Phosphoproteomic Analysis Reveals the Predominating Cellular Processes and the Involved Key Phosphoproteins Essential for the Proliferation of Toxoplasma gondii.

PURPOSE: To explore the essential roles of phosphorylation in mediating the proliferation of T. gondii in its cell lytic life. METHODS: We profiled the phosphoproteome data of T. gondii residing in HFF cells for 2 h and 6 h, representing the early- and late-stages of proliferation (ESP and LSP) within its first generation of division. RESULTS: We identified 70 phosphoproteins, among which 8 phosphoproteins were quantified with the phosphorylation level significantly regulated. While only two of the eight phosphoproteins, GRA7 and TGGT1_242070, were significantly down-regulated at the transcriptional level in the group of LSP vs. ESP. Moreover, GO terms correlated with host membrane component were significantly enriched in the category of cellular component, suggesting phosphoprotein played important roles in acquiring essential substance from host cell via manipulating host membrane. Further GO analysis in the categories of molecular function and biological process and pathway analysis revealed that the cellular processes of glucose and lipid metabolism were regulated by T. gondii phosphoproteins such as PMCAA1, LIPIN, Pyk1 and ALD. Additionally, several phosphoproteins were enriched at the central nodes in the protein-protein interaction network, which may have essential roles in T. gondii proliferation including GAP45, MLC1, fructose-1,6-bisphosphate aldolase, GRAs and so on. CONCLUSION: This study revealed the main cellular processes and key phosphoproteins crucial for the intracellular proliferation of T. gondii, which would provide clues to explore the roles of phosphorylation in regulating the development of tachyzoites and new insight into the mechanism of T. gondii development in vitro.

A uracil auxotroph Toxoplasma gondii exerting immunomodulation to inhibit breast cancer growth and metastasis.

BACKGROUND: Breast cancer is the most common cause of cancer-related death among women, and prognosis is especially poor for patients with triple-negative breast cancer (TNBC); therefore, there is an urgent need for new effective therapies. Recent studies have demonstrated that the uracil auxotroph Toxoplasma gondii vaccine displays anti-tumor effects. Here, we examined the immunotherapy effects of an attenuated uracil auxotroph strain of T. gondii against 4T1 murine breast cancer. METHODS: We constructed a uracil auxotroph T. gondii RH strain via orotidine 5'-monophosphate decarboxylase gene deletion (RH-Δompdc) with CRISPR/Cas9 technology. The strain's virulence in the T. gondii-infected mice was determined in vitro and in vivo by parasite replication assay, plaque assay, parasite burden detection in mice peritoneal fluids and survival analysis. The immunomodulation ability of the strain was evaluated by cytokine detection. Its anti-tumor effect was evaluated after its in situ inoculation into 4T1 tumors in a mouse model; the tumor volume was measured, and the 4T1 lung metastasis was detected by hematoxylin and eosin and Ki67 antibody staining, and the cytokine levels were measured by an enzyme-linked immunosorbent assay. RESULTS: The RH-Δompdc strain proliferated normally when supplemented with uracil, but it was unable to propagate without the addition of uracil and in vivo, which suggested that it was avirulent to the hosts. This mutant showed vaccine characteristics that could induce intense immune responses both in vitro and in vivo by significantly boosting the expression of inflammatory cytokines. Inoculation of RH-Δompdc in situ into the 4T1 tumor inhibited tumor growth, reduced lung metastasis, promoted the survival of the tumor-bearing mice and increased the secretion of Th1 cytokines, including interleukin-12 (IL-12) and interferon-γ (INF-δ), in both the serum and tumor microenvironment (TME). CONCLUSION: Inoculation of the uracil auxotroph RH-Δompdc directly into the 4T1 tumor stimulated anti-infection and anti-tumor immunity in mice, and resulted in inhibition of tumor growth and metastasis, promotion of the survival of the tumor-bearing mice and increased secretion of IL-12 and IFN-γ in both the serum and TME. Our findings suggest that the immunomodulation caused by RH-Δompdc could be a potential anti-tumor strategy.

Toxoplasma gondii SAG1 targeting host cell S100A6 for parasite invasion and host immunity.

Toxoplasma gondii surface antigen 1 (TgSAG1) is a surface protein of tachyzoites, which plays a crucial role in toxoplasma gondii infection and host cell immune regulation. However, how TgSAG1 regulates these processes remains elucidated. We utilized the biotin ligase -TurboID fusion with TgSAG1 to identify the host proteins interacting with TgSAG1, and identified that S100A6 was co-localized with TgSAG1 when T. gondii attached to the host cell. S100A6, either knocking down or blocking its functional epitopes resulted in inhibited parasites invasion. Meanwhile, S100A6 overexpression in host cells promoted T. gondii infection. We further verified that TgSAG1 could inhibit the interaction of host cell vimentin with S100A6 for cytoskeleton organization during T. gondii invasion. As an immunogen, TgSAG1 could promote the secretion of tumor necrosis factor alpha (TNF-α) through S100A6-Vimentin/PKCθ-NF-κB signaling pathway. In summary, our findings revealed a mechanism for how TgSAG1 functioned in parasitic invasion and host immune regulation.

iTRAQ-Based Phosphoproteomic Analysis of Toxoplasma gondii Tachyzoites Provides Insight Into the Role of Phosphorylation for its Invasion and Egress.

The invasion and egress are two key steps in lytic cycle vital to the propagation of Toxoplasma gondii infection, and phosphorylation is believed to play important roles in these processes. However, the phosphoproteome of T. gondii at these two stages has not been characterized. In this study, we profiled the phosphoproteome of tachyzoites at the stages of "just invading" (JI) and "prior to egress" (PE) based on iTRAQ quantitative analysis, in which a total of 46 phosphopeptides, 42 phosphorylation sites, and 38 phosphoproteins were detected. In the comparison of PE vs. JI, 10 phosphoproteins were detected with their phosphorylation level significantly changed, and four of them were demonstrated to be significantly down-regulated at the transcriptional level. Bioinformatic analysis of these identified phosphoproteins suggested that phosphorylation-mediated modulation of protein function was employed to regulate the pathway of toxoplasmosis and metabolism and cellular processes correlated with tachyzoite's binding, location, and metabolism, and thus play vital roles in the parasite lytic cycle. Moreover, cytoskeletal network (CN)-associated Inner Membrane Complex (IMC1, IMC4, IMC6 and IMC12), Intravascular Network (IVN)-related GRAs (GRA2, GRA3, GRA7 and GRA12), and Parasitophorous Vacuole Membrane (PVM)-localized ROP5 were shown to be enriched at the central nodes in the protein interaction network generated by bioinformatic analysis, in which the phosphorylation level of IMC4, GRA2, GRA3, and GRA12 were found to be significantly regulated. This study revealed the main cellular processes and key phosphoproteins crucial for the invasion and egress of T. gondii, which will provide new insights into the developmental biology of T. gondii in vitro and contribute to the understanding of pathogen-host interaction from the parasite perspective.

Strain-specific disruption of interferon-stimulated N-myc and STAT interactor (NMI) function by Toxoplasma gondii type I ROP18 in human cells.

Toxoplasma gondii rhoptry protein TgROP18 is a polymorphic virulence effector that targets immunity-related GTPases (IRGs) in rodents. Given that IRGs are uniquely diversified in rodents and not in other T. gondii intermediate hosts, the role of TgROP18 in manipulating non-rodent cells is unclear. Here we show that in human cells TgROP18I interacts with the interferon-gamma-inducible protein N-myc and STAT interactor (NMI) and that this is a property that is unique to the type I TgROP18 allele. Specifically, when expressed ectopically in mammalian cells only TgROP18I co-immunoprecipitates with NMI in IFN-γ-treated cells, while TgROP18II does not. In parasites expressing TgROP18I or TgROP18II, NMI only co-immunoprecipitates with TgROP18I and this is associated with allele-specific immunolocalization of NMI on the parasitophorous vacuolar membrane (PVM). We also found that TgROP18I reduces NMI association with IFN-γ-activated sequences (GAS) in the IRF1 gene promoter. Finally, we determined that polymorphisms in the C-terminal kinase domain of TgROP18I are required for allele-specific effects on NMI. Together, these data further define new host pathway targeted by TgROP18I and provide the first function driven by allelic differences in the highly polymorphic ROP18 locus.

Current epidemic situation of human toxocariasis in China.

Toxocariasis is a worldwide-distributed helminthic zoonosis, which mainly results from ascarid nematodes Toxocara canis and Toxocara cati. Humans become infected by accidental ingestion of infective eggs, raw or undercooked meat containing larvae. Keeping and contacting cats and dogs, and bad hygiene situations or habits are the main risk factors for Toxocara infection in China. The seroprevalence of Toxocara spp. is reported from 12.14% to 44.83%, and the overall seroprevalence in children was 12.14% in 1993 and elevated to 19.3% in 2015. Among the 103 cases reported in China during 1983-2019, ocular larva migrans (OLM), visceral larva migrans (VLM), and neural larva migrans (NLM) occupied 92.23%, 6.80%, and 0.97% of cases, respectively. The diagnosis of toxocariasis is mainly based on the history of exposure to infective eggs or larvae, clinical manifestations, laboratory examinations, and imaging studies. As most individuals who are infected with larval Toxocara, are unaware of their infections, patients with mild signs as described under covert toxocariasis (CT) can recover spontaneously, and treatment may not be necessary. Albendazole is the preferred treatment for patients with VLM; steroids, such as prednisolone combined with albendazole, are frequently used in treating patients with OLM, and surgery serves as an alternative treatment; thiabendazole is effective in treating patients with NLM. The true number of cases and prevalence of toxocariasis in China seems to be underestimated and neglected because of the lack of population-based epidemiological studies and insufficient clinical awareness of this disease, which are aspects that need to be improved by the Chinese government.

Characteristics of and Public Health Responses to the Coronavirus Disease 2019 Outbreak in China.

In December 2019, cases of unidentified pneumonia with a history of exposure in the Huanan Seafood Market were reported in Wuhan, Hubei Province. A novel coronavirus, SARS-CoV-2, was identified to be accountable for this disease. Human-to-human transmission is confirmed, and this disease (named COVID-19 by World Health Organization (WHO)) spread rapidly around the country and the world. As of 18 February 2020, the number of confirmed cases had reached 75,199 with 2009 fatalities. The COVID-19 resulted in a much lower case-fatality rate (about 2.67%) among the confirmed cases, compared with Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Among the symptom composition of the 45 fatality cases collected from the released official reports, the top four are fever, cough, short of breath, and chest tightness/pain. The major comorbidities of the fatality cases include hypertension, diabetes, coronary heart disease, cerebral infarction, and chronic bronchitis. The source of the virus and the pathogenesis of this disease are still unconfirmed. No specific therapeutic drug has been found. The Chinese Government has initiated a level-1 public health response to prevent the spread of the disease. Meanwhile, it is also crucial to speed up the development of vaccines and drugs for treatment, which will enable us to defeat COVID-19 as soon as possible.

A Review on Dengue Vaccine Development.

Dengue virus (DENV) has become a global health threat with about half of the world's population at risk of infection. Although the disease caused by DENV is self-limiting in the first infection, the antibody-dependent enhancement (ADE) effect increases the mortality in the second infection with a heterotypic virus. Since there is no specific efficient medicine in treatment, it is urgent to develop vaccines to prevent infection and disease progression. Currently, only a live attenuated vaccine, chimeric yellow fever 17D-tetravalent dengue vaccine (CYD-TDV), has been licensed for clinical use in some countries, and many candidate vaccines are still under research and development. This review discusses the progress, strengths, and weaknesses of the five types of vaccines including live attenuated vaccine, inactivated virus vaccine, recombinant subunit vaccine, viral vectored vaccine, and DNA vaccine.

Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection.

Toxoplasma gondii is an intracellular parasite that infects humans and other warm-blooded animals. Exosomes are endocytic-derived vesicles released by cells, representing an important mode of intercellular communication. In exosomes, specific molecules of proteins, lipids, and mRNAs or miRNAs have been detected, some of which are capable of transferring biologically active molecules to recipient cells. Dendritic cells (DCs) are the only antigen-presenting cells (APCs) that activate the initial immune response. In this study, high-throughput sequencing was used to analyze the exosomal miRNA profile of DC2.4 cells infected with Toxoplasma gondii for 28 h, compared with those of uninfected DC2.4 cells. Differential exosomal miRNAs (DEmiRs) from these two cell groups were analyzed. Through high-throughput sequencing, 3434 DEmiRs were obtained, and 12 stably enriched DEmiRNAs were verified by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and selected for further analysis. The target genes of these 12 miRNAs were predicted with online analysis software and subjected to bioinformatics analyses including protein-protein interaction (PPI) network analysis, key driver analysis (KDA), gene ontology (GO) enrichment, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. These DEmiRs were found to be associated with a variety of biological processes and signaling pathways involved in host ubiquitin system, innate immunity, biosynthesis, and transferase activity and could be potential biomarkers for T. gondii infection.

Application of the Scorpion Neurotoxin AaIT against Insect Pests.

Androctonus australis Hector insect toxin (AaIT), an insect-selective toxin, was identified in the venom of the scorpion Androctonus australis. The exclusive and specific target of the toxin is the voltage-gated sodium channels of the insect, resulting in fast excitatory paralysis and even death. Because of its strict toxic selectivity and high bioactivity, AaIT has been widely used in experiments exploring pest bio-control. Recombinant expression of AaIT in a baculovirus or a fungus can increase their virulence to insect pests and diseases vectors. Likewise, transgenic plants expressing AaIT have notable anti-insect activity. AaIT is an efficient toxin and has great potential to be used in the development of commercial insecticides.

Expression of Bacillus thuringiensis toxin Cyt2Ba in the entomopathogenic fungus Beauveria bassiana increases its virulence towards Aedes mosquitoes.

BACKGROUND: The entomopathogenic fungus Beauveria bassiana has been widely used to kill mosquito larvae and adults in the laboratory and field. However, its slow action of killing has hampered its widespread application. In our study, the B. bassiana fungus was genetically modified to express the Bacillus thuringiensis (Bt) toxin Cyt2Ba to improve its efficacy in killing mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: The efficacy of the wild type (WT) of B. bassiana and a transgenic strain expressing Cyt2Ba toxin (Bb-Cyt2Ba) was evaluated against larval and adult Aedes mosquitoes (Aedes aegypti and Aedes albopictus) using insect bioassays. The Bb-Cyt2Ba displayed increased virulence against larval and adult Aedes mosquitoes compared with the WT: for Ae. aegypti adults, the median lethal time (LT50) was decreased by 33% at the concentration of 1× 108 conidia/ml, 19% at 1× 107 conidia/ml and 47% at 1× 106 conidia/ml. The LT50 for Ae. albopictus adults was reduced by 20%, 23% and 29% at the same concentrations, respectively. The LT50 for Ae. aegypti larvae was decreased by 42% at 1× 107 conidia/ml and 25% at 1× 106 conidia/ml, and that for Ae. albopictus larvae was reduced by 33% and 31% at the same concentrations, respectively. In addition, infection with Bb-Cyt2Ba resulted in a dramatic reduction in the fecundity of Aedes mosquitoes. CONCLUSIONS/SIGNIFICANCE: In conclusion, our study demonstrated that the virulence of B. bassiana against mosquitoes can be significantly improved by introducing the Bt toxin gene Cyt2Ba into the genome to express the exogenous toxin in the fungus. The transgenic strain Bb-Cyt2Ba significantly reduced the survival and fecundity of Ae. aegypti and Ae. albopictus compared with the WT strain, which suggested that this recombinant B. bassiana has great potential for use in mosquito control.

Toxoplasma gondii ROP18 inhibits human glioblastoma cell apoptosis through a mitochondrial pathway by targeting host cell P2X1.

BACKGROUND: Apoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed. METHODS: Apoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting. RESULTS: The apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation. CONCLUSIONS: Toxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

iTRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress.

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.

Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection.

Toxoplasma gondii are obligate intracellular protoza, and due to their small genome and limited encoded proteins, they have to exploit host factors for entry, replication, and dissemination. Such host factors can be defined as host dependency factors (HDFs). Though HDFs are inessential for cell viability, they are critical for pathogen infection, and potential ideal targets for therapeutic intervention. However, information about these HDFs required by T. gondii infection is highly deficient. In this study, the genes of human foreskin fibroblast (HFF) cells were comprehensively edited using the lentiviral CRISPR-Cas9-sgRNA library, and then the lentivirus-treated cells were infected with T. gondii at multiplication of infection 1 (MOI = 1) for 10 days to identify HDFs essential for T. gondii infection. The survival cells were harvested and sent for sgRNA sequencing. The sgRNA sequence matched genes or miRNAs were potential HDFs. Some cells in the lentivirus-treated group could survive longer than those in the untreated control group after T. gondii infection. From a pool of 19,050 human genes and 1,864 human pri-miRNAs, 1,193 potential HDFs were identified, including 1,183 genes and 10 pri-miRNAs (corresponding with 17 mature miRNAs). Among them, seven genes and five mature miRNAs were validated with siRNAs, miRNA inhibitors, and mimics, respectively. Bioinformatics analysis revealed that, among the 1,183 genes, 53 potential HDFs were associated with regulation of host actin cytoskeleton and 23 potential HDFs coded immune negative regulators. This result indicated that actin dynamics were indispensable for T. gondii infection, and some host immune negative regulators may be involved in disarming host defenses. Our findings contribute to the current limited knowledge about host factors required by T. gondii infection and provide us with new targets for medication therapy and vaccine exploitation.

Diagnostic Function of 3D Optical Coherence Tomography Images in Diagnosis of Vogt-Koyanagi-Harada Disease at Acute Uveitis Stage.

BACKGROUND This study analyzed the macular 3D-OCT images of Vogt-Koyanagi-Harada disease (VKH) in uveitis, explored the characteristics of 3D-OCT images of the macular region of VKH, and assessed which characteristics contribute most to VKH diagnosis. MATERIAL AND METHODS The 3D-OCT examination of 25 cases of VKH was performed on the macular area, and the image characteristics were analyzed. RESULTS Our study included a total of 50 eyes from 25 cases of VKH patients, 10 males and 15 females, aged 17 to 64 years, mean (39.44±11.60) years old. According to OCT B-scan images, 49 (98%) eyes had ERD, 49 (98%) eyes had nerve retinal edema, 36 (72%) eyes had endometrium-like structure (including cysts), 5 (10%) eyes had RPE folds, 35 (70%) eyes had changes in the internal septum, 49 (98%) eyes had RPE monolayer structure outside the ERD region. In ILM-RPE thickness, 49 (98%) eyes had retinal irregular thickening and 31 (62%) eyes had radial stripe changes. In ILM contour figure, 50 eyes (100%) showed exceptional uplift, 5 (10%) eyes had small focal uplift for PED on the RPE surface, and 48 (96%) eyes had wavy ups and downs. CONCLUSIONS In OCT B-scan imaging, the ERT, retinal edema of the retina, and the RPE monolayer structure outside the range are most likely to occur in VKH. The ILM-RPE thickness chart in 3D reconstruction showed irregular thickening of the retina. The ILM contour graph showed abnormal uplift, and RPE surface wavy ups and downs in VKH most likely to occur.

Genome-Wide Bimolecular Fluorescence Complementation-Based Proteomic Analysis of Toxoplasma gondii ROP18's Human Interactome Shows Its Key Role in Regulation of Cell Immunity and Apoptosis.

Toxoplasma gondii rhoptry protein ROP18 (TgROP18) is a key virulence factor secreted into the host cell during invasion, where it modulates the host cell response by interacting with its host targets. However, only a few TgROP18 targets have been identified. In this study, we applied a high-throughput protein-protein interaction (PPI) screening in human cells using bimolecular fluorescence complementation (BiFC) to identify the targets of Type I strain ROP18 (ROP18I) and Type II strain ROP18 (ROP18II). From a pool of more than 18,000 human proteins, 492 and 141 proteins were identified as the targets of ROP18I and ROP18II, respectively. Gene ontology, search tool for the retrieval of interacting genes/proteins PPI network, and Ingenuity pathway analyses revealed that the majority of these proteins were associated with immune response and apoptosis. This indicates a key role of TgROP18 in manipulating host's immunity and cell apoptosis, which might contribute to the immune escape and successful parasitism of the parasite. Among the proteins identified, the immunity-related proteins N-myc and STAT interactor, IL20RB, IL21, ubiquitin C, and vimentin and the apoptosis-related protein P2RX1 were further verified as ROP18I targets by sensitized emission-fluorescence resonance energy transfer (SE-FRET) and co-immunoprecipitation. Our study substantially contributes to the current limited knowledge on human targets of TgROP18 and provides a novel tool to investigate the function of parasite effectors in human cells.

Roles of Interferons in Pregnant Women with Dengue Infection: Protective or Dangerous Factors.

Dengue infection is a serious public health problem in tropical and subtropical areas. With the recent outbreaks of Zika disease and its reported correlation with microcephaly, the large number of pregnancies with dengue infection has become a serious concern. This review describes the epidemiological characteristics of pregnancy with dengue and the initial immune response to dengue infection, especially in IFNs production in this group of patients. Dengue is much more prevalent in pregnant women compared with other populations. The severity of dengue is correlated with the level of IFNs, while the serum IFN level must be sufficiently high to maintain the pregnancy and to inhibit virus replication.

The Differential Expression and Possible Function of Long Noncoding RNAs in Liver Cells Infected by Dengue Virus.

The function of long noncoding RNAs (lncRNAs) in liver injury resulted by dengue virus (DENV) infection have not yet been explored. The differential expression profiles of lncRNAs (as well as mRNAs) in the L-02 liver cells infected by DENV1, DENV2, or uninfected were compared and analyzed after a high throughput RNA seq. The significantly up-regulated and down-regulated lncRNAs (or mRNAs) resulted by DENV infection were identified with a cutoff value at log2 (ratio) ≥ 1.5 and log2 (ratio) ≤ -1.5 (ratio = the reads of the lncRNAs or mRNAs from the infection groups divided by the reads from the control group). Several differentially expressed lncRNAs were verified with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Target gene analysis, pre-miRNA prediction, and the lncRNA-mRNA co-expression network construction were performed to predict the function of the differentially expressed lncRNAs. The differentially expressed lncRNAs were associated with biosynthesis, DNA/RNA related processes, inhibition of estrogen signaling pathway, sterol biosynthetic process, protein dimerization activity, vesicular fraction in DENV1 infection group; and with protein secretion, methyltransferase process, host cell cytoskeleton reorganization and the small GTPase Ras superfamily, inhibition of cell proliferation, induction of apoptosis in DENV2 infection. LncRNAs might be novel diagnostic markers and targets for further researches on dengue infection and liver injury resulted by dengue virus.

Spatiotemporal Analysis of the Malaria Epidemic in Mainland China, 2004-2014.

The purpose of this study is to characterize spatiotemporal heterogeneities in malaria distribution at a provincial level and investigate the association between malaria incidence and climate factors from 2004 to 2014 in China to inform current malaria control efforts. National malaria incidence peaked (4.6/100,000) in 2006 and decreased to a very low level (0.21/100,000) in 2014, and the proportion of imported cases increased from 16.2% in 2004 to 98.2% in 2014. Statistical analyses of global and local spatial autocorrelations and purely spatial scan statistics revealed that malaria was localized in Hainan, Anhui, and Yunnan during 2004-2009 and then gradually shifted and clustered in Yunnan after 2010. Purely temporal clusters shortened to less than 5 months during 2012-2014. The two most likely clusters detected using spatiotemporal analysis occurred in Anhui between July 2005 and November 2007 and Yunnan between January 2010 and June 2012. Correlation coefficients for the association between malaria incidence and climate factors sharply decreased after 2010, and there were zero-month lag effects for climate factors during 2010-2014. Overall, the spatiotemporal distribution of malaria in China changed from relatively scattered (2004-2009) to relatively clustered (2010-2014). As the proportion of imported cases increased, the effect of climate factors on malaria incidence has gradually become weaker since 2011. Therefore, new warning systems should be applied to monitor resurgence and outbreaks of malaria in mainland China, and quarantine at borders should be reinforced to control the increasingly trend of imported malaria cases.

[Larvicidal activity of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or B.t.i toxin Cyt2Ba against mosquito larvae and formulations for enhancing the effects].

OBJECTIVE: To assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects. METHODS: The AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested. RESULTS: AaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LC50 of 3.00×106 cells/mL and 1.25×107 cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002). CONCLUSION: The B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.