microRNAs (miRNAs) are an extensive class of -22-nucleotide (nt) endogenous noncoding RNAs regulating life activities ofmetazoans through binding to 3'-untranslated regions (3'-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3'-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3'RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3'-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.
3' Untranslated Regions ; Animals ; Bombyx ; genetics ; Cell Line ; Cloning, Molecular ; Female ; Genetic Vectors ; Insect Proteins ; genetics ; Larva ; Metamorphosis, Biological ; MicroRNAs ; genetics ; Pupa

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.
Animals ; Bombyx/metabolism* ; Metamorphosis, Biological/genetics* ; Larva/metabolism* ; Gene Expression ; Insect Proteins/metabolism* ; Chitin

Protozoa of the genus Cryptosporidium are small coccidian parasite known to infect the mucosal epithelium of a variety of animals including human, causing fatal course in immunodeficient patients as well as self-limited illness in healthy individuals. Various life cycle stages including trophozoite, meront, merozoite, gametocyte and oocyst in infected mucosa are a diagnostic feature. Electron microscopy (EM) provides sufficient findings for genus and species identification of this parasitic organism. The authors presented scanning and transmission EM findings of Cryptosporidium parvum infection in two children: one with acute lymphoblastic leukemia and the other without any evidence of immune compromise.
Animals ; Child ; Cryptosporidium parvum ; Cryptosporidium* ; Epithelium ; Humans* ; Intestines* ; Life Cycle Stages ; Merozoites ; Microscopy, Electron ; Mucous Membrane ; Oocysts ; Parasites ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Trophozoites

This study investigated the development characteristics of Dermacentor everestianus under laboratory conditions. The time taken for D. everestianus to complete the whole life cycle was 110.2 days on average, and the average developmental durations of larvae and nymphs were 17.1 days and 29.5 days, respectively. The summation of the prefeeding, feeding, and preoviposition periods of females was 17.8 days, and the oviposition and egg incubation lasted for 18.1 days and 27.7 days, respectively. A highly positive correlation was observed between the weight of engorged female and the number of egg mass laid (r=0.947). The reproductive efficiency index and the reproductive fitness index were 7.1 and 6.1, respectively.
Dermacentor* ; Female ; Genetic Fitness ; Humans ; Larva ; Life Cycle Stages* ; Nymph ; Oviposition ; Ovum

The ascidian Ciona intestinalis is a model organism of developmental and evolutionary biology and may provide crucial clues concerning two fundamental matters, namely, how chordates originated from the putative deuterostome ancestor and how advanced chordates originated from the simplest chordates. In this paper, a whole-life-span culture of C. intestinalis was conducted. Fed with the diet combination of dry Spirulina, egg yolk, Dicrateria sp., edible yeast and weaning diet for shrimp, C. intestinalis grew up to average 59 mm and matured after 60 d cultivation. This culture process could be repeated using the artificially cultured mature ascidians as material. When the fertilized eggs were maintained under 10, 15, 20, 25 degrees C, they hatched within 30 h, 22 h, 16 h and 12 h 50 min respectively experiencing cleavage, blastulation, gastrulation, neurulation, tailbud stage and tadpole stage. The tadpole larvae were characterized as typical but simplified chordates because of their dorsal nerve cord, notochord and primordial brain. After 8 - 24 h freely swimming, the tadpole larvae settled on the substrates and metamorphosized within 1- 2 d into filter feeding sessile juvenile ascidians. In addition, unfertilized eggs were successfully dechorionated in filtered seawater containing 1% Tripsin, 0.25% EDTA at pH of 10.5 within 40 min. After fertilization, the dechorionated eggs developed well and hatched at normal hatching rate. In conclusion, this paper presented feasible methodology for rearing the tadpole larvae of C. intestinalis into sexual maturity under controlled conditions and detailed observations on the embryogenesis of the laboratory cultured ascidians, which will facilitate developmental and genetic research using this model system.
Animals ; Ciona intestinalis ; growth & development ; Embryonic Development ; physiology ; Female ; Male ; Metamorphosis, Biological ; physiology ; Zygote ; growth & development

This article reviews Leptotrombidium deliense, including its discovery and nomenclature, morphological features and identification, life cycle, ecology, relationship with diseases, chromosomes and artificial cultivation. The first record of L. deliense was early in 1922 by Walch. Under the genus Leptotrombidium, there are many sibling species similar to L. deliense, which makes it difficult to differentiate L. deliense from another sibling chigger mites, for example, L. rubellum. The life cycle of the mite (L. deliense) includes 7 stages: egg, deutovum (or prelarva), larva, nymphochrysalis, nymph, imagochrysalis and adult. The mite has a wide geographical distribution with low host specificity, and it often appears in different regions and habitats and on many species of hosts. As a vector species of chigger mite, L. deliense is of great importance in transmitting scrub typhus (tsutsugamushi disease) in many parts of the world, especially in tropical regions of Southeast Asia. The seasonal fluctuation of the mite population varies in different geographical regions. The mite has been successfully cultured in the laboratory, facilitating research on its chromosomes, biochemistry and molecular biology.
Adult ; Asia, Southeastern ; Biochemistry ; Ecology ; Ecosystem ; Host Specificity ; Humans ; Larva ; Life Cycle Stages ; Mites ; Molecular Biology ; Nymph ; Ovum ; Scrub Typhus ; Seasons ; Siblings ; Trombiculidae

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with microRNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3'UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3'UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.
3' Untranslated Regions ; Animals ; Bombyx ; Cloning, Molecular ; DNA, Complementary ; Down-Regulation ; Drosophila melanogaster ; Gene Expression Regulation ; Insect Proteins ; genetics ; metabolism ; Metamorphosis, Biological ; MicroRNAs ; metabolism ; Transcription Factors ; genetics ; metabolism

To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.
Alkaline Phosphatase ; genetics ; immunology ; Animals ; Antibodies ; immunology ; Escherichia coli ; genetics ; metabolism ; Flounder ; physiology ; Metamorphosis, Biological ; physiology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Thyroid Hormone Receptors alpha ; biosynthesis ; genetics ; immunology

This study was performed to investigate the distribution of ticks and the rate of infection with severe fever with thrombocytopenia syndrome (SFTS) virus in ticks collected at Mt. Gwanak and the Seoul National University campus, Korea. Ticks (n = 273) were collected from May to October and included 76 Haemaphysalis longicornis (4 adult females, 72 nymphs), 49 Haemaphysalis flava (9 adult females, 3 adult males, 37 nymphs), and 148 Haemaphysalis spp. larvae. SFTS virus detection was performed by using one-step RT PCR and nested PCR. The SFTS virus was detected in 7 samples (1 Haemaphysalis longicornis nymph, 3 Haemaphysalis flava nymphs, and 3 Haemaphysalis spp. larva). The overall minimum field infection rate was 2.6%, whereas the minimum field infection rates of adult, nymphal, and larval ticks were 0%, 3.2%, and 2.0%, respectively. For a more accurate indication of the prevalence of SFTS virus in Korea, further in-depth investigations of tick species and SFTS virus occurrence over a larger area and longer period are needed.
Adult ; Female ; Fever* ; Humans ; Korea* ; Larva ; Male ; Nymph ; Polymerase Chain Reaction ; Prevalence* ; Seoul ; Thrombocytopenia* ; Ticks*

OBJECTIVETo investigate the changes in plasma matrix metalloproteinases-2 and -9 (MMP2 and MMP9, respectively) levels in patients with different types of coronary heart diseases (CHD), and assess the value of MMP2/MMP9 detection in predicting acute coronary syndrome (ACS).

METHODSAccording to the findings by coronary angiography and the clinical manifestations, 118 patients were divided in ACS group including 30 patients with unstable angina pectoris (UAP) and 19 with acute myocardial infarction (AMI) and non-ACS group including 23 patients with stable angina pectoris (SAP) and 21 with chronic total occlusion (CTO) of the coronary artery. Twenty-five individuals with normal coronary artery (NCA) served as the control group. Plasma levels of MMP9 and MMP2 were determined in these subjects using enzyme-linked immunosorbent assay (ELISA).

RESULTSBoth the ACS and non-ACS groups showed significantly higher MMP9 and MMP2 levels than the NCA group (P<0.05), and MMP2 and MMP9 levels were significantly higher in ACS group than in non-ACS group (P<0.05). Compared with the NCA group, the UAP, AMI and CTO subgroups showed obvious increases in plasma MMP2 and MMP9 levels (P<0.01). Significantly increased MMP9, but not MMP2 level was noted in AMI subgroup in comparison with SAP (P<0.01) and UAP subgroups (P<0.05); both MMP2 and MMP9 levels were elevated in CTO subgroup in comparison with those in SAP (P<0.001), UAP (P<0.01), and AMI subgroups (P<0.05).

CONCLUSIONIncreased MMP2 and MMP9 levels in patients with CHD suggest the instability of the atherosclerotic plaque in correlation to the severity of ACS, and may serve as good indicators for the prediction of ACS and diagnosis of CTO of the coronary artery.

Acute Coronary Syndrome ; blood ; diagnostic imaging ; Aged ; Angina, Unstable ; blood ; diagnostic imaging ; Chronic Disease ; Coronary Angiography ; Coronary Occlusion ; blood ; diagnostic imaging ; Female ; Humans ; Male ; Matrix Metalloproteinase 2 ; blood ; Matrix Metalloproteinase 9 ; blood ; Metamorphosis, Biological ; Middle Aged ; Myocardial Infarction ; blood ; diagnostic imaging