Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection

Gene therapy of retinal diseases using recombinant adeno-associated virus (rAAV) vector-based delivery has shown clinical success, and clinical trials based on rAAV-based optogenetic therapies are currently in progress. Recently, we have developed multi-characteristic opsin (MCO), which has been shown to effectively re-photosensitize photoreceptor-degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO-I) in live samples and post-mortem organs following intraocular delivery in wild-type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO-I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low-dose group 13 weeks after injection and in the faeces of the high-dose group at 3–13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post-injection. Intravitreal injection of vMCO-I resulted in few sporadic off-target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2-based delivery of MCO-I for retinal gene therapy is safe.     

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening

BackgroundInfections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour.Methodology/Principal findingsPrior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK₂ and DMEM supplemented with 10% NCS and seeded on LLC-MK₂ systems.Conclusions/SignificanceThe present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.     

i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats

Using NMR methods, we have resolved the i-motif structures formed by d(AACCCC) and by d(CCCCAA), two versions of the DNA sequence repeated in the telomeric regions of the C-rich strand of tetrahymena chromosomes. Both oligonucleotides form fully symmetrical i-motif tetramers built by intercalation of two hemiprotonated duplexes containing four C•C⁺ pairs. The structures are extremely stable. In the tetramer of d(AACCCC), the outermost C•C⁺ pairs are formed by the cytidines of the 5′ ends of the cytidine tracts. A2 forms an A2•A2 (H6trans–N7) pair stacked to C3•C3⁺ and cross-strand stacked to A1. At 0°C, the lifetimes of the hemiprotonated pairs range from 1 ms for the outermost pair to ~1 h for the innermost pairs. The tetramer of d(CCCCAA) adopts two distinct intercalation topologies in slow conformational exchange. One, whose outermost C•C⁺ pairs are built by the cytidines of the 5′ end and the other by those of the 3′ end. In both topologies, the adenosine bases are fairly well stacked to the adjacent C•C⁺ pairs. They are not paired but form symmetrical pseudo-pairs with their H6cis amino proton and N1 nitrogen pointing towards each other.     

The distribution of aquatic oligochaetes in the south and eastern Mediterranean area

On the basis of the material collected in Morocco, Algeria, Tunisia, Lebanon, Syria and Turkey, and published data, mainly on Israel and Egypt, we try to establish a synthesis of the geographical distribution of the poorly known fauna of the South and Eastern Mediterranean basin. A first analysis shows that the fauna of this region is characterized by a high number of species with a widespread distribution (cosmopolite, holarctic, palaearctic ...) and therefore, with great affinities with the European fauna. Nevertheless, three distinct communities can be distinguished:

- The south western basin (North Africa; Morocco, Algeria, Tunisia), where the community is very poor, and which shows great affinities with south European, but none with the Ethiopian region;

- The south eastern basin (Egypt), with some species of African origin;

- The eastern basin (Near East, mainly Lebanon and Israel) with a very diversified community, partly due to its geographical situation, and partly due to a more intensive collecting pressure. This community includes a group of species with more eastern affinities (East Siberian ...), but none typically Ethiopian or exclusively Sino-Indian.

     

Production of the virus‐like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus‐like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti‐myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high‐performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038–1045, 2016     

Oligochaetes in southern European groundwater: new records and an overview

This work deals with an overview of the faunal composition and presents a general ecological approach to the groundwater oligochaetes in southern Europe. The species richness is high and similar to that of the superficial waters in the same area. Among the 150 species that we found, about 29 are stygobiont, so the total number of stygobiont species in the area studied has been increased to 58. Ten of these species are tubificids with marine phyletic affinities. A list of the most frequently found species and the relative abundance of the different families and genera collected in the main environments investigated are presented. Although enchytraeids often dominated the fauna, lumbriculids and tubificids exhibit a high level of diversity and comprised the majority of stygobiont and stygophilous taxa found. The discovery of members of the family Parvidrilidae in southern European caves is the first outside of North America. This indicates that the family is widely distributed in the Palearctic region and typical of underground waters. Here, we update the previous evaluation of the knowledge about oligochaetes inhabiting groundwater: 96 species of stygobiont freshwater oligochaetes (sensu stricto) are now known in the world and 81 of them have been recorded from the Palearctic region.     

The Mediating Role of Sleep Quality and Quantity in the Link Between Food Insecurity and Obesity Across Race and Ethnicity

Objective

The objective of the study was to examine the mediating role of sleep quality and quantity in the relationship between food insecurity and obesity across races/ethnicities.

Methods

Bivariate negative binomial regression and multinomial logistic regression were used to examine direct associations between food insecurity and sleep quality and quantity among non‐Hispanic Native Hawaiian and other Pacific Islanders (NHPI) relative to non‐Hispanic White, Black, Asian, and Hispanic individuals with obesity. The potential outcomes approach and adjusted Wald test were used to explore the mediating role of sleep quality and quantity and the moderating role of race/ethnicity, respectively.

Results

Among adults with obesity, the study found a positive and direct relationship between food insecurity and trouble falling asleep in each racial/ethnic group. Trouble staying asleep was associated with food insecurity in each racial/ethnic group, except in the Asian group. Positive and direct associations were observed between food insecurity and short/very short sleep. A positive and direct relationship was found between food insecurity and having obesity in each racial/ethnic group, except in the Black group. Sleep quality and sleep quantity partially mediated the relationship between food insecurity and obesity in non‐Hispanic NHPI, White, Asian, and Hispanic individuals. Race/ethnicity moderated the indirect effects of food insecurity on sleep quality and quantity.

Conclusions

Food security and sleep hygiene should be an integral part of the fight against obesity.