Serotype and PCR-fingerprints of Clinical and Environmental isolates of Cryptococcus neoformans in Chiang Mai,Thailand

From May 1999 to April 2000, serotypes of clinical and environmental isolates of Cryptococcus neoformans were studied in Chiang Mai province, northern Thailand. Three hundred and eighty-five environmental samples, of which 100 were dove droppings, 55 pigeon droppings and 230 eucalyptus flower, were collected from 7 Amphoes in Chiang Mai. C. neoformans was isolated from 45 of 100 (45.0%) dove dropping samples, 9 of 55 (16.4%) pigeon dropping samples and 2 of 230 (0.9%) eucalyptus flower samples. Serotypes of 56 environmental isolates and 75 clinical isolates of C. neoformans,obtained during the same period, were determined by the slide agglutination test. Fifty-six environmental and 74 clinical isolates belonged to C. neoformans serotype A (C. neoformans var. grubii), and only one clinical isolate belonged to C. neoformans serotype AD. The isolation of C. neoformans var. grubii from eucalyptus flower samples suggests contamination of avian droppings. PCR-fingerprinting, using (GACA)4 as a primer, discriminated 131 clinical and environmental isolates into 2 groups (group I and II). Seventy-five clinical and 54 environmental isolates were of group I, which had two major specific bands of approximately 1,250 and 960 base pairs. Two environmental isolates, one from pigeon excreta and the other from a eucalyptus flower sample were of group II, which had two major specific bands of approximately 1,180 and 500 base pairs.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

Genome-Wide Distribution of Transposed Dissociation Elements in Maize

The maize (Zea mays) transposable element Dissociation (Ds) was mobilized for large-scale genome mutagenesis and to study its endogenous biology. Starting from a single donor locus on chromosome 10, over 1500 elements were distributed throughout the genome and positioned on the maize physical map. Genetic strategies to enrich for both local and unlinked insertions were used to distribute Ds insertions. Global, regional, and local insertion site trends were examined. We show that Ds transposed to both linked and unlinked sites and displayed a nonuniform distribution on the genetic map around the donor r1-sc:m3 locus. Comparison of Ds and Mutator insertions reveals distinct target preferences, which provide functional complementarity of the two elements for gene tagging in maize. In particular, Ds displays a stronger preference for insertions within exons and introns, whereas Mutator insertions are more enriched in promoters and 5′-untranslated regions. Ds has no strong target site consensus sequence, but we identified properties of the DNA molecule inherent to its local structure that may influence Ds target site selection. We discuss the utility of Ds for forward and reverse genetics in maize and provide evidence that genes within a 2- to 3-centimorgan region flanking Ds insertions will serve as optimal targets for regional mutagenesis.     

Duration and dose-dependency of female sexual receptivity responses to seminal fluid proteins in Aedes albopictus and Ae. aegypti mosquitoes

Male mosquitoes transfer seminal fluid proteins (hereafter ‘SFPs’) during mating. These proteins can have profound effects on female behavior in the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. SFPs are thought to be responsible for female refractoriness to mating in both species. However, only limited information is available about the duration of induced refractoriness or the quantity of SFPs required to be effective in Ae. albopictus. Here, we tested the duration of the effect of SFPs on female refractory behavior for both Aedes species. Additionally, we determined the lowest SFP dose required to induce female refractory behavior in Ae. aegypti. Virgin females were injected intra-thoracically with doses ranging from 0.25 to 0.008 equivalents of one male’s SFP amount. Our results demonstrate high sensitivity of female Ae. aegypti and Ae. albopictus to SFPs of their own species, with the majority of females becoming refractory at doses ? 0.031 male-equivalents after injection into the hemocoel. This effect was long-lasting in both species; none of the injected females were inseminated when presented with males of their own species 30 to 34 days post-injection, whereas most saline-injected control females mated at this time point. These results will aid future work to characterize individual SFPs involved in post-mating refractoriness in these two species. Moreover, they show that as is the situation in the mosquito Anopheles gambiae, and unlike Drosophila melanogaster, sperm are not required for the maintenance of a sexual refractoriness response in Ae. aegypti and Ae. albopictus.     

Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.     

In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds

The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Insulin-like growth factor-I attenuates the inhibitory effect of type I collagen through beta1 integrin receptor

Extracellular matrix and growth factors are the crucial factors that regulate healing and regenerating processes in human periodontal ligament cells. The purpose of this study was to examine the effects of type I collagen and insulin-like growth factor-I (IGF-I) on osteopontin (OPN) expression. The data showed that OPN expression was significantly decreased when cells were cultured on collagen-coated plates. Addition of IGF-I obviously induced OPN expression only in a collagen-coated condition, suggesting an attenuating effect of IGF-I on the decrease of OPN expression. Cells treated with a combination of inhibitory antibody to beta1 integrin and IGF-I showed the same level of OPN expression as those treated with either inhibitory antibody to beta1 integrin or IGF-I alone. These results indicate that IGF-I counteracts with the inhibitory signal from type I collagen through beta1 integrin receptor.