Distribution and Transport of Cholesterol in Caenorhabditis elegans

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1–L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.

Sistemas duplos renais : revisão sistemática

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Revision of Telotrema Ozaki, 1933 (Digenea : Gyliauchenidae Fukui, 1929), including the description of a new species from an acanthurid fish from the Great Barrier Reef, Queensland, Australia

We describe one new species of Telotrema Ozaki, 1933 from the intestine of an acanthurid fish of the Great Barrier Reef. Telotrema brevicaudatum n. sp. is described from 2 mature specimens from the yellowfin surgeonfish, Acanthurus xanthopterus Valenciennes, 1835 ( Acanthuridae), from waters off Lizard Island, Queensland, Australia. This species is distinguished from the type-species, Telotrema caudatum Ozaki, 1933, by the smaller excretory papilla, the massive pars prostatica, the unipartite, globular seminal vesicle, and the intertesticular position of the ovary. The proposal of a new species of Telotrema necessitates re-examination of the generic diagnosis, and the genus is here redefined in light of the morphology of T. brevicaudatum. Telotrema is distinguished from Gyliauchen Nicoll, 1915 by the possession of a ventral sucker which is larger than the pharynx, a straight or sigmoid oesophagus, an extensive and dense vitellarium, and a distinct excretory papilla. We here recognise 3 species and distinguish them in a key. The biogeographical range for species of Telotrema now includes acanthurid and pomacentrid fishes of the western Pacific Ocean.

New Thelastomatoidea (Nematoda : Oxyurida) from Australian burrowing cockroaches (Blaberidae : Geoscapheinae, Panesthiinae)

Four new species and two new genera of thelastomatoid are described from several species of Australian burrowing cockroaches (Blattodea: Panesthiinae; Geoscapheinae). Corpicracens munozae n. g., n. sp., Pseudodesmicola botti n. g., n. sp. and Cephalobellus nolani n. sp. are described from Geoscapheus dilatatus (Blattodea: Geoscapheinae) from Mendooran, New South Wales; one new thelastomatid, Blattophila praelongicauda n. sp., is described from Panesthia cribrata from Lamington National Park, Queensland. Corpicracens munozae n. g., n. sp. is long and slender, with a monodelphic female reproductive system, a clavate corpus with a slight posterior pseudobulb, oval eggs flattened at the poles, and a relatively robust, subulate tail. Pseudodesmicola botti n. g., n. sp. is slightly more robust in body, also has a monodelphic reproductive system, a cylindrical corpus with a posterior pseudobulb, ovoid eggs and a very long, subulate tail. Cephalobellus nolani n. sp. is distinguished from other members of the genus by its relatively short and broad body and egg shape. Lastly, Blattophila praelongicauda n. sp. is distinguished from other members of the genus by having eggs with a single, polar operculum, tail length, and position of the vulva, nerve ring and excretory pore. An additional species, known by a single specimen from Panesthia tryoni tryoni from the same locality is characterised but not named. The species found are all relatively rare parasites of Australian burrowing cockroaches, each having a prevalence of less than 10%.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.