Farnesyl Phosphatase,a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.

Methods

A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.

Conclusions

AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.     

Using GFP video to track 3D movement and conditional gene expression in free-moving flies

Background

In vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.

Results

We report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.

Conclusions

FluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.     

Variability in the precursor proteins of collagen I and III in different stages of COPD

Background

Levels of precursor proteins of collagen I and III are increased in fibrotic pulmonary diseases. This study determined whether the expression of precursors of type I and III collagen proteins would be increased in small and large airways of COPD patients in various stages of the disease reflecting fibrogenesis.

Methods

The levels of precursor proteins of collagen I and III were studied by immunohistochemistry and quantified by image analysis in lung tissue of 16 non-smokers, 20 smokers with normal lung function, 20 smokers with stage I-II COPD and 8 ex-smokers with stage IV COPD.

Results

In large airways, the subepithelial layer which was positive for precursor proteins of collagen I and III was thicker in smokers and in stage I-II COPD compared to non-smokers. Large airways in stage IV COPD showed reduced expression of precursor protein of collagen I whereas precursor of collagen III was increased. The amount of precursor protein of collagen III was increased in small airways of smokers and stage I-II COPD but reduced in stage IV COPD.

Conclusions

Precursor proteins of collagen I and III revealed different expression profiles in large and small airways in various stages of COPD. Smoking enhanced expression of both precursors in large airways with a positive correlation with pack-years.     

Regional Variation in Brain White Matter Diffusion Index Changes following Chemoradiotherapy: A Prospective Study Using Tract-Based Spatial Statistics

Purpose

There is little known about how brain white matter structures differ in their response to radiation, which may have implications for radiation-induced neurocognitive impairment. We used diffusion tensor imaging (DTI) to examine regional variation in white matter changes following chemoradiotherapy.

Methods

Fourteen patients receiving two or three weeks of whole-brain radiation therapy (RT) ± chemotherapy underwent DTI pre-RT, at end-RT, and one month post-RT. Three diffusion indices were measured: fractional anisotropy (FA), radial diffusivity (RD), and axial diffusivity (AD). We determined significant individual voxel changes of diffusion indices using tract-based spatial statistics, and mean changes of the indices within fourteen white matter structures of interest.

Results

Voxels of significant FA decreases and RD increases were seen in all structures (p<0.05), with the largest changes (20–50%) in the fornix, cingula, and corpus callosum. There were highly significant between-structure differences in pre-RT to end-RT mean FA changes (p<0.001). The inferior cingula had a mean FA decrease from pre-RT to end-RT significantly greater than 11 of the 13 other structures (p<0.00385).

Conclusions

Brain white matter structures varied greatly in their response to chemoradiotherapy as measured by DTI changes. Changes in FA and RD related to white matter demyelination were prominent in the cingula and fornix, structures relevant to radiation-induced neurocognitive impairment. Future research should evaluate DTI as a predictive biomarker of brain chemoradiotherapy adverse effects.     

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application

Background

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples.

Methods

The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep.

Conclusions

The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively.

Significance

The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.     

Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

Background

Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C₅ hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects.

Results

We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg²⁺, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP).

Conclusions

AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis.     

Method for Obtaining Committed Adult Mesenchymal Precursors from Skin and Lung Tissue

Aims

The present study reports an easy and efficient method for obtaining adult mesenchymal precursors from different adult mouse tissues.

Materials and Methods

We describe the isolation and expansion of mesenchymal precursors from skin and lung by a non-enzymatic method. Skin and lung mesenchymal precursors isolated by a modified explant technique were characterized in vitro by defined morphology and by a specific gene expression profile and surface markers.

Results and Conclusions

Our results show that these precursors express stem cell and mesenchymal surface markers as well as epithelial markers. However, they are negative for markers of endothelium, cardiac and skeletal muscle or adipose tissue, indicating that they have initiated commitment to the tissues from which were isolated. These precursors can migrate without any stimulus and in response to stimuli as SDF1, MCP1 and TNFα and can be differentiated into epithelial lineages. Based on the properties of these precursors from adult tissues, we propose their use as tools for regenerative biomedicine.     

White matter and cognition in adults who were born preterm

Background and Purpose

Individuals born very preterm (before 33 weeks of gestation, VPT) are at risk of damage to developing white matter, which may affect later cognition and behaviour.

Methods

We used diffusion tensor MRI (DT-MRI) to assess white matter microstructure (fractional anisotropy; FA) in 80 VPT and 41 term-born individuals (mean age 19.1 years, range 17–22, and 18.5 years, range17–22 years, respectively). VPT individuals were part of a 1982–1984 birth cohort which had been followed up since birth; term individuals were recruited by local press advertisement. General intellectual function, executive function and memory were assessed.

Results

The VPT group had reduced FA in four clusters, and increased FA in four clusters relative to the Term group, involving several association tracts of both hemispheres. Clusters of increased FA were associated with more severe neonatal brain injury in the VPT group. Clusters of reduced FA were associated with lower birth weight and perinatal hypoxia, and with reduced adult cognitive performance in the VPT group only.

Conclusions

Alterations of white matter microstructure persist into adulthood in VPT individuals and are associated with cognitive function.     

A Flow Cytometry-Based Quantitative Drug Sensitivity Assay for All Plasmodium falciparum Gametocyte Stages

Background

Malaria elimination/eradication campaigns emphasize interruption of parasite transmission as a priority strategy. Screening for new drugs and vaccines against gametocytes is therefore urgently needed. However, current methods for sexual stage drug assays, usually performed by counting or via fluorescent markers are either laborious or restricted to a certain stage. Here we describe the use of a transgenic parasite line for assaying drug sensitivity in all gametocyte stages.

Methods

A transgenic parasite line expressing green fluorescence protein (GFP) under the control of the gametocyte-specific gene α-tubulin II promoter was generated. This parasite line expresses GFP in all gametocyte stages. Using this transgenic line, we developed a flow cytometry-based assay to determine drug sensitivity of all gametocyte stages, and tested the gametocytocidal activities of four antimalarial drugs.

Findings

This assay proved to be suitable for determining drug sensitivity of all sexual stages and can be automated. A Z’ factor of 0.79±0.02 indicated that this assay could be further optimized for high-throughput screening. The daily sensitivity of gametocytes to three antimalarial drugs (chloroquine, dihydroartemisinin and pyronaridine) showed a drastic decrease from stage III on, whereas it remained relatively steady for primaquine.

Conclusions

A drug assay was developed to use a single transgenic parasite line for determining drug susceptibility of all gametocyte stages. This assay may be further automated into a high-throughput platform for screening compound libraries against P. falciparum gametocytes.     

Surface-Based Analysis on Shape and Fractional Anisotropy of White Matter Tracts in Alzheimer's Disease

Background

White matter disruption has been suggested as one of anatomical features associated with Alzheimer''s disease (AD). Diffusion tensor imaging (DTI), which has been widely used in AD studies, obtains new insights into the white matter structure.

Methods

We introduced surface-based geometric models of the deep white matter tracts extracted from DTI, allowing the characterization of their shape variations relative to an atlas as well as fractional anisotropy (FA) variations on the atlas surface through large deformation diffeomorphic metric mapping (LDDMM). We applied it to assess local shapes and FA variations of twenty-three deep white matter tracts in 13 patients with AD and 19 healthy control subjects.

Results

Our results showed regionally-specific shape abnormalities and FA reduction in the cingulum tract and the sagittal stratum tract in AD, suggesting that disruption in the white matter tracts near the temporal lobe may represent the secondary consequence of the medial temporal lobe pathology in AD. Moreover, the regionally-specific patterns of FA and shape of the white matter tracts were shown to be of sufficient sensitivity to robustly differentiate patients with AD from healthy comparison controls when compared with the mean FA and volumes within the regions of the white matter tracts. Finally, greater FA or deformation abnormalities of the white matter tracts were associated with lower MMSE scores.

Conclusion

The regionally-specific shape and FA patterns could be potential imaging markers for differentiating AD from normal aging.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.     

Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Diffusion Tensor Magnetic Resonance Imaging of the Brain in APP Transgenic Mice: A Cohort Study

Introduction

Fast in-vivo high resolution diffusion tensor imaging (DTI) of the mouse brain has recently been shown to enable cohort studies by the combination of appropriate pulse sequences and cryogenically cooled resonators (CCR). The objective of this study was to apply this DTI approach at the group level to β-amyloid precursor protein (APP) transgenic mice.

Methods

Twelve mice (5 wild type, 7 APP transgenic tg2576) underwent DTI examination at 156²×250 µm³ spatial resolution with a CCR at ultrahigh field (11.7 T). Diffusion images were acquired along 30 gradient directions plus 5 references without diffusion encoding with a total acquisition time of 35 minutes. Fractional anisotropy (FA) maps were statistically compared by whole brain-based spatial statistics (WBSS) at the group level vs. wild type controls.

Results

FA-map comparison showed characteristic regional patterns of differences between the groups with localizations associated with Alzheimer’s disease in humans, such as the hippocampus, the entorhinal cortex, and the caudoputamen.

Conclusion

In this proof-of-principle study, regions associated with amyloid-β deposition could be identified by WBSS of FA maps in APP transgenic mice vs. wild type mice. Thus, DTI in the mouse brain acquired at 11.7 T by use of a CCR was demonstrated to be feasible for cohort studies.     

Peroxynitrite (ONOO?) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress

Background and Aims

Peroxisomes are subcellular compartments involved in multiple cellular metabolic pathways. Peroxynitrite (ONOO⁻) is a nitric oxide-derived molecule which is a nitrating species that causes nitration of proteins. This study used cell biology techniques to explore the potential presence of peroxynitrite in peroxisomes and evaluated its content under stress conditions (excess cadmium).

Methods

Peroxynitrite, nitric oxide and superoxide anion were studied using cell-permeable specific fluorescent probes by confocal laser scanning microscopy in Arabidopsis thaliana transgenic plants expressing cyan fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo.

Key Results

When no stress was applied, peroxynitrite was clearly localized in the peroxisomes of roots and stomatal guard cells. Under cadmium (150 μm) stress, the generation of peroxynitrite, nitric oxide and the superoxide anion (O₂^·–) increased and was localized in peroxisomes and the cytosol, participating in the generation of nitro-oxidative stress.

Conclusions

The results show that peroxisomes are an endogenous source of peroxynitrite, which is over-produced under cadmium stress, suggesting that the metabolism of reactive nitrogen species in peroxisomes could participate in the mechanism of the response to this heavy metal.     

Molecular Determinants of Juvenile Hormone Action as Revealed by 3D QSAR Analysis in Drosophila

Background

Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH). While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive.

Methodology/Principal Findings

To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen) at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 Å or longer than 13.5 Å, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity.

Conclusions/Significance

The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.     

Cellular Mechanism Underlying Formaldehyde-Stimulated Cl? Secretion in Rat Airway Epithelium

Background

Recent studies suggest that formaldehyde (FA) could be synthesized endogeneously and transient receptor potential (TRP) channel might be the sensor of FA. However, the physiological significance is still unclear.

Methodology/Principal Findings

The present study investigated the FA induced epithelial Cl^- secretion by activation of TRPV-1 channel located in the nerve ending fiber. Exogenously applied FA induced an increase of I _SC in intact rat trachea tissue but not in the primary cultured epithelial cells. Western blot and immunofluorescence analysis identified TRPV-1 expression in rat tracheal nerve ending. Capsazepine (CAZ), a TRPV-1 specific antagonist significantly blocked the I _SC induced by FA. The TRPV-1 agonist capsaicin (Cap) induced an increase of I _SC, which was similar to the I _SC induced by FA. L-703606, an NK-1 specific inhibitor and propranolol, an adrenalin β receptor inhibitor significantly abolished the I _SC induced by FA or Cap. In the ion substitute analysis, FA could not induce I _SC in the absence of extracelluar Cl^-. The I _SC induced by FA could be blocked by the non-specific Cl^- channel inhibitor DPC and the CFTR specific inhibitor CFTR_i-172, but not by the Ca²⁺-activated Cl^- channel inhibitor DIDS. Furthermore, both forskolin, an agonist of adenylate cyclase (AC) and MDL-12330A, an antagonist of AC could block FA-induced I _SC.

Conclusion

Our results suggest that FA-induced epithelial I _SC response is mediated by nerve, involving the activation of TRPV-1 and release of adrenalin as well as substance P.     

Diffusion Tensor and Volumetric Magnetic Resonance Measures as Biomarkers of Brain Damage in a Small Animal Model of HIV

Background

There are currently no widely accepted neuro-HIV small animal models. We wanted to validate the HIV-1 Transgenic rat (Tg) as an appropriate neuro-HIV model and then establish in vivo imaging biomarkers of neuropathology, within this model, using MR structural and diffusion tensor imaging (DTI).

Methods

Young and middle-aged Tg and control rats were imaged using MRI. A subset of middle-aged animals underwent longitudinal repeat imaging six months later. Total brain volume (TBV), ventricular volume (VV) and parenchymal volume (PV = TBV–VV) were measured. Fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum (CC) were calculated from DTI data.

Results

TBV and PV were smaller in Tg compared to control rats in young and middle-aged cohorts (p<0.0001). VV increased significantly (p = 0.005) over time in the longitudinal Tg cohort. There were lower FA (p<0.002) and higher MD (p<0.003) values in the CC of middle-aged Tg rats compared to age-matched controls. Longitudinally, MD significantly decreased over time in Tg rats (p<0.03) while it did not change significantly in the control cohort over the same period of time (p>0.05).

Conclusions

We detected brain volume loss in the Tg rat, probably due to astrocytic dysfunction/loss, loss of structural/axonal matrix and striatal neuronal loss as suggested by immunofluorescence. Increased MD and decreased FA in the CC probably reflect microstructural differences between the Tg and Control rats which could include increased extracellular space between white matter tracts, demyelination and axonal degeneration, among other pathologies. We believe that the Tg rat is an adequate model of neuropathology in HIV and that volumetric MR and DTI measures can be potentially used as biomarkers of disease progression.