Specific inhibition of TET1 in the spinal dorsal horn alleviates inflammatory pain in mice by regulating synaptic plasticity.

DNA demethylation mediated by ten-eleven translocation 1 (TET1) is a critical epigenetic mechanism in which gene expression is regulated via catalysis of 5-methylcytosine to 5-hydroxymethylcytosine. Previously, we demonstrated that TET1 is associated with the genesis of chronic inflammatory pain. However, how TET1 participates in enhanced nociceptive responses in chronic pain remains poorly understood. Here, we report that conditional knockout of Tet1 in dorsal horn neurons via intrathecal injection of rAAV-hSyn-Cre in Tet1fl/fl mice not only reversed the inflammation-induced upregulation of synapse-associated proteins (post-synaptic density protein 95 (PSD95) and synaptophysin (SYP)) in the dorsal horn but also ameliorated abnormalities in dendritic spine morphology and alleviated pain hypersensitivities. Pharmacological blockade of TET1 by intrathecal injection of a TET1-specific inhibitor-Bobcat 339-produced similar results, as did knockdown of Tet1 by intrathecal injection of siRNA. Thus, our data strongly suggest that increased TET1 expression during inflammatory pain upregulates the expression of multiple synapse-associated proteins and dysregulates synaptic morphology in dorsal horn neurons, suggesting that Tet1 may be a potential target for analgesic strategies.

DNA N6-methyladenine methylase N6AMT1 controls neuropathic pain through epigenetically modifying Kcnj16 in dorsal horn neurons.

ABSTRACT: Nerve injury-induced aberrant changes in gene expression in spinal dorsal horn neurons are critical for the genesis of neuropathic pain. N6-methyladenine (m 6 A) modification of DNA represents an additional layer of gene regulation. Here, we report that peripheral nerve injury significantly decreased the level of m 6 A-specific DNA methyltransferase 1 ( N6amt1 ) in dorsal horn neurons. This decrease was attributed, at least partly, to a reduction in transcription factor Nr2f6 . Rescuing the decrease in N6amt1 reversed the loss of m 6 A at the promoter for inwardly rectifying potassium channel subfamily J member 16 ( Kcnj16 ), mitigating the nerve injury-induced upregulation of Kcnj16 expression in the dorsal horn and alleviating neuropathic pain hypersensitivities. Conversely, mimicking the downregulation of N6amt1 in naive mice erased DNA m 6 A at the Kcnj16 promoter, elevated Kcnj16 expression, and led to neuropathic pain-like behaviors. Therefore, decreased N6amt1 caused by NR2F6 is required for neuropathic pain, likely through its regulation of m 6 A-controlled KCNJ16 in dorsal horn neurons, suggesting that DNA m 6 A modification may be a potential new target for analgesic and treatment strategies.

Spinal-Specific Super Enhancer in Neuropathic Pain.

Dysfunctional gene expression in nociceptive pathways plays a critical role in the development and maintenance of neuropathic pain. Super enhancers (SEs), composed of a large cluster of transcriptional enhancers, are emerging as new players in the regulation of gene expression. However, whether SEs participate in nociceptive responses remains unknown. Here, we report a spinal-specific SE (SS-SE) that regulates chronic constriction injury (CCI)-induced neuropathic pain by driving Ntmt1 and Prrx2 transcription in dorsal horn neurons. Peripheral nerve injury significantly enhanced the activity of SS-SE and increased the expression of NTMT1 and PRRX2 in the dorsal horn of male mice in a bromodomain-containing protein 4 (BRD4)-dependent manner. Both intrathecal administration of a pharmacological BRD4 inhibitor JQ1 and CRISPR-Cas9-mediated SE deletion abolished the increased NTMT1 and PRRX2 in CCI mice and attenuated their nociceptive hypersensitivities. Furthermore, knocking down Ntmt1 or Prrx2 with siRNA suppressed the injury-induced elevation of phosphorylated extracellular-signal-regulated kinase (p-ERK) and glial fibrillary acidic protein (GFAP) expression in the dorsal horn and alleviated neuropathic pain behaviors. Mimicking the increase in spinal Ntmt1 or Prrx2 in naive mice increased p-ERK and GFAP expression and led to the genesis of neuropathic pain-like behavior. These results redefine our understanding of the regulation of pain-related genes and demonstrate that BRD4-driven increases in SS-SE activity is responsible for the genesis of neuropathic pain through the governance of NTMT1 and PRRX2 expression in dorsal horn neurons. Our findings highlight the therapeutic potential of BRD4 inhibitors for the treatment of neuropathic pain.SIGNIFICANCE STATEMENT SEs drive gene expression by recruiting master transcription factors, cofactors, and RNA polymerase, but their role in the development of neuropathic pain remains unknown. Here, we report that the activity of an SS-SE, located upstream of the genes Ntmt1 and Prrx2, was elevated in the dorsal horn of mice with neuropathic pain. SS-SE contributes to the genesis of neuropathic pain by driving expression of Ntmt1 and Prrx2 Both inhibition of SS-SE with a pharmacological BRD4 inhibitor and genetic deletion of SS-SE attenuated pain hypersensitivities. This study suggests an effective and novel therapeutic strategy for neuropathic pain.

DHX9/DNA-tandem repeat-dependent downregulation of ciRNA-Fmn1 in the dorsal horn is required for neuropathic pain.

Circular RNAs (ciRNAs) are emerging as new players in the regulation of gene expression. However, how ciRNAs are involved in neuropathic pain is poorly understood. Here, we identify the nervous-tissue-specific ciRNA-Fmn1 and report that changes in ciRNA-Fmn1 expression in spinal cord dorsal horn neurons play a key role in neuropathic pain after nerve injury. ciRNA-Fmn1 was significantly downregulated in ipsilateral dorsal horn neurons after peripheral nerve injury, at least in part because of a decrease in DNA helicase 9 (DHX9), which regulates production of ciRNA-Fmn1 by binding to DNA-tandem repeats. Blocking ciRNA-Fmn1 downregulation reversed nerve-injury-induced reductions in both the binding of ciRNA-Fmn1 to the ubiquitin ligase UBR5 and the level of ubiquitination of albumin (ALB), thereby abrogating the nerve-injury-induced increase of ALB expression in the dorsal horn and attenuating the associated pain hypersensitivities. Conversely, mimicking downregulation of ciRNA-Fmn1 in naïve mice reduced the UBR5-controlled ubiquitination of ALB, leading to increased expression of ALB in the dorsal horn and induction of neuropathic-pain-like behaviors in naïve mice. Thus, ciRNA-Fmn1 downregulation caused by changes in binding of DHX9 to DNA-tandem repeats contributes to the genesis of neuropathic pain by negatively modulating UBR5-controlled ALB expression in the dorsal horn.

The Cytidine N-Acetyltransferase NAT10 Participates in Peripheral Nerve Injury-Induced Neuropathic Pain by Stabilizing SYT9 Expression in Primary Sensory Neurons.

RNA N4-acetylcytidine (ac4C) modification is increasingly recognized as an important layer of gene regulation; however, the involvement of ac4C in pain regulation has not been studied. Here, we report that N-acetyltransferase 10 protein (NAT10; the only known ac4C "writer") contributes to the induction and development of neuropathic pain in an ac4C-dependent manner. Peripheral nerve injury increases the levels of NAT10 expression and overall ac4C in injured dorsal root ganglia (DRGs). This upregulation is triggered by the activation of upstream transcription factor 1 (USF1), a transcription factor that binds to the Nat10 promoter. Knock-down or genetic deletion of NAT10 in the DRG abolishes the gain of ac4C sites in Syt9 mRNA and the augmentation of SYT9 protein, resulting in a marked antinociceptive effect in nerve-injured male mice. Conversely, mimicking NAT10 upregulation in the absence of injury evokes the elevation of Syt9 ac4C and SYT9 protein and induces the genesis of neuropathic-pain-like behaviors. These findings demonstrate that USF1-governed NAT10 regulates neuropathic pain by targeting Syt9 ac4C in peripheral nociceptive sensory neurons. Our findings establish NAT10 as a critical endogenous initiator of nociceptive behavior and a promising new target for treating neuropathic pain.SIGNIFICANCE STATEMENT The cytidine N4-acetylcytidine (ac4C), a new epigenetic RNA modification, is crucial for the translation and stability of mRNA, but its role for chronic pain remains unclear. Here, we demonstrate that N-acetyltransferase 10 (NAT10) acts as ac4C N-acetyltransferase and plays an important role in the development and maintenance of neuropathic pain. NAT10 was upregulated via the activation of the transcription factor upstream transcription factor 1 (USF1) in the injured dorsal root ganglion (DRG) after peripheral nerve injury. Since pharmacological or genetic deleting NAT10 in the DRG attenuated the nerve injury-induced nociceptive hypersensitivities partially through suppressing Syt9 mRNA ac4C and stabilizing SYT9 protein level, NAT10 may serve as an effective and novel therapeutic target for neuropathic pain.

Cholesterol Metabolic Markers for Differential Evaluation of Patients with Hyperlipidemia and Familial Hypercholesterolemia.

The cholesterol metabolism in humans can be indirectly reflected by measuring cholesterol metabolism marker levels. We aimed to investigate the association of cholesterol homeostasis markers on standard lipid profiling components in familial hypercholesteremia and hyperlipidemia patients. A total of 69 hyperlipidemia patients, 25 familial hypercholesteremia (FHC) patients, and 64 healthy controls were enrolled in this study. We performed routine testing of blood lipid water. Gas chromatography was used to determine the changes in the concentration of cholesterol synthesis (squalene, desmosterol, and lathosterol) and absorption markers (campesterol, sitosterol, and stigmasterol) in the blood. Baseline hyperlipidemia patients displayed significantly higher total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels in comparison to the control group, which was reflected in the increased levels of squalene, desmosterol, campesterol, and sitosterol observed (P < 0.05) in the hyperlipidemia patients. The desmosterol, lathosterol, campesterol, stigmasterol, and sitosterol were statistically different in the FHC group than the hyperlipidemic group (P < 0.05). The proportions of squalene/cholesterol, lathosterol/cholesterol, stigmasterol/cholesterol, and sitosterol/cholesterol in the FHC group were lower than those in the hyperlipidemic group; only desmosterol/cholesterol was higher than that in the hyperlipidemic group. Correlation studies between lipid metabolic factors showed that the proportion of moderate and strong correlations was much higher in the FHC group than in the other two groups (76.92% vs. 32.50% and 31.25%). Logistic regression analysis showed that the concentrations of glucose, LDL-C, lactosterol, and sitosterol were all independent risk factors for developing hyperlipidemia. This result was further confirmed by the ROC curve. These results indicated that the study of cholesterol synthesis and decomposition markers can serve as a reference index for related diseases caused by changes in its concentration.

Natural infection of pangolins with human respiratory syncytial viruses.

Respiratory syncytial virus (RSV) is an enveloped non-segmented negative sense RNA virus that belongs to Orthopneumovirus genus of the Pneumoviridae family in the order Mononegavirales. The virus is the leading cause of severe respiratory disease in children under two years of age and is responsible for substantial disease burden in infants and elder people in both developed and developing countries1,2. RSV is only known to circulate among humans, though it was first isolated from chimpanzees3. The virus can experimentally infect mice, rats, cotton rats, ferrets, and hamsters, but does not naturally circulate in these animal populations4. We found that Malayan pangolins (Manis javanica) were naturally infected with RSVs that have 99.4-99.8% genomic identity with strains circulating in humans. Phylogenetic analyses revealed that five RSVs in pangolins were RSV-A ON1 and seven were RSV-B BA genotypes, both of which are currently prevalent in humans worldwide. These findings suggest that humans might transmit their viruses to endangered wildlife.

Gas Chromatography and Flame-Ionization Detection of Non-Cholesterol Sterols as Indicators of Cholesterol Absorption and Synthesis in 158 Chinese Individuals with Normolipidemia, Hyperlipidemia, and Familial Hypercholesterolemia.

BACKGROUND There are limited studies on the effects of cholesterol homeostasis in populations at high risk for cardiovascular disease. We aimed to use gas chromatography and flame-ionization detection (GC-FID) of non-cholesterol sterols as indicators of cholesterol absorption and synthesis. Sterol indicators of cholesterol absorption included campesterol, stigmasterol, and sitosterol. Sterol indicators of cholesterol synthesis included squalene, 7-lathosterol, and desmosterol. MATERIAL AND METHODS A total of 158 participants were enrolled in 3 groups: healthy control (n=64), hyperlipidemia (n=69), and familial hypercholesterolemia (FH, n=25). Age, sex, blood pressure, blood glucose, and lipoprotein were collected, and cholesterol absorption and synthesis markers were determined by GC-FID. RESULTS All 6 cholesterol concentration indicators, except squalene, were significantly different among the 3 groups (all P<0.05); whereas in the ratio to cholesterol (%, sterols/cholesterol), only desmosterol and lathosterol were significantly different (P<0.05). Multifactorial regression analysis showed that triglycerides, total cholesterol, and desmosterol were independent risk factors affecting the development of hyperlipidemia (P<0.05). The efficacy of the ROC curve for the diagnosis of dyslipidemia was also higher for all 3 indices (Model 1, AUC=0.960). Model 1 was superior to Model 2 for the 6 indicators of cholesterol. For the FH and dyslipidemia groups, the 6-indicator model (Model 3) was shown to have a good diagnostic value (AUC=1.000). CONCLUSIONS The 6 sterol indicators of cholesterol absorption and synthesis had a dynamic course in all study participants. Desmosterol was an indicator of dyslipidemia. The combined use of the 6 sterol indicators differentiated between healthy individuals and patients with dyslipidemia and FH.

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus.

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-ßeta (IFN-ß), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

Zoonotic origins of human coronavirus 2019 (HCoV-19 / SARS-CoV-2): why is this work important?

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by infection with human coronavirus 2019 (HCoV-19 / SARS-CoV-2 / 2019-nCoV), is a global threat to the human population. Here, we briefly summarize the available data for the zoonotic origins of HCoV-19, with reference to the other two epidemics of highly virulent coronaviruses, SARS-CoV and MERS-CoV, which cause severe pneumonia in humans. We propose to intensify future efforts for tracing the origins of HCoV-19, which is a very important scientific question for the control and prevention of the pandemic.

Transcription factor networks involved in cell death in the dorsal root ganglia following peripheral nerve injury.

The peripheral nervous system has the potential to regenerate after nerve injury owing to the intrinsic regrowth ability of neurons and the permissive microenvironment. The regenerative process involves numerous gene expression changes, in which transcription factors play a critical role. Previously, we profiled dysregulated genes in dorsal root ganglion neurons at different time points (0, 3 and 9 hours, and 1, 4 and 7 days) after sciatic nerve injury in rats by RNA sequencing. In the present study, we investigated differentially expressed transcription factors following nerve injury, and we identified enriched molecular and cellular functions of these transcription factors by Ingenuity Pathway Analysis. This analysis revealed the dynamic changes in the expression of transcription factors involved in cell death at different time points following sciatic nerve injury. In addition, we constructed regulatory networks of the differentially expressed transcription factors in cell death and identified some key transcription factors (such as STAT1, JUN, MYC and IRF7). We confirmed the changes in expression of some key transcription factors (STAT1 and IRF7) by quantitative reverse transcription-polymerase chain reaction. Collectively, our analyses provide a global overview of transcription factor changes in dorsal root ganglia after sciatic nerve injury and offer insight into the regulatory transcription factor networks involved in cell death.

miR-30c promotes Schwann cell remyelination following peripheral nerve injury.

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

The Characteristics of Staphylococcus aureus Small Colony Variant Isolated from Chronic Mastitis at a Dairy Farm in Yunnan Province, China.

Staphylococcus aureus is a major causative agent leading to bovine mastitis and has specific phonotypical characteristics including small colony, slow growth, and decreased hemolysis, therefore named as the small colony variants (SCVs). Out of 30 tested samples of the chronic S. aureus cases, one strain of SCVs (S. aureus SCV22) was isolated along with its parental strains (S. aureus11). S. aureus SCV22 showed a slow growth rate when it is compared with the parental strain. However, their resistant patterns were similar. Meanwhile, S. aureus SCV22 depicted the lower rate of apoptosis in bovine mammary epithelial cells. These findings of the present study presented the unique characteristics of S. aureus SCV22 for the first time in Yunnan province, which provided a prophase foundation for further study about the pathogenesis of S. aureus SCVs in chronic mastitis.

Change in cholesterol absorption and synthesis markers in patients with coronary heart disease after combination therapy with simvastatin plus ezetimibe.

BACKGROUND: Statins and ezetimibe have been reported to change the balance of cholesterol metabolism, but few studies have been performed on Chinese patients. The aim of this study was to evaluate changes in cholesterol metabolism markers in patients with coronary heart disease. METHODS: Forty-five patients with coronary heart disease were treated with 20 mg/d of simvastatin for four weeks. Subjects were then divided into two different therapy groups according to whether they reached the target values for total cholesterol and low density lipoprotein cholesterol level. Patients who reached the target values remained on simvastatin and those who did not reach the target values took a combination of simvastatin plus 10 mg/d ezetimibe until the 12th week. The concentrations of cholesterol synthesis markers (lathosterol and desmosterol) and absorption markers (campesterol and sitosterol) were measured on the 1st, 4th, and 12th week of the study by gas chromatography. RESULTS: After treatment with simvastatin for four weeks, the levels of total cholesterol and low density lipoprotein cholesterol decreased significantly compared to levels measured during the 1st week (P < 0.05). On the 12th week the levels of total cholesterol and low density lipoprotein cholesterol had decreased significantly (P < 0.001) compared to levels during the 4th week. By the 12th week the levels of campesterol and sitosterol in the combination group had decreased significantly (P < 0.05) compared with levels measured during the 4th week. CONCLUSIONS: Coronary heart disease patients with high cholesterol synthesis at baseline might gain a greater benefit from simvastatin treatment. Combination therapy with simvastatin plus ezetimibe in patients with low cholesterol synthesis at baseline might increase the success rate of lipid-lowering through decreasing the absorption of cholesterol.

Gas chromatography analysis of serum cholesterol synthesis and absorption markers used to predict the efficacy of simvastatin in patients with coronary heart disease.

OBJECTIVES: We investigated the changes in cholesterol absorption and synthesis markers before and after simvastatin therapy in Chinese patients with coronary heart disease. DESIGN AND METHOD: We developed a gas chromatography method to identify cholesterol synthesis and absorption markers and measured them in patients with coronary heart disease. We then tested their use in predicting the efficacy of simvastatin in lowering cholesterol. Serum samples from 45 patients and 38 healthy humans (controls) were analyzed in a gas chromatography-flame ionization detector. RESULTS: Squalene and five non-cholesterol sterols--desmosterol and lathosterol (synthesis markers) and campesterol, stigmasterol, and sitosterol (absorption markers)--were detected. The recovery rates of the markers were 95-102%. After simvastatin treatment for four weeks, the total cholesterol and low-density lipoprotein cholesterol levels had significantly decreased from the baseline values (p<0.05). The baseline lathosterol level was significantly higher in good responders than in poor responders (p<0.05), and the stigmasterol level was significantly lower in good responders than in poor responders (p<0.05). CONCLUSIONS: This method should be suitable for the detection of serum squalene and non-cholesterol markers and can be used to predict the efficacy of simvastatin in patients with coronary heart disease.

CYP3A4*1B polymorphism and cancer risk: a HuGE review and meta-analysis.

CYP450 3A4 (CYP3A4), encoded by the CYP3A4 gene, is a major enzyme catalyzing the metabolism of both endogenous and exogenous agents that may play a role in the etiology of carcinogenesis. Several potentially functional polymorphisms of the CYP3A4 gene have been implicated in cancer risk, but individually published studies have shown inconclusive results. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis was to investigate the association between CYP3A4*1B (rs2740574 A > G) polymorphism and cancer risk. Eleven studies were included with a total of 3,810 cancer patients and 3,173 healthy controls. We found that the G allele and GG genotype of CYP3A4*1B polymorphism were associated with increased risk of cancers using the fixed effects model (allele model: odds ratio (OR) = 1.24, 95 %CI: 1.09-1.42, P = 0.001; recessive model: OR = 1.77, 95 %CI: 1.30-2.41, P < 0.001; homozygous model: OR = 1.72, 95 %CI: 1.19-2.47, P = 0.004). Subgroup analyses by cancer type showed that the G allele and G carrier (AG + GG) of CYP3A4*1B polymorphism had significant associations with increased risk of prostate cancer, but not with breast cancer, leukemia, or other cancers. With further subgroup analysis based on different ethnicities, the results indicated that the GG genotype of CYP3A4*1B polymorphism might increase the risk of cancer among African populations. However, similar associations were not observed among Caucasian and Asian populations. Results from the current meta-analysis indicate that the G allele and GG genotype of CYP3A4*1B polymorphism might be associated with increased cancer risk, especially for prostate cancer among African populations.

Effects of Tat peptide on intracellular delivery of arsenic trioxide albumin microspheres.

The current study was designed to evaluate the ability of cell-penetrating peptides to deliver arsenic trioxide albumin microspheres (AsAMs) into bladder cancer cells. The transactivating transcriptional activator (Tat) peptide was labeled with the enhanced green fluorescent protein (EGFP) using eukaryotic vector construction and fusion gene expression techniques. Arsenic trioxide albumin mirospheres were prepared using the chemical crosslink and solidification method. The conjugate, Tat-EGFP-As2O3-AMs (TEAsAMs), was synthesized using the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio) propionate and verified by electrophoresis under reducing conditions and fluorescence microscopy. The intracellular delivery of TEAsAMs was evaluated by laser confocal microscopy and transmission electron microscopy. The arsenic content in the bladder cancer EJ cells was assayed to evaluate the efficiency of delivery. Gene sequencing showed that the pET-Tat-EGFP expression vector was constructed successfully. The expression of the Tat-EGFP fusion protein was verified by matrix-assisted laser desorption/ionization-time of flight analysis, and the protein was transduced into cell cytoplasm as observed under a fluorescence microscope. Electrophoresis under reducing conditions demonstrated the covalent linkage between Tat-EGFP and AsAMs. Under a laser confocal microscope and a transmission electron microscope, TEAsAMs surrounded by green fluorescence were shown to enter the cells faster than EGFP-As2O3-AMs, with an increase in the intracellular arsenic content being observed in cells treated with TEAsAMs compared with those treated with EGFP-As2O3-AMs. These results suggest that Tat peptide promotes the cellular uptake of large albumin microspheres with encapsulated arsenicals.

[Mechanism of the effect of LPSp on the production of anti-HBs in mice].

AIM: To investigate the mechanism of the effect of avirulent Pantoea agglomerans lipopolysaccharide (LPSp) as the adjuvant of HBsAg on the production of anti-HBs in mice. METHODS: The peritoneal macrophages of BALB/c mice were pulsed in vitro by HBsAg+LPSp, HBsAg, LPSp, and NS respectively. The levels of anti-HBs IgG in serum were determined by ELISA. The variation of phagocytosis of the macrophages was detected. The culture supernatants were harvested and assayed for TNF-alpha and NO. HBsAg-pulsed peritoneal macrophages in the presence or absence of LPSp were injected in the hind footpads of syngeneic mice. Lymph node cells were harvested and cultured by HBsAg. IL-4 and IFN-gamma in the supernatants were quantitated by ELISA. RESULTS: The titer of anti-HBs IgG from the mice immunized with HBsAg-pulsed peritoneal macrophages treated with LPSp was higher than that of the mice immunized in the absence of LPSp. The phagocytosis rate of the LPSp-treated peritoneal macrophages was higher than that of the untreated macrophages. The level of TNF-alpha and NO of the LPSp-treated peritoneal macrophages was higher than that of the untreated macrophages. HBsAg-pulsed peritoneal cells in the presence of LPSp can induced higher production of IL-4 than those in the absence of LPSp. CONCLUSION: LPSp can act as the adjuvant of HBsAg and effectively increase the anti-HBS in the mice immunized by HBsAg. LPSp can activate macrophages and stimulate HBsAg-pulsed macrophages to induce the production of IL-4 of lymph node cells.

[Transformation of Populus tomentosa with anti-PLD gene].

Antisense Phospholipase Dgamma( PLDgamma) gene was introduced into Populus tomentosa mediated by Agrobactrium tumefaciens. The young leaves of triploid populus were used as the material and the regeneration system of high frequency has been established. We have developed the traditional transgene method by Agrobactrium trmefaciens and obtained many transgenic plants of anti-PLDgamma gene. Tests showed that the transgenic plants can grow well on the culture medium with 0.7% NaCl.