2-Methyl-4'-(methylthio)-2-morpholinopropiophenone: A commercial photoinitiator being used as a new psychoactive substance.

Synthetic cathinones, which are novel psychoactive substances, have caused major social problems worldwide. A substance called 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP), which is employed as a commercial industrial photoinitiator for triggering polymerization, has a basic cathinone backbone; however, few reports regarding MMMP have been published. In the current study, three potential metabolites of MMMP-namely hydroxy-MMMP (HO-MMMP), HO-MMMP-sulfoxide (HO-MMMP-SO), and HO-MMMP-sulfone (HO-MMMP-SO2)-were successfully synthesized, and MMMP and these three potential metabolites were used as standards to establish an analytic method based on liquid chromatography-tandem mass spectrometry for the quantitative analysis of urine. This analytic method and related parameters-including dynamic range, limit of quantification, selectivity, precision, accuracy, carryover effect, matrix effect, interference, and dilution integrity-were optimized and validated. Forty urine samples from 1,691 individuals who abused drugs were determined to contain MMMP, HO-MMMP, HO-MMMP-SO, or HO-MMMP-SO2; the results of this study indicate that approximately 2.37 % of drug abusers in Taiwan consumed MMMP in 2023. These 40 urine samples were analyzed to investigate the metabolism of MMMP in humans. The results indicate that HO-MMMP-SO is the main metabolite in human urine. This study recommends HO-MMMP-SO with a concentration of 2 ng/mL as a target and cutoff value, respectively, for identifying individuals who have consumed MMMP.

Enhancing Zn2+ Storage Performance by Constructing the Interfaces Between VO2 and Co-N-C Layers.

Vanadium oxides have aroused attention as cathode materials in aqueous zinc-ion batteries (AZIBs) due to their low cost and high safety. However, low ion diffusion and vanadium dissolution often lead to capacity decay and deteriorating stability during cycling. Herein, vanadium dioxides (VO2) nanobelts are coated with a single-atom cobalt dispersed N-doped carbon (Co-N-C) layer via a facile calcination strategy to form Co-N-C layer coated VO2 nanobelts (VO2@Co-N-C NBs) for cathodes in AZIBs. Various in-/ex situ characterizations demonstrate the interfaces between VO2 layers and Co-N-C layers can protect the VO2 NBs from collapsing, increase ion diffusion, and enhance the Zn2+ storage performance. Additional density functional theory (DFT) simulations demonstrate that Co─O─V bonds between VO2 and Co-N-C layers can enhance interfacial Zn2+ storage. Moreover, the VO2@Co-N-C NBs provided an ultrahigh capacity (418.7 mAh g-1 at 1 A g-1), outstanding long-term stability (over 8000 cycles at 20 A g-1), and superior rate performance.

A new process of ketamine synthesis from 2-(2-chlorophenyl)-2-nitrocyclohexanone proposed by analyzing drug materials and chemicals seized in Taiwan.

Because of its hallucinogenic and dissociative effects, ketamine is often abused for recreational purposes. Thus, the seizure of ketamine manufacturing units is crucial for preventing drug abuse. The precursors popularly used for ketamine synthesis include 1-[(2-chlorophenyl)(methylimino)methyl]cyclopentanol hydrochloride and 2-(2-chlorophenyl)-2-nitrocyclohexanone (2-CPNCH). Herein, we report a case of the seizure of a ketamine manufacturing unit by law enforcement officers. The seized materials were sent to our laboratory for confirmation. We found that 2-CPNCH was used as the precursor. Using zinc powder and formic acid, 2-CPNCH was reduced to norketamine. Through the Eschweiler-Clarke reaction, norketamine was reacted with formaldehyde and formic acid to synthesize ketamine; the advantages of this process are a short duration of reaction and the requirement of small amounts of chemicals. We further identified an impurity (N-methyl ketamine), which was used as a marker to validate this new process of ketamine synthesis. To the best of our knowledge, this study is the first to report illegal ketamine synthesis through the Eschweiler-Clarke reaction when using 2-CPNCH as the precursor. Our findings inform law enforcement officers and forensic practitioners about this new process of ketamine synthesis.

Profile of the RNA in exosomes from astrocytes and microglia using deep sequencing: implications for neurodegeneration mechanisms.

Glial cells play an important role in signal transduction, energy metabolism, extracellular ion homeostasis and neuroprotection of the central nervous system. However, few studies have explained the potential effects of exosomes from glial cells on central nervous system health and disease. In this study, the genes expressed in exosomes from astrocytes and microglia were identified by deep RNA sequencing. Kyoto Encyclopedia of Genes and Genomes analysis indicated that several pathways in these exosomes are responsible for promoting neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Gene ontology analysis showed that extracellular exosome, mitochondrion and growth factor activity were enriched in exosomes from the unique astrocyte group, while extracellular exosome and mitochondrion were enriched in exosomes from the unique microglia group. Next, combined with the screening of hub genes, the protein-protein interaction network analysis showed that exosomes from astrocytes influence neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance, whereas exosomes from microglia influence neurodegenerative diseases through immune inflammation and oxidative stress. Although there were differences in RNA expression between exosomes from astrocytes and microglia, the groups were related by the hub genes, ubiquitin B and heat shock protein family A (Hsp70) member 8. Ubiquitin B appeared to be involved in pleiotropic regulatory functions, including immune regulation, inflammation inhibition, protein catabolism, intracellular protein transport, exosomes and oxidative stress. The results revealed the clinical significance of exosomes from glia in neurodegenerative diseases. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. S20180102-152) on January 2, 2018.

Silencing the enhancer of zeste homologue 2, Ezh2, represses axon regeneration of dorsal root ganglion neurons.

Recovery from injury to the peripheral nervous system is different from that of the central nervous system in that it can lead to gene reprogramming that can induce the expression of a series of regeneration-associated genes. This eventually leads to axonal regeneration of injured neurons. Although some regeneration-related genes have been identified, the regulatory network underlying axon regeneration remains largely unknown. To explore the regulator of axon regeneration, we performed RNA sequencing of lumbar L4 and L5 dorsal root ganglion (DRG) neurons at different time points (0, 3, 6, 12 hours, 1, 3 and 7 days) after rat sciatic nerve crush. The isolation of neurons was carried out by laser capture microscopy combined with NeuN immunofluorescence staining. We found 1228 differentially expressed genes in the injured sciatic nerve tissue. The hub genes within these differentially expressed genes include Atf3, Jun, Myc, Ngf, Fgf2, Ezh2, Gfap and Il6. We verified that the expression of the enhancer of zeste homologue 2 gene (Ezh2) was up-regulated in DRG neurons after injury, and this up-regulation differed between large- and small-sized dorsal root ganglion neurons. To investigate whether the up-regulation of Ezh2 impacts axonal regeneration, we silenced Ezh2 with siRNA in cultured DRG neurons and found that the growth of the newborn axons was repressed. In our investigation into the regulatory network of Ezh2 by interpretive phenomenal analysis, we found some regulators of Ezh2 (including Erk, Il6 and Hif1a) and targets (including Atf3, Cdkn1a and Smad1). Our findings suggest that Ezh2, as a nerve regeneration-related gene, participates in the repair of the injured DRG neurons, and knocking down the Ezh2 in vitro inhibits the axonal growth of DRG neurons. All the experimental procedures approved by the Administration Committee of Experimental Animals of Jiangsu Province of China (approval No. S20191201-201) on March 21, 2019.

Proteolysis targeting chimera technology: a novel strategy for treating diseases of the central nervous system.

Neurological diseases such as stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease are among the intractable diseases for which appropriate drugs and treatments are lacking. Proteolysis targeting chimera (PROTAC) technology is a novel strategy to solve this problem. PROTAC technology uses the ubiquitin-protease system to eliminate mutated, denatured, and harmful proteins in cells. It can be reused, and utilizes the protein destruction mechanism of the cells, thus making up for the deficiencies of traditional protein degradation methods. It can effectively target and degrade proteins, including proteins that are difficult to identify and bind. Therefore, it has extremely important implications for drug development and the treatment of neurological diseases. At present, the targeted degradation of mutant BTK, mHTT, Tau, EGFR, and other proteins using PROTAC technology is gaining attention. It is expected that corresponding treatment of nervous system diseases can be achieved. This review first focuses on the recent developments in PROTAC technology in terms of protein degradation, drug production, and treatment of central nervous system diseases, and then discusses its limitations. This review will provide a brief overview of the recent application of PROTAC technology in the treatment of central nervous system diseases.

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.

Autophagy plays a protective role against apoptosis induced by toxicarioside N via the Akt/mTOR pathway in human gastric cancer SGC-7901 cells.

Toxicarioside N (Tox N), a natural product extract from Antiaris toxicaria, has been reported to induce apoptosis in human gastric cancer cells. However, the mechanism and actual role of autophagy in Tox N-induced apoptosis of human gastric cancer cells remains poorly understood. In the current study, we demonstrated that Tox N could induce autophagy by inhibiting the Akt/mTOR signaling pathway in SGC-7901 cells. Moreover, we found that the inhibition of autophagy by 3-methyladenine, an autophagy inhibitor, enhanced Tox N-induced apoptotic cell death. However, the stimulation of autophagy by rapamycin, an autophagy activator, remarkably suppressed Tox N-induced apoptosis, suggesting that autophagy plays a protective role in Tox N-induced apoptosis. Thus, the results from this study suggested that Tox N combination with an autophagy inhibitor might be a promising strategy to enhance the anticancer activity of Tox N for the treatment of human gastric cancer.

Toxicarioside N induces apoptosis in human gastric cancer SGC-7901 cell by activating the p38MAPK pathway.

Natural plant compounds with potent proliferation inhibition and apoptosis induction properties have been screened as novel anticancer drugs. Toxicarioside N (Tox N) was isolated from the seeds of the tropical plant Antiaris toxicaria in Hainan province, China. To our knowledge, the effects that Tox N has on the apoptosis of SGC-7901 cells and its potential mechanism have never been investigated. In this study, we detected the anticancer activities of Tox N and explored the potential mechanism in the human gastrointestinal cancer cell line SGC-7901. Here, we found that Tox N inhibited SGC-7901 cell growth in a dose- and time-dependent manner and induced apoptosis in cells based on cell morphology and flow cytometry analyses. Additionally, the SGC-7901 cell treated with Tox N up-regulated the expression level of cleaved caspase-3/9 and PARP, increased the Bax/Bcl-2 ratio, and led to the release of cytochrome c into the cytoplasm. In addition, Tox N treatment led to the phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, partially attenuated Tox N induced apoptosis by preventing the activation of caspase-3/9 and PARP. Our results indicated for the first time that Tox N can induce SGC-7901 cells apoptosis by activating the p38MAPK pathway.

[Effect of Sanbitang recipe in treatment of rheumatoid arthritis with kidney empty and cold-dampness symptom].

To explore the clinical effect of Sanbitang recipe in treatment for the rheumatoid arthritis (RA) with kidney empty and cold-dampness symptom and its safety. A total 168 cases eligible patients were randomly divided into the traditional Chinese medicine (TCM) group, the chemical medicine group and the TCM combined with chemical medicine group, with 56 cases in each group. The TCM group was treated with Sanbitang recipe; The chemical medicine group was given methotrexate tablets; And Sanbitang recipe and methotrexate tablets was adopted in the TCM combined with chemical medicine group. A course of treatment was 16 weeks. Health assessment questionnaire (HAQ), disease activity scores 28-joint counts (DAS28), visual analogue scale (VAS), TCM symptom, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), cyclic citrullinated peptides (CCP) and rheumatoid factor (RF) were detected. The efficiencies and incidence of adverse reactions in the three groups were compared. The total effective rate of the TCM combined with chemical medicine group was 92.7%, which was higher than 79.2% of the TCM group and 82.4% of the chemical medicine group (P<0.05). There was no statistically significant difference between the TCM group and the chemical medicine group. This suggested that Sanbitang recipe was effective in treating rheumatoid arthritis (RA) with kidney empty and cold-dampness symptom. After treatment, the scores of HAQ, DAS28, VAS, ESR, CRP, CCP and RF of the TCM combined with chemical medicine group were significantly higher (P<0.05) among the three groups. There was no statistically significant difference between the TCM group and the chemical medicine group. This indicated that Sanbitang recipe could effectively alleviate the clinical symptoms of rheumatoid arthritis (RA) with kidney empty and cold-dampness symptom. In terms of efficiency and incidence of adverse reactions, the order from low to high was that the TCM group (3.8%, 2/53)

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.

Penicilazaphilone C, a new antineoplastic and antibacterial azaphilone from the Marine Fungus Penicillium sclerotiorum.

Two azaphilonidal derivatives [penicilazaphilones B (1) and C (2)], have been isolated from the fermented products of marine fungus strain Penicillium sclerotiorum M-22, penicilazaphilones C was a new compound. The compound's structures were identified by the analysis of spectroscopic data including 1D and 2D NMR techniques (1H-NMR, 13C-NMR, COSY, HMQC, and HMBC). Biological evaluation revealed that penicilazaphilones B and C showed selective cytotoxicity against melanoma cells B-16 and human gastric cancer cells SGC-7901 with IC50 values of 0.291, 0.449 and 0.065, 0.720 mM, respectively, while exhibiting no significant toxicity to normal mammary epithelial cells M10 at the same concentration. Moreover, penicilazaphilones C also exhibited strong antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia and Escherichia coli with MIC values 0.037-0.150 mM, while penicilazaphilones B's bacteriostatic action was weaker.

miR-148b-3p promotes migration of Schwann cells by targeting cullin-associated and neddylation-dissociated 1.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively adjust gene expression in multifarious biological processes. However, the regulatory effects of miRNAs on Schwann cells remain poorly understood. Previous microarray analysis results have shown that miRNA expression is altered following sciatic nerve transaction, thereby affecting proliferation and migration of Schwann cells. This study investigated whether miR-148b-3p could regulate migration of Schwann cells by directly targeting cullin-associated and neddylation-dissociated 1 (Cand1). Up-regulated expression of miR-148b-3p promoted Schwann cell migration, whereas silencing of miR-148b-3p inhibited Schwann cell migration in vitro. Further experiments confirmed that Cand1 was a direct target of miR-148b-3p, and Cand1 knockdown reversed suppression of the miR-148b-3p inhibitor on Schwann cell migration. These results suggested that miR-148b-3p promoted migration of Schwann cells by directly targeting Cand1 in vitro.

Microencapsulation of tumor lysates and live cell engineering with MIP-3α as an effective vaccine.

The combination of several potential strategies so as to develop new tumor vaccines is an attractive field of translational medicine. Pulsing tumor lysates with dendritic cells (DCs), in-vivo attraction of DCs by macrophage inflammatory protein 3α (MIP-3α), and reversion of the tumor suppressive microenvironment have been tested as strategies to develop tumor vaccines. In this study, we generated an alginate microsphere (named PaLtTcAdMIP3α) that encapsulated tumor lysates, live tumor cells engineering with a recombinant MIP-3α adenovirus and BCG. We used PaLtTcAdMIP3α as a model vaccine to test its antitumor activities. Our results showed that PaLtTcAdMIP3α expressed and excreted MIP-3α, which effectively attracted DCs ex vivo and in vivo. Injection of PaLtTcAdMIP3α into tumor-bearing mice effectively induced both therapeutic and prophylactic antitumor immunities in CT26, Meth A, B16-F10 and H22 models, but without any ensuing increase in adverse effects. Both tumor-specific cellular and humoral immune responses, especially the CD8(+) T cell-dependent cytotoxic T immunity, were found in the mice injected with PaLtTcAdMIP3α. The anti-tumor activity was abrogated completely by depletion of CD8(+) and partially by CD4(+) T lymphocytes. In addition, the number of IFN-γ-producing CD8(+) T cells in spleen and tumor tissues was significantly increased; but the number of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) in tumor tissues was decreased. These data strongly suggest that a combination of multi-current-using strategies such as the novel approach of using our PaLtTcAdMIP3α microspheres could be an effective tumor model vaccine.

Strophalloside induces apoptosis of SGC-7901 cells through the mitochondrion-dependent caspase-3 pathway.

Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.

Sanpao herbs inhibit development of atopic dermatitis in Balb/c mice.

OBJECTIVE: To explore the effects of SANPAOCAO (SPC), a compound traditional Chinese folk medicine, on chronic dermatitis/eczema in mice induced by 2, 4-dinitrochlorobenzene (DNCB). METHODS: Thirty-three Balb/c mice were randomly divided into a negative control group, a positive control group, a prednisolone treatment group, an SPC ethanol extract treatment group, a Cardiospermum halicacabum ethanol extract treatment group, a Physalis minima ethanol extract treatment group, and a Jussiaea repens ethanol extract treatment group. Mice in the six treatment groups had twenty-five microliters of 0.1% DNCB in acetone/olive oil (3: 1) applied to each side of their right ears and dorsal skin three times a week, over a 5 week period. They were treated with prednisolone or the various kinds of ethanol extract after each challenge. The weight difference between the two ears, pathological changes in the right ears, dermal inflammatory cell numbers, and total serum Ig E levels were used to assess the effects of the drugs. RESULTS: after the 5 weeks of challenges, the weight differences of the ears in the SPC group and the prednisolone group were significantly less than those in the other groups. There was evidence of significant suppression of the development of dermatitis, as determined by a histological examination and the serum Ig E levels. CONCLUSION: SPC has beneficial effects when used in the treatment of chronic dermatitis-eczema in mice.

Antigen 43/Fcε3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine.

The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and ß subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules.

Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis.

Various angiogenesis-related self-molecules have been considered to be therapeutic targets. However, the direct use of self-molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a ß subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self-molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43') expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis-related endoglin gene was then subcloned into plasmid pETAg43', resulting in a recombinant plasmid pETAg43'/END(e) which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43'/END(e) ) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43'/END(e) as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43'/END(e) chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis-related self-molecules for cancer therapy.

Cytochalasin D promotes pulmonary metastasis of B16 melanoma through expression of tissue factor.

Cytochalasin D (CytD) targets actin, a ubiquitous protein in eukaryotic cells. Previous studies have focused mainly on the antitumor effects of CytD. We previously found CytD to promote lung metastasis in B16 melanoma cells, which we had not anticipated, and, therefore, in the present study we investigated the possible underlying mechanisms. B16 melanoma cells were co-cultured with CytD and other agents and used to establish a lung metastatic model. In this B16 melanoma metastatic model, significantly increased lung metastasis and lung weight were found in CytD-treated mice, which was almost completely suppressed by tissue factor (TF) RNA interference expressed via lentivirus. The results of northern and western blot, and real-time RT-PCR analysis showed that the expression of TF was significantly upregulated in B16 cells treated with CytD but was significantly inhibited by TF RNA interference. In addition, upregulation and phosphorylation of mitogen-activated protein kinase p38 were also found in the metastatic lung tissues treated with CytD and in the B16 cells co-cultured with CytD and factor VIIa (FVIIa), but not in cells cultured with CytD, dimethyl sulfoxide or FVIIa alone. These results indicate that CytD stimulates the expression of TF in B16 melanoma cells, activating both coagulation-dependent and -independent pathways via binding to FVIIa, eventually promoting lung metastasis. TF interference is a potential approach to the prevention of B16 melanoma metastasis.

[Immune response in mice induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii].

OBJECTIVE: To observe the immune response induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii in mice. METHODS: The recombinant eukaryotic expression plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were constructed and identified by PCR, restriction enzyme digestion, and sequencing. The three recombinant plasmids were transfected into HeLa cells to express in vitro and identified by Western blotting analysis. Seventy Kunming mice were randomly divided into 5 groups with 14 each, i.e. pcSAG1 group, pcROP5 group, pcSAG1-ROP5 group, blank plasmid group and PBS control group. The mice were immunized intramuscularly with pcSAG1, pcROP5, pcSAG1-ROP5, pcDNA3.1, and PBS, respectively, every two weeks for three times. Sera were collected before each injection and 2 weeks after the last immunization. The titer of mice serum in pcSAG1-ROP5 group combined with recombinant protein SAG1, ROP5 and SAG1-ROP5 and the level of IgG against T. gondii in 5 groups were determined by ELISA. Three weeks after the last immunization, ten mice of each group were challenged with 10(3) tachyzoites of the virulent T. gondii RH strain to observe the survival time. One week later, the rest four mice in each group were sacrificed and the supernatant of cultured splenocytes was collected for the detection of IFN-gamma and IL-4. RESULTS: Western blotting showed that the recombinant plasmids pcSAG1, pcROP5 and pcSAG1-ROP5 were expressed in HeLa cells with M(r) 31 000, 57 000, and 88 000, respectively. The serum titer in pcSAG1-ROP5 group combined with SAG1, ROP5 and SAG1-ROP5 was 1:320, 1:160, and 1:2560, respectively. The IgG level kept rising in pcSAG1, pcROP5 and pcSAG1-ROP5 groups. Two weeks after the last immunization, the IgG level in pcSAG1-ROP5 group was higher than those in other groups (P<0.05). After a lethal challenge of T. gondii RH strain, the survival time of the mice in pcSAG1-ROP5 group was (288 +/- 7) h, which was 48 h and 96h longer than the groups of pcSAG1 and pcROP5, respectively (P< 0.05). Four weeks after the last immunization, IFN-gamma in splenocyte culture of pcSAG1-ROP5 group [(908.52 +/- 6.31) pg/ml] was higher than other groups (P<0.05), with no significant difference in IL-4 (P>0.05). CONCLUSION: Compared with the single gene vaccines pcSAG1 and pcROP5, higher levels of IgG and IFN-gamma and longer survival time are observed in mice immunized with pcSAG1-ROP5.