Anopheles gambiae laminin interacts with the P25 surface protein of Plasmodium berghei ookinetes

Laminin is a major constituent of the basal lamina surrounding the midgut of the malaria vectors that has been implicated in the development of the Plasmodium oocyst. In this report we describe the cloning of the Anopheles gambiae gene encoding the laminin gamma 1 polypeptide and follow its expression during mosquito development. To further investigate the putative role of laminin in the transmission of the malaria parasite we studied the potential binding of the P25 surface protein of Plasmodium berghei using a yeast two-hybrid system. Heterodimer formation was observed and does not require any additional protein factors since purified fusion proteins can also bind each other in vitro. Laminin gamma 1 also interacts with the paralogue of P25, namely P28, albeit more weakly, possibly explaining why the two parasite proteins can substitute for each other in deletion mutants. This represents the first direct evidence for molecular interactions between a surface protein of the Plasmodium parasite with an Anopheles protein; the strong interplay between laminin gamma 1 and P25 suggests that this pair of proteins may function as a receptor/ligand complex regulating parasite development in the mosquito vector.     

Cyclical transmission of Plasmodium berghei (Coccidiida: plasmodiidae) by Anopheles omorii (Diptera: Culicidae).

Anopheles omorii, a tree-hole breeding anopheline collected in Japan, transmitted a rodent malaria, Plasmodium berghei ANKA strain, in the laboratory. Mice with 0.2-15% parasitemia, 0.01-1.5% gametocytemia, and 0.001-0.5% exflagellations were used as infective hosts. Oocyst numbers ranged from 2 to 171 (mean 44) on the midgut 8-16 d after the blood meal. Several to hundreds of sporozoites were detected in the salivary glands 14 d after feeding. The mosquitoes were infective to mice from 13 to 40 d after feeding. An. omorii occurs naturally at temperatures less than 25 degrees C and therefore is a suitable laboratory vector for the cyclic transmission of P. berghei, a malaria parasite that completes sporogony at 18-21 degrees C.     

Bypassing the midgut results in development of Plasmodium berghei oocysts in a refractory strain of Anopheles gambiae (Diptera: Culicidae)

The L35 strain of Anopheles gambiae Giles was genetically selected for its ability to melanize and kill malaria parasites. A wide range of Plasmodium species are subject to this response when orally ingested, including the rodent malaria, P. berghei. However, when we directly injected P. berghei into the hemocoel, we found that parasites developed normally to the oocyst stage. This work suggests that the parasite melanization response depends on the interaction of the ookinetes and the midgut. This result is surprising because it contrasts with a genetically validated model system, where injection of CM-Sephadex beads directly into the hemocoel results in bead melanization.     

Transcriptome analysis of the ependymal barrier during murine neurocysticercosis

ABSTRACT: BACKGROUND: Central nervous system (CNS) barriers play a pivotal role in the protection and homeostasis of the CNS by enabling the exchange of metabolites while restricting the entry of xenobiotics, blood cells and blood-borne macromolecules. While the blood-brain barrier and blood-cerebrospinal fluid barrier (CSF) control the interface between the blood and CNS, the ependyma acts as a barrier between the CSF and parenchyma, and regulates hydrocephalic pressure and metabolic toxicity. Neurocysticercosis (NCC) is an infection of the CNS caused by the metacestode (larva) of Taenia solium and a major cause of acquired epilepsy worldwide. The common clinical manifestations of NCC are seizures, hydrocephalus and symptoms due to increased intracranial pressure. The majority of the associated pathogenesis is attributed to the immune response against the parasite. The properties of the CNS barriers, including the ependyma, are affected during infection, resulting in disrupted homeostasis and infiltration of leukocytes, which correlates with the pathology and disease symptoms of NCC patients. RESULTS: In order to characterize the role of the ependymal barrier in the immunopathogenesis of NCC, we isolated ependymal cells using laser capture microdissection from mice infected or mock-infected with the closely related parasite Mesocestoides corti, and analyzed the genes that were differentially expressed using microarray analysis. The expression of 382 genes was altered. Immune response-related genes were verified by real-time RT-PCR. Ingenuity Pathway Analysis (IPA) software was used to analyze the biological significance of the differentially expressed genes, and revealed that genes known to participate in innate immune responses, antigen presentation and leukocyte infiltration were affected along with the genes involved in carbohydrate, lipid and small molecule biochemistry. Further, MHC class II molecules and chemokines, including CCL12, were found to be upregulated at the protein level using immunofluorescence microcopy. This is important, because these molecules are members of the most significant pathways by IPA analyses. CONCLUSION: Thus, our study indicates that ependymal cells actively express immune mediators and likely contribute to the observed immunopathogenesis during infection. Of particular interest is the major upregulation of antigen presentation pathway-related genes and chemokines/cytokines. This could explain how the ependyma is a prominent source of leukocyte infiltration into ventricles through the disrupted ependymal lining by way of pial vessels present in the internal leptomeninges in murine NCC.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Transcriptome analysis of fowl adenovirus serotype 4 infection in chickens

Virus Genes - Fowl adenovirus serotype 4 (FAdV-4) is a causative agent of inclusion body hepatitis and hydropericardium–hepatitis syndrome. These diseases cause considerable economic losses...     

对约氏疟原虫不同易感性的大劣按蚊和斯氏按蚊基因组RAPD序列比较

目的分析对约氏疟原虫不易感的大劣按蚊和易感的斯氏按蚊基因组RAPD谱带并测序，探讨媒介按蚊基因型与疟原虫基因型间的相互关系。方法用已筛选的一条随机引物，随机扩增大劣按蚊和斯氏按蚊成蚊的基因组DNA，扩增产物经琼脂糖凝胶电泳后，对相同迁移率的DNA条带克隆、测序，并采用相关在线程序及软件进行序列比较分析。结果大劣按蚊和斯氏按蚊RAPD谱带具有明显的种间差异，但有4对相同迁移率的条带。序列分析显示4对DNA条带在序列长度和组成上呈现多态性，序列的GC含量和简单重复序列存在差异，序列相似性介于48％～52％之间。结论大劣按蚊和斯氏按蚊之间存在不同的遗传背景，其基因多态性可能与产生对约氏疟原虫的易感性不同有关，为进一步研究按蚊与疟原虫之间的相互作用奠定了基础。     

Transcriptome analysis of salt-stressed Deinococcus radiodurans and characterization of salt-sensitive mutants

Deinococcus radiodurans is a bacterium best known for its extreme resistance to high levels of ionizing radiation. Gene expression profiles of D. radiodurans exposed to 0.3 M NaCl revealed that at least 389 genes were induced and 415 were repressed by twofold or more. A general down-regulation of the central metabolic pathways and a strong decrease of nrd gene expression, which encodes proteins necessary for DNA synthesis, likely reflect the growth retardation induced by NaCl stress. The expression of rsbRSTX, which encodes sigma B (σ^B) activity regulators, was also reduced by NaCl stress even though D. radiodurans does not have σ^B. The mutation of rsbX (drB0027) decreased the tolerance of D. radiodurans to NaCl, suggesting the possible role of the Rsb module in NaCl response. On the other hand, NaCl stress activated genes associated with osmoprotectant accumulation: the pstSCAB operon, which encodes a high affinity phosphate transporter, and DRA0135 and DR1438, which are components of transporters of glycine betaine and trehalose. Survival analysis of mutant strains lacking DR0392 (membrane-binding protein) and DR1115 (S-layer protein), whose expressions were highly activated by NaCl, showed a reduction in NaCl tolerance. In addition, the Δdr0392 strain showed sensitivity to γ-irradiation compared to the wild type. These results suggest that DR0392 plays a role in the resistance of D. radiodurans to NaCl and γ-irradiation.     

Description and morphometric analysis of the eggs of Anopheles (Anopheles) vestitipennis (Diptera: Culcidae) from southern Mexico

Light and scanning electron microscopy were used to compare the eggs of Anopheles vestitipennis Dyar & Knab females collected from human and animal baits in 9 villages of southern Mexico. An. vestitipennis eggs are boat-shaped, with lateral floats extending the length of the egg. Both the deck and dorsal surface are covered with hexagonal and pentagonal chorionic cells that contain round tubercles in the cell field. Crowns that enclose 3-5 lobed tubercles are present at both egg poles. By light microscopy, the mean length/width ratio of eggs of females caught at human bait were statistically different from those of females caught in horse-baited animal traps. In a regression tree model that included 19 egg attributes, the same egg groups could be separated by their posterior crown length/width ratio and mean anterior cell deck form factor (an index of their roundness). These findings support of the possible existence of 2 An. vestitipennis subpopulations with different host preferences in southern Mexico.     

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry

Anopheles gambiae is a major mosquito vector responsible for malaria transmission, whose genome sequence was reported in 2002. Genome annotation is a continuing effort, and many of the approximately 13,000 genes listed in VectorBase for Anopheles gambiae are predictions that have still not been validated by any other method. To identify protein-coding genes of An. gambiae based on its genomic sequence, we carried out a deep proteomic analysis using high-resolution Fourier transform mass spectrometry for both precursor and fragment ions. Based on peptide evidence, we were able to support or correct more than 6000 gene annotations including 80 novel gene structures and about 500 translational start sites. An additional validation by RT-PCR and cDNA sequencing was successfully performed for 105 selected genes. Our proteogenomic analysis led to the identification of 2682 genome search-specific peptides. Numerous cases of encoded proteins were documented in regions annotated as intergenic, introns, or untranslated regions. Using a database created to contain potential splice sites, we also identified 35 novel splice junctions. This is a first report to annotate the An. gambiae genome using high-accuracy mass spectrometry data as a complementary technology for genome annotation.     

Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts

Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner and that there were genes whose expression was changed independently of the enzyme treatment time, amount and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.