In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

The effect of irradiance on long-term skeletal growth and net photosynthesis in Galaxea fascicularis under four light conditions

The relation between irradiance, skeletal growth and net photosynthesis was studied for the scleractinian coral Galaxea fascicularis to provide experimental evidence for mediation of light-enhanced calcification through photosynthesis. The hypothesis was tested that skeletal growth and photosynthesis are linearly correlated.A long-term experiment was performed in a closed-circuit aquarium system, in which four series of nine nubbins (single polyp clones of a coral colony) of Galaxea fascicularis were exposed to four light treatments (10L:14D): 144 W T8 fluorescent lighting providing an irradiance of 68 µE/m²/s and 70, 250 and 400 W Metal Halide lighting providing an irradiance of 38 µE/m²/s, 166 µE/m²/s and 410 µE/m²/s, respectively. Growth of these nubbins was measured as buoyant weight at different time intervals in a 294 day experiment. A light-saturation curve for photosynthesis was measured in a respirometric flow cell using a 54 week Galaxea fascicularis colony grown at 60 µE/m²/s.No saturation of net photosynthesis of Galaxea fascicularis was found at the irradiances tested. The specific growth rate (µ, in day^- 1) of the coral nubbins increased with irradiance. Whereas irradiance varied 11-fold (38 to 410 µE/m²/s), buoyant weight (increase after 294 days) increased 5.7 times (2243 to 12374 mg), specific growth rate (1-294 days) increased 1.6 times (0.0103 to 0.0161 day^- 1), while net photosynthetic rate increased 8.9 times (0.009 µmol O₂/min/cm² to 0.077 µmol O₂/min/cm²). The increase of specific growth rate with irradiance was less than expected based on the increase in net photosynthetic rate with irradiance. This discrepancy between potential energy produced in photosynthesis and energy used for skeletal growth indicates that skeletal growth is not limited by photosynthetic potential at high irradiance levels.     

Phylogenetic relationships of Malaysia’s long-tailed macaques,Macaca fascicularis,based on cytochrome b sequences

Phylogenetic relationships among Malaysia’s long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo’s population was distinguished from Peninsula’s population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia’s M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.     

Relationship between the expression level of SLA-DQA and Escherichia coli F18 infection in piglets

The expression of SLA-DQA was assayed by Real-time PCR to analyze the differential expression between ETEC F18-resistant and -sensitive post-weaning piglets, and then to compare the expression levels of SLA-DQA in 11 different tissues from 8-, 18-, 30- and 35-day-old ETEC F18-resistant piglets, which aimed at discussing the role of SLA-DQA in resistance to ETEC F18. The results showed that SLA-DQA is broadly expressed in 11 tissues with the highest expression level in lymph nodes, and a relatively higher expression level in lung, spleen, jejunum, and duodenum. In tissues of lymph node, lung, spleen, jejunum, and duodenum, the mRNA expression of SLA-DQA in resistant individuals was significantly higher than that in sensitive ones (P < 0.05). In most tissues, the expression of SLA-DQA increased from 8 to 18 and 30 days (weaning day), and increased persistently to 35 days of post-weaning. Expression levels of SLA-DQA on 35 days in most tissues were significant higher than that on 8, 18 and 30 days (P < 0.05). The results demonstrated that the resistance to ETEC F18 in post-weaning piglets is related to up-regulation of mRNA expression of SLA-DQA to a certain extent. The analysis suggested that SLA-DQA may be not the direct immune factor that resisted the Escherichia coli F18, but perhaps enhanced humoral immunity and cell immunity to reduce the transmembrane signal transduction of ETEC F18 bacterial LPS and then led to the resistance to ETEC F18 in piglets.     

Downregulation of microRNA-214 and overexpression of FGFR-1 contribute to hepatocellular carcinoma metastasis

miR-214 is one of the most significantly downregulated microRNAs (miRNAs) in hepatocellular carcinoma (HCC). Fibroblast growth factor receptor 1 (FGFR-1) is a miR-214 target gene implicated in the progression of HCC. However, the roles of miR-214 and FGFR-1 in HCC are not fully understood. Here, we analyzed the expression of miR-214 and FGFR-1 in 65 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR and Western blot (WB), respectively. Our data indicated that miR-214 was downregulated and FGFR-1 was overexpressed in HCC compared to the paired non-neoplastic tissues. The low miR-214 expression was correlated with portal vein invasion (p = 0.016) and early recurrence (p = 0.045) in HCC patients. Moreover, the low miR-214 expression was correlated with high positive rate of FGFR-1 in HCC cases (p = 0.020). Our data further demonstrated that miR-214 overexpression in SK-HEP1 and HepG2 cells downregulated FGFR-1 expression and inhibited liver cancer cell invasion. The Luciferase assay results further demonstrated the targeted regulation of FGFR-1 by miR-214. In conclusion, our data indicate that the downregulation of miR-214 in HCC and the upregulation of its target gene FGFR-1 is associated with HCC progression. Therefore, miR-214 and FGFR-1 are potential prognostic markers and therapeutic targets in HCC.     

A cytosolic glutathione s-transferase,GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P < 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.     

Null genotypes of GSTM1 and GSTT1 contribute to increased risk of diabetes mellitus: A meta-analysis

Diabetes mellitus (DM) is a common disease which results from various causes including genetic and environmental factors. Glutathione S-Transferase M1 (GSTM1) and Glutathione S-Transferase T1 (GSTT1) genes are polymorphic in human and the null genotypes lead to the absence of enzyme function. Many studies assessed the associations between GSTM1/GSTT1 null genotypes and DM risk but reported conflicting results. In order to get a more precise estimate of the associations of GSTM1/GSTT1 null genotypes with DM risk, we performed this meta-analysis. Published literature from PubMed, Embase and China Biology Medicine (CBM) databases was searched for eligible studies. Pooled odds ratios (OR) and corresponding 95% confidence intervals (95%CI) were calculated using a fixed- or random-effects model. 11 publications (a total of 2577 cases and 4572 controls) were finally included into this meta-analysis. Meta-analyses indicated that null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 were all associated with increased risk of DM (GSTM1: OR _{random-effects} = 1.60, 95%CI 1.10–2.34, P_OR = 0.014; GSTT1: OR _{random-effects} = 1.47, 95%CI 1.12–1.92, P_OR = 0.005; GSTM1–GSTT1: OR _{fixed-effects} = 1.83, 95%CI 1.30–2.59, P_OR = 0.001). Subgroup by ethnicity suggested significant associations between null genotypes of GSTM1 and GSTT1 and DM risk among Asians (GSTM1: OR _{random-effects} = 1.77, 95%CI 1.24–2.53, P_OR = 0.002; GSTT1: OR _{random-effects} = 1.58, 95%CI 1.09–2.27, P_OR = 0.015). This meta-analysis suggests null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are all associated with increased risk of DM, and null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are potential biomarkers of DM.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.