Computational analysis of small RNA cloning data

Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides--and tens of millions of small RNAs--from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.     

Angling-induced cardiac disturbance of free-swimming largemouth bass (Micropterus salmoides) monitored with heart rate telemetry

The sub‐lethal effects of catch‐and‐release angling have been poorly studied because of the difficulties in monitoring physiological parameters in free‐swimming fish. Laboratory studies provide the opportunity to examine sub‐lethal effects in controlled environments, but do not incorporate site‐specific characteristics. In this study we angled free‐swimming largemouth bass (Micropterus salmoides) equipped with heart rate transmitters to exhaustion using rod and reel, and exposed fish to air for 30 s. Experiments were repeated at four water temperatures (13, 17, 21, and 25°C). These field data were compared with published findings from largemouth bass collected at the same water temperatures in a controlled laboratory setting using Doppler flow probes. Field collected heart rate data increased with increasing water temperatures (Q₁₀ values 1.30–1.37). Pre‐disturbance heart rates were ～30% higher for free‐swimming fish in the field than previously collected laboratory data at the same water temperatures. Fish angled in the field exhausted ～40% more rapidly than fish chased in the laboratory. Maximal heart rate was ～15% higher for free‐swimming fish in the field than for data collected from laboratory restrained fish, but scope for heart rate was reduced by up to 20% in the field, especially at higher water temperatures. Heart rate in free‐swimming fish was highly variable at all times, obscuring clear recovery patterns. Conversely, laboratory cardiac parameters exhibited less variable patterns, peaking clearly following disturbances and recovering in about 135 min, independent of water temperature. Based upon these findings, we suggest that comprehensive studies incorporating both laboratory and field experiments are needed for truly understanding the effect of catch‐and‐release angling on fish.     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

Irreversible Electroporation of Malignant Hepatic Tumors - Alterations in Venous Structures at Subacute Follow-Up and Evolution at Mid-Term Follow-Up

Purpose

To evaluate risk factors associated with alterations in venous structures adjacent to an ablation zone after percutaneous irreversible electroporation (IRE) of hepatic malignancies at subacute follow-up (1 to 3 days after IRE) and to describe evolution of these alterations at mid-term follow-up.

Materials and Methods

43 patients (men/women, 32/11; mean age, 60.3 years) were identified in whom venous structures were located within a perimeter of 1.0 cm of the ablation zone at subacute follow-up after IRE of 84 hepatic lesions (primary/secondary hepatic tumors, 31/53). These vessels were retrospectively evaluated by means of pre-interventional and post-interventional contrast-enhanced magnetic resonance imaging or computed tomography or both. Any vascular changes in flow, patency, and diameter were documented. Correlations between vascular change (yes/no) and characteristics of patients, lesions, and ablation procedures were assessed by generalized linear models.

Results

191 venous structures were located within a perimeter of 1.0 cm of the ablation zone: 55 (29%) were encased by the ablation zone, 78 (41%) abutted the ablation zone, and 58 (30%) were located between 0.1 and 1.0 cm from the border of the ablation zone. At subacute follow-up, vascular changes were found in 19 of the 191 vessels (9.9%), with partial portal vein thrombosis in 2, complete portal vein thrombosis in 3, and lumen narrowing in 14 of 19. At follow-up of patients with subacute vessel alterations (mean, 5.7 months; range, 0 to 14 months) thrombosis had resolved in 2 of 5 cases; vessel narrowing had completely resolved in 8 of 14 cases, and partly resolved in 1 of 14 cases. The encasement of a vessel by ablation zone (OR = 6.36, p<0.001), ablation zone being adjacent to a portal vein (OR = 8.94, p<0.001), and the usage of more than 3 IRE probes (OR = 3.60, p = 0.035) were independently associated with post-IRE vessel alterations.

Conclusion

Venous structures located in close proximity to an IRE ablation zone remain largely unaffected by this procedure, and thrombosis is rare.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

MKR mice are resistant to the metabolic actions of both insulin and adiponectin: discordance between insulin resistance and adiponectin responsiveness

Most rodent models of insulin resistance are accompanied by decreased circulating adiponectin levels. Adiponectin treatment improves the metabolic phenotype by increasing fatty acid oxidation in skeletal muscle and suppressing hepatic glucose production. Muscle IGF-I receptor (IGF-IR)-lysine-arginine (MKR) mice expressing dominant-negative mutant IGF-IRs in skeletal muscle are diabetic with insulin resistance in muscle, liver, and adipose tissue. Adiponectin levels are elevated in MKR mice, suggesting an unusual discordance between insulin resistance and adiponectin responsiveness. Therefore, we investigated the metabolic actions of adiponectin in MKR mice. MKR and ob/ob mice were treated both acutely (28 microg/g) and chronically (for 2 wk) with full-length adiponectin. Acute hypoglycemic effects of adiponectin were evident only in ob/ob mice but not in MKR mice. Chronic adiponectin treatment significantly improved both insulin sensitivity and glucose tolerance in ob/ob but not in MKR mice. Adiponectin receptor mRNA levels and adiponectin-stimulated phosphorylation of AMPK in skeletal muscle and liver were similar among MKR, wild-type, and ob/ob mice. Thus MKR mice are adiponectin resistant despite normal expression of adiponectin receptors and normal AMPK phosphorylation in muscle and liver. MKR mice may be a useful model for dissecting relationships between insulin resistance and adiponectin action in regulation of glucose homeostasis.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.