Reliability of Intra-Retinal Layer Thickness Estimates

Purpose

Measurement of intra-retinal layer thickness using optical coherence tomography (OCT) has become increasingly prominent in multiple sclerosis (MS) research. Nevertheless, the approaches used for determining the mean layer thicknesses vary greatly. Insufficient data exist on the reliability of different thickness estimates, which is crucial for their application in clinical studies. This study addresses this lack by evaluating the repeatability of different thickness estimates.

Methods

Studies that used intra-retinal layer segmentation of macular OCT scans in patients with MS were retrieved from PubMed. To investigate the repeatability of previously applied layer estimation approaches, we generated datasets of repeating measurements of 15 healthy subjects and 13 multiple sclerosis patients using two OCT devices (Cirrus HD-OCT and Spectralis SD-OCT). We calculated each thickness estimate in each repeated session and analyzed repeatability using intra-class correlation coefficients and coefficients of repeatability.

Results

We identified 27 articles, eleven of them used the Spectralis SD-OCT, nine Cirrus HD-OCT, two studies used both devices and two studies applied RTVue-100. Topcon OCT-1000, Stratus OCT and a research device were used in one study each. In the studies that used the Spectralis, ten different thickness estimates were identified, while thickness estimates of the Cirrus OCT were based on two different scan settings. In the simulation dataset, thickness estimates averaging larger areas showed an excellent repeatability for all retinal layers except the outer plexiform layer (OPL).

Conclusions

Given the good reliability, the thickness estimate of the 6mm-diameter area around the fovea should be favored when OCT is used in clinical research. Assessment of the OPL was weak in general and needs further investigation before OPL thickness can be used as a reliable parameter.     

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

Background

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods

We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results

The 50% effective inhibitory concentration (IC₅₀) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions

To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.     

A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactones

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.     

Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

Hybridization among cryptic species of the cellar fungus Coniophora puteana (Basidiomycota)

In this study we have analysed the genetic variation and phylogeography in a global sample of the cellar fungus Coniophora puteana, which is an important destroyer of wooden materials indoor. Multilocus genealogies of three DNA regions (beta tubulin, nrDNA ITS and translation elongation factor 1alpha) revealed the occurrence of three cryptic species (PS1-3) in the morphotaxon C. puteana. One of the lineages (PS3) is apparently restricted to North America while the other two (PS1-2) have wider distributions on multiple continents. Interspecific hybridization has happened between two of the lineages (PS1 and PS3) in North America. In three dikaryotic isolates, two highly divergent beta tubulin alleles coexisted, one derived from PS1 and one from PS3. Furthermore, one isolate included a recombinant ITS sequence, where ITS1 resembled the ITS1 version of PS3 while ITS2 was identical to a frequent PS1 ITS2 version. This pattern must be due to hybridization succeeded by intralocus recombination in ITS. The results further indicated that introgression has happened between subgroups appearing in PS1. We hypothesize that the observed reticulate evolution is due to previous allopatric separation followed by more recent reoccurrence in sympatry, where barriers to gene flow have not yet evolved. A complex phylogeographical structure is observed in the morphotaxon C. puteana caused by (i) cryptic speciation; (ii) the interplay between natural migration and distribution patterns and probably more recent human mediated dispersal events; and (iii) hybridization and introgression.     

Compartmentalised expression of <Emphasis Type="Italic">Delta-like 1</Emphasis> in epithelial somites is required for the formation of intervertebral joints

Background  

Expression of the mouse Delta-like 1 (Dll1) gene in the presomitic mesoderm and in the caudal halves of somites of the developing embryo is required for the formation of epithelial somites and for the maintenance of caudal somite identity, respectively. The rostro-caudal polarity of somites is initiated early on within the presomitic mesoderm in nascent somites. Here we have investigated the requirement of restricted Dll1 expression in caudal somite compartments for the maintenance of rostro-caudal somite polarity and the morphogenesis of the axial skeleton. We did this by overexpressing a functional copy of the Dll1 gene throughout the paraxial mesoderm, in particular in anterior somite compartments, during somitogenesis in transgenic mice.     

Akt signalling in health and disease

Akt (also known as protein kinase B or PKB) comprises three closely related isoforms Akt1, Akt2 and Akt3 (or PKBα/β/γ respectively). We have a very good understanding of the mechanisms by which Akt isoforms are activated by growth factors and other extracellular stimuli as well as by oncogenic mutations in key upstream regulatory proteins including Ras, PI3-kinase subunits and PTEN. There are also an ever increasing number of Akt substrates being identified that play a role in the regulation of the diverse array of biological effects of activated Akt; this includes the regulation of cell proliferation, survival and metabolism. Dysregulation of Akt leads to diseases of major unmet medical need such as cancer, diabetes, cardiovascular and neurological diseases. As a result there has been substantial investment in the development of small molecular Akt inhibitors that act competitively with ATP or phospholipid binding, or allosterically. In this review we will briefly discuss our current understanding of how Akt isoforms are regulated, the substrate proteins they phosphorylate and how this integrates with the role of Akt in disease. We will furthermore discuss the types of Akt inhibitors that have been developed and are in clinical trials for human cancer, as well as speculate on potential on-target toxicities, such as disturbances of heart and vascular function, metabolism, memory and mood, which should be monitored very carefully during clinical trial.     

Lack of CCR7 induces pulmonary hypertension involving perivascular leukocyte infiltration and inflammation

The chemokine receptor CCR7 regulates lymphocyte trafficking, and CCR7 deficiency induces infiltration of T and B cells adjacent to vessels in mouse lungs. Perivascular infiltration of T and B cells has also been found in human pulmonary arterial hypertension, and downregulation of the CCR7 receptor in circulating leukocytes of such patients has been observed. To investigate whether changes in the CCR7 system contribute to the pathogenesis of pulmonary hypertension, we utilized mice deficient of the CCR7 receptor. The cardiopulmonary and inflammatory responses of CCR7 depletion were evaluated in CCR7-deficient and wild-type mice. Measurements of cytokines upregulated in the animal model were also performed in patients with pulmonary hypertension and controls and in vascular smooth muscle cells. We found that mice lacking CCR7 had increased right ventricular systolic pressure, reduced pulmonary artery acceleration time, increased right ventricular/tibial length ratio, Rho kinase-mediated pulmonary vasoconstriction, and increased muscularization of distal arteries, indicating pulmonary hypertension. These mice also showed increased perivascular infiltration of leukocytes, consisting mainly of T and B cells, and increased mRNA levels of the inflammatory cytokines interleukin-12 and CX3CL1 within pulmonary tissue. Increased serum levels of interleukin-12 and CX3CL1 were also observed in patients with pulmonary hypertension, particularly in those with pulmonary hypertension associated with connective tissue disorder. In smooth muscle cells, interleukin-12 induced secretion of the angiogenic cytokine interleukin-8. We conclude that these results suggest a role for CCR7 in the development of pulmonary arterial hypertension, at least in some subgroups, possibly via pulmonary infiltration of lymphocytes and secretion of interleukin-12 and CX3CL1.