Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.     

Performance comparison of four exome capture systems for deep sequencing

Background

Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample.

Results

Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content.

Conclusions

We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-449) contains supplementary material, which is available to authorized users.     

Structural analysis of three prokaryotic 5S rRNA species and selected 5S rRNA--ribosomal-protein complexes by means of Pb(II)-induced hydrolysis.

Lead ions have been applied to the structural analysis of 5S rRNA from Thermus thermophilus, Bacillus stearothermophilus and Escherichia coli. Based on the distribution of Pb(II)-induced cleavages, some minor modifications of the consensus secondary structure model of 5S rRNA are proposed. They include the possible base pairing between nucleotides at positions 11 and 109, as well as changes in secondary interactions within the helix B region. The 'prokaryotic arm' region is completely resistant to hydrolysis in the three RNA species, suggesting that it is a relatively stable, highly ordered structure. Hydrolysis of E. coli 5S rRNA complexed with ribosomal protein L18 shows, besides the shielding effect of the bound protein, a highly enhanced cleavage between A108 and A109. It supports the concept that the major L18-induced conformational change involves the junction of helices A, B and D.     

Lack of anion effect on volume expansion natriuresis in the developing canine kidney

The renal response to volume expansion with sodium chloride or sodium bicarbonate was studied in 15 newborn and 13 adult dogs. Proximal and distal nephron function were estimated using the technique of distal nephron blockade. Fractional sodium reabsorption was 99.0 +/- 0.3% in newborn and 96.6 +/- 0.06% in adult during the NaCl expansion (P less than 0.01) and 98.1 +/- 0.7% in the newborn and 93.2 +/- 0.7% in the adult during NaHCO3 expansion (P less than 0.001). With either anion the higher fractional sodium reabsorption in the newborn was due to reabsorption of a greater fraction of the load presented to the distal nephron segment. The percent of the distal sodium load that was reabsorbed was 98.0 +/- 0.6% in the newborn and 92.2 +/- 1.0% in the adult during NaCl expansion, and 96.1 +/- 1.3% in the newborn and 81.5 +/- 2.4% in the adult during NaHCO3 expansion. Differences in distal nephron chloride, potassium and bicarbonate reabsorption among the groups support the hypothesis that the enhanced distal sodium reabsorption in the newborn occurred largely in the ascending loop of Henle with NaCl expansion, while it occurred in the late distal and cortical collecting tubules with NaHCO3 expansion. There was no difference between the natriuretic responses to NaCl or NaHCO3 in the newborn (P greater than 0.20); however, the natriuretic response to NaCl was less than that to NaHCO3 in the adult (P less than 0.001). This suggests that the bulk of the sodium that escaped reabsorption in Henle's loop during NaHCO3 expansion was reabsorbed in the late distal tubule in the newborn, but not in the adult.     

Reproductive Biology of the Introduced Mayan cichlid, Cichlasoma urophthalmus, Within an Estuarine Mangrove Habitat of Southern Florida

We investigated the reproductive biology of the Mayan cichlid, Cichlasoma urophthalmus, for twenty consecutive months in a southern Florida mangrove creek and compared it to that of available published accounts where this species is native. Mayan cichlid reproduction occurred during a period of the year when physical conditions facilitated the best growth and survival of the young, and was influenced more by changes in water level and salinity than by temperature. Most nests were observed along mangrove shorelines in April. The majority of reproduction and parental care of the young followed in May, and was completed by June when rising water levels flooded shallow habitats and dispersed young-of-year fish. Mayan cichlids in Florida became reproductively mature at a greater size than in native regions, but produced roughly the same number of eggs at a given body mass. The similarity of environmental conditions between southern Florida and areas where Mayan cichlids naturally occur imparts a very favorable reproductive potential for this species in southern Florida, and facilitates their continued presence within the ichthyofauna of the region.     

Description of a Well Preserved Fetus of the European Eocene Equoid Eurohippus messelensis

The early Middle Eocene locality of Grube Messel, near Darmstadt (Germany), is famous for its complete vertebrate skeletons. The degree of preservation of soft tissues, such as body silhouettes, internal organs and gut contents, is frequently remarkable. The present specimen was analyzed for remnants of the reproductive system. Classic anatomy and osteology and high-resolution micro-x-ray were applied to describe the fetus of the European Eocene equoid Eurohippus messelensis. Scanning electronic microscopy (SEM) was used for determination of soft tissue remnants. The fetus is the earliest and best-preserved fossil specimen of its kind. The postcranial fetal skeleton is almost complete and largely articulated, allowing the conclusion that the pregnant mare was in late gestation. The apparent intrauterine position of the fetus is normal for the phase of pregnancy. Death of mare and fetus were probably not related to problems associated with parturition. Soft tissue interpreted as the uteroplacenta and a broad uterine ligament are preserved due to bacterial activity and allow considerations on the evolutionary development of the structures.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.