High-resolution whole-genome association study of Parkinson disease

We performed a two-tiered, whole-genome association study of Parkinson disease (PD). For tier 1, we individually genotyped 198,345 uniformly spaced and informative single-nucleotide polymorphisms (SNPs) in 443 sibling pairs discordant for PD. For tier 2a, we individually genotyped 1,793 PD-associated SNPs (P<.01 in tier 1) and 300 genomic control SNPs in 332 matched case-unrelated control pairs. We identified 11 SNPs that were associated with PD (P<.01) in both tier 1 and tier 2 samples and had the same direction of effect. For these SNPs, we combined data from the case-unaffected sibling pair (tier 1) and case-unrelated control pair (tier 2) samples and employed a liberalization of the sibling transmission/disequilibrium test to calculate odds ratios, 95% confidence intervals, and P values. A SNP within the semaphorin 5A gene (SEMA5A) had the lowest combined P value (P=7.62 x 10(-6)). The protein encoded by this gene plays an important role in neurogenesis and in neuronal apoptosis, which is consistent with existing hypotheses regarding PD pathogenesis. A second SNP tagged the PARK11 late-onset PD susceptibility locus (P=1.70 x 10(-5)). In tier 2b, we also selected for genotyping additional SNPs that were borderline significant (P<.05) in tier 1 but that tested a priori biological and genetic hypotheses regarding susceptibility to PD (n=941 SNPs). In analysis of the combined tier 1 and tier 2b data, the two SNPs with the lowest P values (P=9.07 x 10(-6); P=2.96 x 10(-5)) tagged the PARK10 late-onset PD susceptibility locus. Independent replication across populations will clarify the role of the genomic loci tagged by these SNPs in conferring PD susceptibility.     

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.     

Notes on the Laboulbeniales (Ascomycota) parasitic on Diptera from Portugal and other countries

Eighteen species of Stigmatomyces are reported for the first time from continental Portugal and/or from the Azores. These are Stigmatomyces asteiae W. Rossi et Cesari, Stigmatomyces athyroglossae W. Rossi et Cesari, Stigmatomycescanzonerii W. Rossi et Cesari, Stigmatomyces ceratophorus Whisler, Stigmatomyces constrictus Thaxt., Stigmatomyces crassicollis Thaxt., Stigmatomyces divergatus Thaxt., Stigmatomyces discocerinae Thaxt., Stigmatomyces ensinae Thaxt., Stigmatomyces cf. ephydrae L. Mercier et R. A. Poiss., Stigmatomycesgeomyzae W. Rossi et Cesari, Stigmatomyces limnophorae Thaxt., Stigmatomyces majewskii H. L. Dainat, Manier et Balazuc, Stigmatomyces papuanus Thaxt., Stigmatomycesplatensis Speg., Stigmatomyces ptylomyiae Thaxt. and Stigmatomyces purpureus Thaxt., Stigmatomyces rugosus Thaxt. New records of these species are also reported from Australia, Canada, Canary Islands (Spain), Costa Rica, Cuba, Ecuador, Finland, Germany, Great Britain, Greece, Kenya, Hungary, Israel, Kyrgyzstan, Lebanon, Morocco, Saudi Arabia, Sierra Leone, Taiwan, Thailand, Turkey, Uganda, United Arab Emirates, USA, Yemen and Zimbabwe. The new records increase, sometimes considerably, information about distribution of these parasites. Two synonymies are also established: Stigmatomyces autriquei Balazuc = Stigmatomyces ensinae Thaxt.; Stigmatomyces psilopae Thaxt. var. camarguensis H. L. Dainat et J. Dainat = S. rugosus Thaxt.     

Photosensitization in cattle grazing on pastures of Brahciaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis

Aspects of photosensitization in bovines grazing on pastures of Brachiaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures 45(2):117-136, 1978. This paper reports experimental studies on photosensitization in bovines grazing on different pastures of Brachiaria decumbens Stapf in the "Cerrados" region (Planaltina, DF). Climatic conditions, zinc content and occurence of fungi on pastures were investigated. Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures examined. Photosensitization was observed in one animal maintained on a pasture of B. decumbens formed with seeds from Australia. Clinical and necropsy data were similar to those related in literature for sporidesmin-intoxicated animals. An isolate of P. chartarum and samples of bovine bile were assayed for sporidesmin presence.     

Giant Cicada Emergence,Protandry and Chorus Centers Formation as Revealed by Studies Using a Sound Trap

The giant cicada, Quesada gigas (Olivier) (Hemiptera: Cicadidae), is an important coffee pest and information about the behavior and reproduction of this species, e.g. emergence, senescence and ovarian maturation status, can be valuable to understand giant cicada ecology and to improve the use of a sound trap as a control method. A great number of Q. gigas adult males and females was captured using a sound trap and a protandrous type of emergence possibly associated with chorus centers formation was observed. All giant cicadas collected until 14–15 days after the beginning of male emergence (DAME) had immature ovaries at two different years of evaluation. On the other hand, the majority of cicadas collected from 20 until 48 DAME had mature ovaries with visible chorionated oocytes. Despite the use of the sound trap to collect insects for ecological studies, we believe that next generations of Q. gigas can be reduced by using this sound trap to hinder the formation of giant cicada chorus centers, to reduce male availability to copulate with females and to reduce the number of females to oviposit in coffee plants.     

Larvicidal potential of cell wall degrading enzymes from Trichoderma asperellum against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and β-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.     

Identification of Bioactive Compounds against Aedes aegypti (Diptera: Culicidae) by Bioassays and in Silico Assays

Most of the hematophagous insects act as disease vectors, including Aedes aegypti, responsible for transmitting some of the most critical arboviruses globally, such as Dengue. The use of repellents based on natural products is a promising alternative for personal protection compared to industrial chemical repellents. In this study, the repellent effect of essential oils extracted from Lippia thymoides, Lippia alba, Cymbopogon winterianus, and Eucalyptus globulus leaves was evaluated. Essential oils used showed repellent activity against Ae. aegypti in laboratory bioassays, obtaining protection rates above 70 % from 3.75 mg/mL and higher concentration for all analyzed oils. GC/MS identified 57 constituents, which were used in the ligand-based pharmacophore model to expose compounds with requirements for repellents that modulate mosquitoes behavior through odorant-binding protein 1 Ae. aegypti. Ligand-based pharmacophore model approach results suggested that repellent activity from C. winterianus, L. alba, and L. thymoides essential oils’ metabolites is related to Citronelal (QFIT=26.77), Citronelol (QFIT=11.29), Citronelol acetate (QFIT=52.22) and Geranil acetate (QFIT=10.28) with synergistic or individual activity. E. globulus essential oil's repellent activity is associated with Ledol (0.94 %; QFIT=41.95). Molecular docking was applied to understand the binding mode and affinity of the essential oils’ data set at the protein binding site. According to molecular docking, Citronelol (ChemPLP=60.98) and geranyl acetate (ChemPLP=60.55) were the best-classified compounds compared to the others and they can be explored to develop new repellents.     

Stem water storage and diurnal patterns of water use in tropical forest canopy trees

Stem water storage capacity and diurnal patterns of water use were studied in five canopy trees of a seasonal tropical forest in Panama. Sap flow was measured simultaneously at the top and at the base of each tree using constant energy input thermal probes inserted in the sapwood. The daily stem storage capacity was calculated by comparing the diurnal patterns of basal and crown sap flow. The amount of water withdrawn from storage and subsequently replaced daily ranged from 4 kg d^–1 in a 0·20-m-diameter individual of Cecropia longipes to 54 kg d^–1 in a 1·02-m-diameter individual of Anacardium excelsum, representing 9–15% of the total daily water loss, respectively. Ficus insipida, Luehea seemannii and Spondias mombin had intermediate diurnal water storage capacities. Trees with greater storage capacity maintained maximum rates of transpiration for a substantially longer fraction of the day than trees with smaller water storage capacity. All five trees conformed to a common linear relationship between diurnal storage capacity and basal sapwood area, suggesting that this relationship was species-independent and size-specific for trees at the study site. According to this relationship there was an increment of 10 kg of diurnal water storage capacity for every 0·1 m² increase in basal sapwood area. The diurnal withdrawal of water from, and refill of, internal stores was a dynamic process, tightly coupled to fluctuations in environmental conditions. The variations in basal and crown sap flow were more synchronized after 1100 h when internal reserves were mostly depleted. Stem water storage may partially compensate for increases in axial hydraulic resistance with tree size and thus play an important role in regulating the water status of leaves exposed to the large diurnal variations in evaporative demand that occur in the upper canopy of seasonal lowland tropical forests.     

Ornamentation of achene silica walls and its contribution to the systematics of <Emphasis Type="Italic">Eleocharis</Emphasis> (Cyperaceae)

Micromorphology of the achene surface of 26 Brazilian species of Eleocharis was studied by scanning electron microscopy in order to evaluate its usefulness in the taxonomy of the genus. The results point out two patterns of cell organization according to silica structures of achenes. The first corresponds to a group of species (group A) that have small to medium cells arranged vertically. The second is found in those species (group B) with medium to large cells arranged horizontally. These data were useful in separating species of Eleocharis subgenus Scirpidium and E. subgenus Limnochloa (group B) from E. subgenus Eleocharis (group A). However, group A shows considerable variation in silica wall arrangement. Eleocharis squamigera, previously considered as part of E. subgenus Eleocharis, shows features rather similar to those of Scirpidium, confirming recent phylogenies. The subgenus Limnochloa was clearly distinguished from others by achenes with large cells (over 55 μm width), presence of crenate or repand anticlinal walls, and some orifices near the wall in some species. The silica wall ornamentation seems to be a useful morphological tool for studying relationships between subgenera and distinguishes Limnochloa from the other subgenera.     

Cardiolipin, a lipid found in mitochondria, hydrogenosomes and bacteria was not detected in Giardia lamblia

Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.