Vigour-related traits and immunity in hybrids of evolutionary divergent cyprinoid species: advantages of hybrid heterosis?

Hybrid advantage, described as the superiority of hybrids in some traits over their parents and termed the “heterosis effect,” is widely documented in the case of reciprocal crosses of parental species (i.e., hybrids representing the F1 generation). In fish, high survival, fast growth and better health status have been widely documented in F1 hybrids. Nonetheless, the effects of interspecific hybridization on vigour, physiology and immunity-related traits in fish are largely unknown, especially concerning native systems of coexisting parental and hybrid genomes in the same habitat. The present study examined the potential physiological and immune aspects of hybrid heterosis by comparing condition status (measured especially by indexes), haematological profile, glucose concentration and selected parameters of non-specific and specific immunity between the evolutionarily divergent non-congeneric cyprinoid species Abramis brama and Rutilus rutilus and their hybrids representing the F1 generation, all of them caught in nature. Clear differences were documented for vigour-related, physiological and immune parameters between the two divergent species. Hybrids generally tended to express intermediate characters of the measured traits, likely generated by the evolutionary divergence of the hybridizing species; nonetheless, for some traits, hybrids exhibited a character that was more similar to one parental species than to the other. This was interpreted as the heterozygote advantage for F1 hybrids. It is suggested that a maternally inherited genetic background may potentially influence the expression of some branches of non-specific immunity or other aspects related to the fish health status.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Data exploration tools for the Gene Ontology database

MOTIVATION: To improve the ability of biologists (both researchers and students) to ask biologically interesting questions of the Gene Ontology (GO) database and to explore the ontologies by seeing large portions of the ontology graphs in context, along with details of individual terms in the ontologies. RESULTS: GoGet and GoView are two new tools built as part of an extensible web application system based on Java 2 Enterprise Edition technology. GoGet has a user interface that enables users to ask biologically interesting questions, such as (1) What are the DNA binding proteins involved in DNA repair, but not in DNA replication? and (2) Of the terms containing the word triphosphatase, which have associated gene products from mouse, but not fruit fly? The results of such queries can be viewed in a collapsed tabular format that eases the burden of getting through large tables of data. GoView enables users to explore the large directed acyclic graph structure of the ontologies in the GO database. The two tools are coordinated, so that results from queries in GoGet can be visualized in GoView in the ontology in which they appear, and explorations started from GoView can request details of gene product associations to appear in a result table in GoGet. AVAILABILITY: Free access to the GoGet query tool and free download of the GoView ontology viewer are provided to all users at http://db.math.macalester.edu/goproject. In addition, source code for the GoView tool is also available from this site, along with a user manual for both tools.     

Two new species of Choanocotyle Jue Sue and Platt, 1998 (Digenea: Choanocotylidae) from an Australian freshwater turtle (Testudines: Pleurodira: Chelidae)

Choanocotyle hobbsi n. sp. and Choanocotyle juesuei n. sp. are described from the small intestine of the oblong turtle Chelodina oblonga from the vicinity of Perth, Western Australia. These are the third and fourth species referred to Choanocotyle. Choanocotyle hobbsi is most similar to Choanocotyle nematoides but differs in the size and shape of the oral sucker and the absence of a median loop in the cirrus sac. Choanocotyle juesuei is most similar to Choanocotyle elegans but differs in the size of the oral sucker and other morphometric criteria. Comparative analysis of the sequences of different nuclear ribosomal deoxyribonucleic acid regions of C. nematoides and C. hobbsi has confirmed that they are closely related but distinct species.     

On the systematic position of Ophiosacculus Macy, 1935 (digenea: Lecithodendriidae), with the erection of the Ophiosacculinae n. subfam

The phylogenetic relationships and systematic position of the digenean genus Ophiosacculus Macy, 1935 has been controversial and opinions of different authors on its systematic position and content are contradictory. Molecular analysis based on the partial sequences of the large subunit ribosomal DNA gene of the type and only valid species of the genus, Ophiosacculus mehelyi (Mödlinger, 1930), as well as previously published sequences of members of several families of Plagiorchiata (including the Allassogonoporidae, Lecithodendriidae and Pleurogenidae as potential relatives of Ophiosacculus) has shown that Ophiosacculus forms a clade with the typical representatives of the Lecithodendriidae from bats. Ophiosacculus is basal to the cluster containing Lecithodendrium, Prosthodendrium and Pycnoporus and has quite pronounced differences in the sequenced fragment compared to these genera. Based on the results of the molecular study, morphological characteristics of Ophiosacculus (in particular, possession of a seminal vesicle lying freely in parenchyma) and the fact that the type-specimen of Gyrabascus brevigastrus Macy, 1835 (type-species of the monotypic genus Gyrabascus and type-genus of the subfamily Gyrabascinae) belongs to Allassogonoporus, a new subfamily, the Ophiosacculinae, with Ophiosacculus as the type-genus, is established within the Lecithodendriidae. Molecular study did not support a close phylogenetic relationship between Allassogonoporus and Ophiosacculus, although several authors previously allocated both these genera to the Allassogonoporidae. Morphological study revealed the position of the genital pore in O. mehelyi to be at the posterior margin of the ventral sucker. An amended diagnosis of Ophiosacculus and a diagnosis of Ophiosacculinae n. subfam. are given.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.     

Identification of Tumor-Associated Antigens from Medullary Breast Carcinoma by a Modified SEREX Approach

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.