Discovery of Novel Non-Nucleoside Inhibitors Interacting with Dizinc Ions of CD73.

High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73, resulting in potent immunosuppression to inhibit T cell and natural killer (NK) cell function. CD73, as the determining enzyme for adenosine production, has been shown to correlate with poor clinical tumor prognosis. Conventional inhibitors as analogues of adenosine 5'-monophosphate (AMP) may have a risk of further metabolism to adenosine analogues. Here, we report a new series of malonic acid non-nucleoside inhibitors coordinating with zinc ions of CD73. Compound 12f was found to be a superior CD73 inhibitor (IC50 = 60 nM) by structural optimization, and its pharmacokinetic properties were investigated. In mouse tumor models, compound 12f showed excellent efficacy and reversal of immunosuppression in combination with chemotherapeutic agents or checkpoint inhibitors, suggesting that it deserves further development as a novel CD73 inhibitor.

ATG5-regulated CCL2/MCP-1 production in myeloid cells selectively modulates anti-malarial CD4+ Th1 responses.

Parasite-specific CD4+ Th1 cell responses are the predominant immune effector for controlling malaria infection; however, the underlying regulatory mechanisms remain largely unknown. This study demonstrated that ATG5 deficiency in myeloid cells can significantly inhibit the growth of rodent blood-stage malarial parasites by selectively enhancing parasite-specific CD4+ Th1 cell responses. This effect was independent of ATG5-mediated canonical and non-canonical autophagy. Mechanistically, ATG5 deficiency suppressed FAS-mediated apoptosis of LY6G- ITGAM/CD11b+ ADGRE1/F4/80- cells and subsequently increased CCL2/MCP-1 production in parasite-infected mice. LY6G- ITGAM+ ADGRE1- cell-derived CCL2 selectively interacted with CCR2 on CD4+ Th1 cells for their optimized responses through the JAK2-STAT4 pathway. The administration of recombinant CCL2 significantly promoted parasite-specific CD4+ Th1 responses and suppressed malaria infection. Conclusively, our study highlights the previously unrecognized role of ATG5 in modulating myeloid cells apoptosis and sequentially affecting CCL2 production, which selectively promotes CD4+ Th1 cell responses. Our findings provide new insights into the development of immune interventions and effective anti-malarial vaccines.Abbreviations: ATG5: autophagy related 5; CBA: cytometric bead array; CCL2/MCP-1: C-C motif chemokine ligand 2; IgG: immunoglobulin G; IL6: interleukin 6; IL10: interleukin 10; IL12: interleukin 12; MFI: mean fluorescence intensity; JAK2: Janus kinase 2; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; pRBCs: parasitized red blood cells; RUBCN: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; STAT4: signal transducer and activator of transcription 4; Th1: T helper 1 cell; Tfh: follicular helper cell; ULK1: unc-51 like kinase 1.

Dietary melatonin positively impacts the immune system of crayfish, Cherax destructor, as revealed by comparative proteomics analysis.

Melatonin, an indoleamine with various biological activities, is being used increasingly in the aquaculture industry for its broad immune effects. Cherax destructor is an emerging economically cultured crayfish that faces many problems in the breeding process. Previous work found that dietary melatonin has positive effects on the growth and immunity of C. destructor, but the specific mechanism involved remained unclear. In this study, proteomics was used to determine the mechanism of action of melatonin in C. destructor. Results showed that dietary melatonin resulted in decreased levels of hydrogen peroxide, alanine aminotransferase, and aspartate aminotransferase, but increased levels of glutathione peroxidase, acid phosphatase, and glutathione S-transferases. In total, 608 proteins were differentially expressed (418 upregulated and 190 downregulated), and were enriched in three main categories: innate immunity (B cell receptor signaling pathway and natural killer cell-mediated cytotoxicity), glucose metabolism (pentose phosphate pathway, pentose and glucuronate interconversions, and propionate metabolism), and amino acid metabolism (valine, leucine, and isoleucine degradation, and cysteine and methionine metabolism). In addition, dietary melatonin was also involved in the regulation of the mTOR signaling pathway, and upregulated the expression of genes encoding key factors, such as Ras-related GTP-binding protein A/B, eukaryotic initiation factor 4E, eukaryotic initiation factor 4E-binding protein, and p70 ribosomal S6 kinase. Overall, this study demonstrates the role of melatonin in the physiological regulation of C. destructor, laying the foundation for the development of melatonin as a feed additive in the aquaculture of this species.

Haloxyfop-P-methyl induces immunotoxicity and glucose metabolism disorders and affects the Nrf2/ARE pathway mediated antioxidant system in Chiromantes dehaani.

Haloxyfop-P-methyl is used extensively in agricultural production, and its metabolites in soil have potentially toxic effects on aquatic ecosystems. In this study, we explored the toxicity of haloxyfop-P-methyl on Chiromantes dehaani. The results of the 21-day toxicity test showed that haloxyfop-P-methyl decreased the weight gain (WG), specific growth rate (SGR) and hepatosomatic index (HSI). In glucose metabolism, haloxyfop-P-methyl reduced pyruvate, lactate, lactate dehydrogenase and succinate dehydrogenase, but enhanced glucose-6-phosphate dehydrogenase and hexokinase. Furthermore, expression of glucose metabolism-related genes was upregulated. We cloned the full-length CdG6PDH gene, which contains a 1587 bp ORF that encoded a 528 amino acid polypeptide. In antioxidant system, haloxyfop-P-methyl increased glutathione, thioredoxin reductase and thioredoxin peroxidase activities and activated the Nrf2/ARE pathway through upregulation of ERK, JNK, PKC and Nrf2. In immunity, low concentrations haloxyfop-P-methyl, or short-term exposure, upregulated the expression of immune-related genes and enhanced immune-related enzymes activity, while high concentrations or long-term exposure inhibited immune function. In summary, haloxyfop-P-methyl inhibited the growth performance, disrupted glucose metabolism, activated the antioxidant system, and led to immunotoxicity. The results deepen our understanding of the toxicity mechanism of haloxyfop-P-methyl and provide basic biological data for the comprehensive assessment of the risk of haloxyfop-P-methyl to the environment and humans.

Effects of dietary melatonin on growth performance, antioxidant capacity, and nonspecific immunity in crayfish, Cherax destructor.

Melatonin (MT) is an indole hormone widely found in plants and animals. Many studies have shown that MT promotes the growth and immunity of mammals, fish, and crabs. However, the effect on commercial crayfish has not been demonstrated. The purpose of this study was to evaluate the effects of dietary MT on growth performance and innate immunity of Cherax destructor from three aspects of individual level, biochemical level, and molecular level after 8 weeks of culture. In this study, we found that MT supplementation increased weight gain rate, specific growth rate, and digestive enzyme activity in C. destructor compared to the control group. Dietary MT not only promoted the activity of T-AOC, SOD, and GR, increased the content of GSH, and decreased the content of MDA in the hepatopancreas, but also increased the content of hemocyanin and copper ions and AKP activity in hemolymph. Gene expression results showed that MT supplementation at appropriate doses increased the expression of cell cycle-regulated genes (CDK, CKI, IGF, and HGF) and non-specific immune genes (TRXR, HSP60, and HSP70). In conclusion, our study showed that adding MT to the diet improved growth performance, enhanced the antioxidant capacity of hepatopancreas, and immune parameters of hemolymph in C. destructor. In addition, our results showed that the optimal dietary supplementation dose of MT in C. destructor is 75-81 mg/kg.

Effects of dietary soluble ß-1,3-glucan on the growth performance, antioxidant status, and immune response of the river prawn (Macrobrachium nipponense).

The effects of dietary ß-1,3-glucan on the growth performance, body composition, hepatopancreas tissue structure, antioxidant activities, and immune response of the river prawn (Macrobrachium nipponense) were investigated. In total, 900 juvenile prawns were fed one of five diets with different contents of ß-1,3-glucan (0%, 0.1%, 0.2%, and 1.0%) or 0.2% curdlan for 6 weeks. The growth rate, weight gain rate, specific growth rate, specific weight gain rate, condition factor, and hepatosomatic index of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those fed 0% ß-1,3-glucan and 0.2% curdlan (p < 0.05). The whole-body crude lipid content of prawns supplemented with curdlan and ß-1,3-glucan was significantly higher than that of the control group (p < 0.05). The antioxidant and immune enzyme activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), catalase (CAT), lysozyme (LZM), phenoloxidase (PO), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the hepatopancreas of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those of the control and 0.2% curdlan groups (p < 0.05), and tended to increase and then decrease with increasing dietary ß-1,3-glucan. The highest malondialdehyde (MDA) content was observed in juvenile prawns without ß-1,3-glucan supplementation. The results of real-time quantitative PCR indicated that dietary ß-1,3-glucan promoted expression of antioxidant and immune-related genes. Binomial fit analysis of weight gain rate and specific weight gain rate showed that the optimum ß-1,3-glucan requirement of juvenile prawns was 0.550%-0.553%. We found that suitable dietary ß-1,3-glucan improved juvenile prawns growth performance, antioxidant capacity, and non-specific immunity, which provide reference for shrimp healthy culture.

Melanoma stem cell vaccine induces effective tumor immunity against melanoma.

Melanoma stem cells (MSCs)-based vaccine strategies have been a potent immunotherapeutic approach for melanoma treatment, which aimed at inducing specific anti-tumor immunity and targeting cancer stem-like cells. As the main cancer-fighting immune cells, CD8+T cells play an important role in vaccine-induced antitumor immunity. Here, we developed a novel MSC vaccine that induces CD8+T cells to target melanoma stem cells specifically. The MSC vaccine was prepared for our study in order to determine the effectiveness of antitumor immunity. The proportion and activity of CD8+T cells were examined in the spleen after immunization, in particular, the expression and cytotoxicity of the immune checkpoint of spleen lymphocytes were detected by flow cytometry and ELISA, moreover, tumor size and the number of lung metastasis nodules were observed and the specific killing effect of the vaccine was evaluated in immunized mice. We found that the MSC vaccine could promote DCs maturation, activate CD8+T cells, suppress the expression of CTLA-4, PD-1, and Tim-3, and increase the expression of IFN-γ and GzmB of CD8+T cells. Melanoma growth and metastasis were inhibited by the vaccine's specific targeted killing effect. The vaccines based on melanoma stem cells (MSCs) delay the progression of melanoma by inducing anti-tumor immune responses in CD8+T cells.

Drug Repurposing of Quisinostat to Discover Novel Plasmodium falciparum HDAC1 Inhibitors with Enhanced Triple-Stage Antimalarial Activity and Improved Safety.

Our previous work found that the clinical histone deacetylase (HDAC) inhibitor quisinostat exhibited a significant antimalarial effect but with severe toxicity. In this work, 35 novel derivatives were designed and synthesized based on quisinostat as the lead compound, and their in vitro antimalarial activities and cytotoxicities were systematically evaluated. Among them, JX35 showed potent inhibition against both wild-type and multidrug-resistant parasite strains and displayed a significant in vivo killing effect against all life cycles of parasites, including the blood stage, liver stage, and gametocyte stage, indicating its potential for the simultaneous treatment, chemoprevention, and blockage of malaria transmission. Compared with quisinostat, JX35 exhibited stronger antimalarial efficacy, more adequate safety, and good pharmacokinetic properties. Additionally, mechanistic studies via molecular docking studies, induced PfHDAC1/2 knockdown assays, and PfHDAC1 enzyme inhibition assays jointly indicated that the antimalarial target of JX35 was PfHDAC1. In summary, we discovered the promising candidate PfHDAC1 inhibitor JX35, which showed stronger triple-stage antimalarial effects and lower toxicity than quisinostat.

Discovery of Novel Plasmodium falciparum HDAC1 Inhibitors with Dual-Stage Antimalarial Potency and Improved Safety Based on the Clinical Anticancer Drug Candidate Quisinostat.

Previously, we identified the clinical anticancer drug candidate quisinostat as a novel and potent antimalarial lead compound. To further enhance the antimalarial effect and improve safety, 31 novel spirocyclic hydroxamic acid derivatives were synthesized based on the structure of quisinostat, and their antimalarial activities and cytotoxicity were evaluated. Among them, compound 11 displayed broad potency in vitro against several multiresistant malarial parasites, especially two artemisinin-resistant clinical isolates. Moreover, 11 could eliminate both liver and erythrocytic parasites in vivo, kill all morphological erythrocytic parasites with specific potency against schizonts, and show acceptable metabolic stability and pharmacokinetic properties. Western blot analysis, PfHDAC gene knockdown, and enzymatic inhibition experiments collectively confirmed that PfHDAC1 was the target of 11. In summary, 11 is a structurally novel PfHDAC1 inhibitor with the potential to prevent and cure malaria, overcome multidrug resistance, and provide a prospective prototype for antimalarial drug research.

Blood-stage malaria parasites manipulate host innate immune responses through the induction of sFGL2.

Malaria parasites suppress host immune responses to facilitate their survival, but the underlying mechanism remains elusive. Here, we found that blood-stage malaria parasites predominantly induced CD4+Foxp3+CD25+ regulatory T cells to release soluble fibrinogen-like protein 2 (sFGL2), which substantially enhanced the infection. This was attributed to the capacity of sFGL2 to inhibit macrophages from releasing monocyte chemoattractant protein-1 (MCP-1) and to sequentially reduce the recruitment of natural killer/natural killer T cells to the spleen and the production of interferon-γ. sFGL2 inhibited c-Jun N-terminal kinase phosphorylation in the Toll-like receptor 2 signaling pathway of macrophages dependent on FcγRIIB receptor to release MCP-1. Notably, sFGL2 were markedly elevated in the sera of patients with malaria, and recombinant FGL2 substantially suppressed Plasmodium falciparum from inducing macrophages to release MCP-1. Therefore, we highlight a previously unrecognized immune suppression strategy of malaria parasites and uncover the fundamental mechanism of sFGL2 to suppress host innate immune responses.

Temporal dynamics of earthworm (Eisenia fetida) microbial communities after cadmium stress based on a compound mathematical model.

Soil cadmium (Cd) pollution has received increasing attention from scholars. In the field of Cd pollution remediation, there is an urgent need to study the combined bioremediation technology of earthworms and microbes. In this paper, a short-term stress test and a long-term stress test were conducted. Eisenia fetida were inoculated into artificial soil that was contaminated with Cd. After different Cd stress times, the regulation process between the microbial communities in the earthworms and in the soil was studied. Canonical correlation analysis and the TOPSIS method were combined to establish a mathematical model for data analysis, and the changes in the carbon source utilization intensity by microbes were analysed. The results showed that in the short-term stress tests, the regulation process could be divided into five stages. Specifically, after 1-3 days of stress, the microbial community in the earthworms regulated the soil microbial community, but on the 3rd day, the regulation was weakened. On the 4th day, the soil microbial community was affected not only by the microbes in the earthworms but also by the increasing intensity of Cd stress. After 5 days of stress, the microbial communities in the earthworms and the soil were both greatly affected by Cd poisoning, and the microbes transitioned from stable to declining. At 6-7 days, the microbes in the earthworms gained control over those in the soil once again, and the Cd-tolerant microbes began to appear and proliferate. At 8-10 days, the regulation of the soil microbes by the earthworm microbes weakened, while the Cd-tolerant population in the soil microbial communities gradually evolved at this stage to adapt to the increasing Cd stress. The long-term stress tests showed that the difference between the microbial communities in the soil and in the earthworms increased, and there was almost no regulation between them.

Attenuated plasmodium sporozoite expressing MAGE-A3 induces antigen-specific CD8+ T cell response against lung cancer in mice.

OBJECTIVE: Cancer vaccines that rely on tumor antigen-specific CD8+ T cell responses, are promising anti-cancer adjuvant immunotherapies. This study investigated whether genetically attenuated Plasmodium sporozoite (GAS) could be used as a novel vector to induce antigen-specific CD8+ T cell responses against lung cancer. METHODS: We constructed GAS/MAGE-A3, a recombinant GAS engineered to express the lung cancer-specific antigen, melanoma-associated antigen 3 (MAGE-A3), and assessed its therapeutic effects against lung cancer. RESULTS: Robust parasite-specific CD8αlowCD11ahigh and CD49dhighCD11ahigh CD4+ T cell responses as well as a MAGE-A3-specific CD8+ T cell response were induced in GAS/MAGE-A3-immunized mice. Adoptive transfer of GAS/MAGE-A3-induced CD8+ T cells from HLA-A2 transgenic mice into lung cancer-bearing nude mice inhibited tumor growth and prolonged survival. CONCLUSIONS: These findings demonstrate that GAS/MAGE-A3 induces a strong MAGE-A3-specific CD8+ T cell response against lung cancer in vivo, and indicate that GAS is a novel and efficacious antigen delivery vector for antitumor immunotherapy.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB.

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

ß-elemene enhances anticancer and anti-metastatic effects of osteosarcoma of ligustrazine in vitro and in vivo.

The present study aimed to determine the anticancer effects of the combination of ß-elemene and ligustrazine in vitro as well as in in vivo. Following evaluation using an MTT assay, ß-elemene, ligustrazine and the ß-elemene-ligustrazine combination treatments all exhibited the capacity to inhibit the growth of OS-732 cells, with inhibitory rates of 43.3, 54.4, and 75.0%, respectively. Using a flow cytometry assay, it was determined that the ß-elemene-ligustrazine combination possessed the highest apoptotic rate (30.6%). Furthermore, ß-elemene-ligustrazine combination treatment resulted in the highest downregulation of G protein-coupled receptor 124, vascular endothelial growth factor, matrix metallopeptidase (MMP)-2 and MMP-9 mRNA, and protein expression levels. In addition, the combined treatment led to an increase in the mRNA and protein expression of endostatin, TIMP metallopeptidase inhibitor (TIMP)-1 and TIMP-2 in OS-732 cells. Additionally, ß-elemene-ligustrazine caused a decrease in nuclear factor-κB, interleukin-8, C-X-C motif chemokine receptor 4 and urokinase-type plasminogen activator mRNA expression, as well as an increase in caspase-3, caspase-8, and caspase-9 mRNA expression. In vivo, the ß-elemene-ligustrazine combination was able to reduce the weight and the bulk of the tumor in BALB/c-nu/nu nude mice compared with any other group. All the results described above regarding changes to mRNA and protein expression were further confirmed in vivo in the tumor tissue of mice. The results of the present study have suggested that the combination of ß-elemene-ligustrazine exhibits greater anticancer effects compared with ß-elemene- or ligustrazine-alone treatment.

The rodent malaria liver stage survives in the rapamycin-induced autophagosome of infected Hepa1-6 cells.

It has been reported that non-selective autophagy of infected hepatocytes could facilitate the development of malaria in the liver stage, but the fate of parasites following selective autophagy of infected hepatocytes is still not very clear. Here, we confirmed that sporozoite infection can induce a selective autophagy-like process targeting EEFs (exo-erythrocytic forms) in Hepa1-6. Rapamycin treatment greatly enhanced this process in EEFs and non-selective autophagy of infected Hepa1-6 cells and enhanced the development of the malaria liver stage in vivo. Although rapamycin promoted the fusion of autophagosomes containing the malaria parasite with lysosomes, some parasites inside the autophagosome survived and replicated normally. Further study showed that the maturation of affected autolysosomes was greatly inhibited. Therefore, in addition to the previously described positive role of rapamycin-induced nonselective autophagy of hepatocytes, we provide evidence that the survival of EEFs in the autophagosome of the infected hepatocytes also contributes to rapamycin-enhanced development of the malaria liver stage, possibly due to the suppression of autolysosome maturation by EEFs. These data suggest that the inhibition of autolysosome maturation might be a novel escape strategy used by the malaria liver stage.

Establishment of a murine model of cerebral malaria in KunMing mice infected with Plasmodium berghei ANKA.

Malaria remains one of the most devastating diseases. Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection resulting in high mortality and morbidity worldwide. Analysis of precise mechanisms of CM in humans is difficult for ethical reasons and animal models of CM have been employed to study malaria pathogenesis. Here, we describe a new experimental cerebral malaria (ECM) model with Plasmodium berghei ANKA infection in KunMing (KM) mice. KM mice developed ECM after blood-stage or sporozoites infection, and the development of ECM in KM mice has a dose-dependent relationship with sporozoites inoculums. Histopathological findings revealed important features associated with ECM, including accumulation of mononuclear cells and red blood cells in brain microvascular, and brain parenchymal haemorrhages. Blood-brain barrier (BBB) examination showed that BBB disruption was present in infected KM mice when displaying clinical signs of CM. In vivo bioluminescent imaging experiment indicated that parasitized red blood cells accumulated in most vital organs including heart, lung, spleen, kidney, liver and brain. The levels of inflammatory cytokines interferon-gamma, tumour necrosis factor-alpha, interleukin (IL)-17, IL-12, IL-6 and IL-10 were all remarkably increased in KM mice infected with P. berghei ANKA. This study indicates that P. berghei ANKA infection in KM mice can be used as ECM model to extend further research on genetic, pharmacological and vaccine studies of CM.

Small-molecule xenomycins inhibit all stages of the Plasmodium life cycle.

Widespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity against Plasmodium asexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells. In vitro studies show potent activity of Xenomycins against Plasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

The Plasmodium circumsporozoite protein, a novel NF-κB inhibitor, suppresses the growth of SW480.

The blocking of NF-κB activation is a promising strategy for the treatment of colorectal cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was recently reported to block NF-κB activation in hepatocytes. Thus, we investigated the effect of the CSP on the growth of the human colon cancer cell line, SW480. We demonstrated that transfection with a recombinant plasmid expressing CSP inhibited the proliferation of SW480 in a dose-dependent manner and induced the apoptosis of SW480. A NF-κB gene reporter assay showed that both the CSP and its nuclear localization signal (NLS) motif could equally suppress the activation of NF-κB following the stimulation with human recombinant TNF-α in the SW480. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but could sharply inhibit the nuclear translocation of NF-κB in TNF-α stimulated SW480. Hence, our data suggest that the CSP might be explored as a new NF-κB inhibitor for the treatment of colorectal cancer.

Tailless-like (TLX) protein promotes neuronal differentiation of dermal multipotent stem cells and benefits spinal cord injury in rats.

Spinal cord injury (SCI) remains a formidable challenge in the clinic. In the current study, we examined the effects of the TLX gene on the proliferation and neuronal differentiation of dermal multipotent stem cells (DMSCs) in vitro and the potential of these cells to improve SCI in rats in vivo. DMSCs were stably transfected with TLX-expressing plasmid (TLX/DMSCs). Cell proliferation was examined using the MTT assay, and neuronal differentiation was characterized by morphological observation combined with immunocytochemical/immunofluorescent staining. The in vivo functions of these cells were evaluated by transplantation into rats with SCI, followed by analysis of hindlimb locomotion and post-mortem histology. Compared to parental DMSCs, TLX/DMSCs showed enhanced proliferation and preferential differentiation into NF200-positive neurons in contrast to GFAP-positive astrocytes. When the undifferentiated cells were transplanted into rats with SCI injury, TLX/DMSCs led to significant improvement in locomotor recovery and healing of SCI, as evidenced by reduction in scar tissues and cavities, increase in continuous nerve fibers/axons and enrichment of NF200-positive neurons on the histological level. In conclusion, TLX promotes the proliferation and neuronal differentiation of DMSCs and thus, may serve as a promising therapy for SCI in the clinic.

Involvement of prophenoloxidases in the suppression of Plasmodium yoelii development by Anopheles dirus.

Anopheles dirus is refractory to a rodent malaria parasite, Plasmodium yoelii, and melanized oocysts are manifested in infected mosquitoes. Prophenoloxidase (PPO) is a zymogen whose active form mediates melanotic encapsulation of invading pathogens in mosquitoes. In this study, we cloned cDNA fragments of four An. dirus PPOs, that are orthologs of Anopheles gambiae PPO2, PPO4, PPO5 and PPO6. AdPPO4 expression in hemocytes was induced in response to P. yoelii infection. RNA interference using double stranded RNA of AdPPO4 led to depletion of its mRNA and other PPO transcripts. This depletion increased P. yoelii infection prevalence and oocyst intensity, and abolished the melanization of oocysts as well. Therefore, An. dirus PPOs may play a role in the refractoriness to P. yoelii.