±0.03) , the expression of NEF in BMSCs of OVX group was down-regulated (0.23±0.01) , and miR-21 was up-regulated (2.96±0.05) ( P<0.05) . Compared with OVX group (1.23±0.15, 5.20±0.31) , NEF in BMSCs cells of Pirse2-nef group was significantly up-regulated (6.83±0.35) ( P<0.05) , while miR-21 was down-regulated (0.29±0.11) ( P<0.05) .NEF has a direct binding base site with miR-21.The levels of IFN-γ (3.25±0.21) , IL-2 (2.44±0.06) and Th17/Treg ratio (3.18±0.65) in peripheral blood of mice in OVX group were significantly higher than those in Sham group (1.03±0.02, 1.00±0.01, 0.86±0.09) (all P<0.05) . The levels of IL-4 (0.45±0.02) , IL-13 (0.43±0.07) , PD-1 (0.24±0.03) and PD-1L (0.51±0.06) were significantly lower than those of Sham group (1.00±0.04, 1.00±0.02, 1.00±0.03, 1.00±0.00) ( P<0.05) ; Compared with OVX, IFN-γ (2.02±0.06) , IL-2 (0.88±0.01) and Th17/Treg ratio (1.43±0.22) in Pirse2-nef group were decreased. The levels of IL-4 (0.87±0.03) , IL-13 (0.84±0.07) , PD-1 (0.79±0.06) and PD-1L (0.77±0.06) were increased (all P<0.05) ; Compared with Pirse2-nef group, IFN-γ (2.89±0.06) , IL-2 (2.07±0.07) and Th17/Treg ratio (2.39±0.38) were increased in Pirse2-nef+ PD-L1-in-1 group. The levels of IL-4 (0.68±0.03) , IL-13 (0.76±0.08) , PD-1 (0.52±0.02) and PD-1L (0.83±0.04) were decreased (all P<0.05) . Moreover, the pIRSE2-NEF+ mimic group had the same adjustment effect as the pIRSE2-NEF+ PD-L1-IN-1 group. Conclusion:lncRNA-NEF improves immune imbalance and PD-1/PD-1L-mediated Treg-Th17 cell balance in postmenopausal osteoporosis mice by sponging miR-21.

Progress in research on neurotoxicity of HIV-1 Nef protein / 中华实验和临床病毒学杂志

Human immunodeficiency virus-1(HIV-1)is the causative agent of acquired immunodeficiency syndrome(AIDS), which can invade the central nervous system and cause a series of neurological disorders such as HIV-associated neurocognitive disorders(HAND). Nef protein is one of the early proteins of HIV-1 virus, it is found that the neurotoxicity of HIV-1 mutant lacking nef gene is less neurotoxic than that of wild strain with nef gene, indicating that Nef protein plays an important role in HIV-1 neuropathogenicity.In this paper, neurotoxicity and mechanism of Nef are reviewed from three aspects, including the effects of Nef on the blood-brain barrier permeability and the direct and indirect damage to neurons.

Identification of Nef-HIV-1 domains involved in p22-phox interaction and superoxide production / Identificación del dominio de Nef-VIH-1 involucrado en la interacción con p22-phox y producción de superoxido

Nef -HIV-1 has been shown to be involved in NADPH complex interaction and superoxide production. The aim of this work was to study the domains involved in the interaction between Nef and p22-phox. Two approaches were used: 1) in silico modelling, to determine the potential binding motifs and design Nef truncated forms and 2) functional assays. The results showed that GFPVT 68-72, FPDW 121-124 and REVLE 179-183 on Nef are critical for p22-phox (RPQIG 142-146 and PGGP 181-184) docking. However, only the region containing FPDW 121-124 on Nef is able to induce superoxide production. Understanding the molecular mechanisms involved in generating oxidative stress during HIV infection, is critical for therapeutic intervention, in order to minimize viral replication and dissemination.

Se ha evidenciado que Nef-VIH-1 está involucrado en la interacción con el complejo NADPH y la producción de superóxido. El objetivo de este trabajo fue identificar los dominios implicados en la interacción entre Nef y p22-phox. Se utilizaron dos estrategias: 1) análisis in silico para determinar los posibles motivos de unión y el diseño Nef formas truncadas y 2) ensayos funcionales. Los resultados mostraron que GFPVT 68-72, FPDW 121 a 124 y 179 a 183 REVLE de Nef son críticos para su unión con p22-phox (RPQIG 142-146 y 181-184 PGGP). Sin embargo, sólo la región que contiene FPDW 121-124 en Nef, es capaz de inducir la producción de superóxido. La comprensión de los mecanismos moleculares implicados en la generación de estrés oxidativo durante la infección por VIH, es crítico para la intervención terapéutica, con el fin de minimizar la replicación y la propagación viral.

Research progress on the role of transcription factor Nrf 2 in diabetic nephropathy / 中国糖尿病杂志

Oxidative stress and inflammation are two main factors in the progress of diabetic nephropathy(DN) and its complications. NF‐E2 p45‐related factor (Nrf2) is a crucial transcriptional factor which manipulates downstream genes that encode some antioxidant enzymes and phase Ⅱ detoxifying enzymes ,to maintain the redox homeostasis and cellular detoxification response. Therefore ,more and more researchers are focusing on the role of Nrf2 in DN. In this review ,the detailed role of Nrf2 in DN will be discussed. Hopefully ,our work can epitomize recent research progress and provide novel clues for diabetic nephropathy prevention and treatment.

HIV-1 Nef promotes KSHV vIL-6-induced angiogenesis by regulating PTEN/PI3K signaling pathway / 中华微生物学和免疫学杂志

Objective To investigate whether HIV-1 Nef could promote the angiogenesis and tu-morigenesis induced by KSHV vIL-6 through regulating PTEN/PI3K signaling pathway .Methods Lipo-some transfection was used to transfect cDNA of pPTEN , dominant-negative ( DN) construct of PI3K and control vector into endothelial cells , which stably express KSHV vIL-6 and HIV-1 Nef.Microtubule forma-tion assay and chicken chorioallantoic membrane ( CAM) assay were used to evaluate microtubule formation and angiogenesis , respectively .Expressions of PTEN and PI 3 K were measured by Western blot .Results Both overexpression of PTEN and inhibited expression of PI 3K suppressed the vIL-6-induced microtubule for-mation and angiogenesis in CAM mediated by Nef .Conclusion HIV-1 Nef enhances vIL-6-induced angio-genesis and tumorigenesis through regulating PTEN/PI3K signaling pathway .

Study on gene polymorphism of HIV-1 B' nef and its influence on disease progression in northern China / 中华微生物学和免疫学杂志

Objective To explore the polymorphism of nef gene and conservation level of functionally important domains of nef as well as their influences on HIV-1 disease progression of HIV-1 B'infected individuals in northern China.Methods 30 long term nonprogressors(LTNPs)and 42 typical progressors (TPs)were selected.Provirus DNA was extracted from whole blood sample.The full nef gene was amplified by nested-PCR.PCR product was sequenced directly after purification.Phylogenetic analysis and amino acid sequence mutation was applied on nef sequences to explore the differences between LTNPs and TPs.Results At position 15,the S15R/K/N substitution was detected.The frequency of TPs(64.29%)wsa higher than LTNPs(33.33%,P<0.01,0R=3.60);R21K/E/H/I.Q,TPs and LTNPs mutation frequency was 59.52%and 93.33%(P<0.005,OR=0.11);At position 39,K39R/E/N was only detected in TPs(23.81%,P<0.005).Conclusion No significant deletion or defect associated with disease progression wsa detected in nef gene of HIV-1 B'.But it suggested that K/E/H/I/Q mutation at 21 st amino acid of nef associated the disease nonprogression.R/K/N at 15 th amino acid of nef and R/E/N mutation at 39th amino acid of nef associated the disease progression in.HIV-1 B'.All domaias of nef amino acids sequences were comparatively conservative.

Regulation of the Expression of NEF3 in RA Induced Differentiation of SH-SYSY Neuroblastoma Cells / 医学研究杂志

Objective To analyze the regulation of NEF3 expression in SH - SY5Y after RA induction. Methods The expression of NEF3 and Brg1 genes was individually detected by realtime RT - PCR and Western blot analyses. Then, the CAT reporter driven by 5' flanking region ofNEF3 gene (pBLCAT3 -2. 8k - NEF3) was co - transfccted with eukaryotic expression plasmids of pBJ5 - Brgl or pBJ5 - Brg1 - NTP into SH - SY5Y respectively. Promoter activity of the NEF3 gene was detected by competitive RT - PCR assay. Results Similar changes in the mRNA expression of NEF3 and Brg1 and that of the protein expression of Brgl were detected in Sh - SY5Y cells af-ter RA treatment for 12 -24h. Wild type Brgl can promote the expression of pBLCAT3 -2.8k -NEF3 promoter after RA induction, but not mutant Brg1. Conclusion Brgl helpes regulating the expression of NEF3 gene possibly via changes in chromatin conformation in the SH -SY5Y cells after RA induction.

Analysis of Nef-specific interferon-γ secretion responses in HIV-1 B/C recombinant virus infectors / 中华检验医学杂志

Objective To analyze characteristics of Nef-specific T lymphocyte responses in Chinese HIV-1 recombinant subtype B/C infectors. Methods 19 HIV-1 recombinant subtype B/C infectors infected within 1 year, 40 chronic infectors infected for more than 3 years were enrolled in this cohort study. Elispot assay was used to observe HIV-1 specific T lymphocyte responses in HIV-1 recombinant subtype B/C infectors. Results Nef-specific T lymphocyte responses of interferon-gamma secretion were identified in 15 Chinese HIV-1 recombinant subtype B/C infectors infected within 1 year. The specific T lymphocytes were mainly targeted at four peptides which span from Nef 83 to 135: EVA7081.1, EVAT081.5, EVA7081.6 and EVAT081.48. Responses were identified in 29(75. 2%) infectors with more than 3 years of infection and the specific T lymphocytes were mainly targeted at three peptides which span from Nef 63 to 101 : EVA7081.43, EVA7081.44, EVAT081.45, EVA7081.47, EVA7081.48 and EVA7081.49. The average magnitude of response in infectors with less than 1 year of infection was 284. 13 SFC/106 PBMC. The average magnitude of response in infectors with more than 3 years of infection was 152. 44 SFC/106 PBMC. There was a significant difference between the two groups (U = 91. 000, P = 0. 002). Conclusions HIV-1recombinant subtype B/C infectors at different stages of diseases (less than 1 year and more thank 3 years) can recognize central region of Nef. The magnitude of Nef-specific IFN-γ secretion T lymphocyte responses in this cohort gradually decrease with disease progression.

Nef-specific T lymphocyte responses in Chinese HIV-1 recombinant subtype C/B' and subtype B' in-fectors / 中华微生物学和免疫学杂志

Objective To analyze character of Nef-specific T lymphocyte responses in Chinese HIV-1 recombinant subtype C/B' and subtype B' infectors and to identify the common immunodominant re-gions recognized by these infectors. Methods Fifty-nine HIV-1 recombinant subtype C/B' infectors and 27 subtype B' infectors were tested by IFN-γ enzyme linked immunospot (ELISPOT) assay using HIV-1 C/B' Nef overlapping peptides. Results Nef-specific T-cell responses of IFN-γ secretion were identified in 44 (74.58%) out of 59 HIV-1 recombinant subtype C/B' infectors. Ten peptides, EVA7081.1,5, 6, 7,43, 44, 45, 47, 48, 49 were mainly recognized. Amino acid position was from Nef63 to 115 and 117 to 139. Twenty of 27 (74.07%) HIV-1 subtype B' infectors recognized peptides. EVA7081.1,2, 43 and 49 were mainly recognized. Amino acid position was from Nef 63 to 77 and 87 to 119. There was no correlation be-tween the Nef-specific IFN-production of HIV-1-specific T cells responses and viral load or CD4 T cell count in both subtype infectors. Conclusion The immunodominant regions, from Nef63 to 77 and 87 to 115 were recognized by both Chinese HIV-1 recombinant subtype C/B' infectors and subtype B' infectors. These re-gions could be used in design of vaccine.

Analysis of HIV-1 Clade B Nef specific CD8~+ T cell inter-clade cross-reactivity to Clade B and C antigen in Chinese population / 医学研究生学报

Objective: To analyse the character of HIV1 Clade B Nef specific CD8~(+)T cell inter-clade cross-reactivity to clade B and C antigen in Chinese population. Methods: PBMC from 51 HIV-1 Clade B infected donors were separated by Ficoll-Hypaque density gradient centrifugation.HIV-1 Nef specific CD8~(+)T cell responses were analyzed by gamma interferon(IFN-?) enzyme-linked immunospot(ELISpot) assay using 54 synthetic peptides spanning all HIV-1B and C Nef proteins. Results:(82.9%)(29/35)of the individuals who had the Nef specific CD8~(+)T cell responses could recognize both Clade B and Clade C peptides.There was no significant difference between the two Clades on number of the recognized peptides and magnitude of the responses(P=0.529,P=0.754).70.4% Nef peptides either of Clade B or of Clade C could be recognized by HIV-1 B Nef specific CD8~(+)T cell.The sequence homology of peptides which could be cross-recognized was significantly higher than that of peptides which could be single-recognized. Conclusion: There are broadly cross-reactive T cells in HIV-1 Clade B infected individuals which can recognize both Clade B and Clade C Nef antigen.These cross-reactive T cells recognize peptides with higher inter-clade homology.

Reactivation of neferine on mice brain cholinesterase inhibited by organophosphate / 中草药

Object To study the reactivation of neferine (Nef) and pralidoxime chloride (2-PAM?Cl) on mice brain cholinestrase (ChE) inhibited by organophosphate. Methods Micro-DTNB method was used to determine the ChE activity of mice brain inhibited by DDVP in vitro and in vivo, then the inhibitory effect of DDVP (0.001-0.03 mg/L) on mice brain ChE in vitro was observed. The reactivative effect of Nef and 2-PAM?Cl on brain ChE of poisoned mice with DDVP in vivo and in vitro was compared. Results In vitro, the inhibitory effect of DDVP at different concentrations on mice brain ChE showed a concentration-effect relationship. The IC_(50) was 0.003 mg/L. The reactivative effect of Nef (2.4, 4.8 mg/L) and 2-PAM?Cl (5, 12.5 mg/L) on brain ChE inhibited by DDVP (0.02 mg/L) enhanced with increasing their concentrations. In vivo, after 30 min of treated with DDVP (10 mg/L, sc), the mice were given (ip) with Nef (15, 30 mg/kg) or 2-PAM?Cl (25, 50 mg/kg), respectively. The ChE activity rate in these two treated groups were (41.6?10.9) %, (56.5?12.4) % and (24.1?14.3) %, (28.4?11.9) %, respectively. The difference between poisoned group (sc DDVP) and Nef treated group was significant (P0.05). Conclusion The results suggest that Nef have a more remarkable reactivative effect on inhibited brain ChE in vitro and in vivo than 2-PAM?Cl. This may be contributed to that Nef can penetrate the blood-brain barrier.