Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.     

Open membranes are the precursors for assembly of large DNA viruses

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double‐stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open‐eight’ conformation with a characteristic diameter of 90 nm for Mimi‐ and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.     

The effect of a traditional dance training program on dynamic balance of individuals with mental retardation

The purpose of this study was to evaluate the influence of a Greek traditional dance training program on the dynamic balance of individuals with mental retardation (MR). A total of 17 individuals participated in this study. Ten individuals with mild or moderate MR and 7 individuals with mild or moderate MR who studied in special schools were assigned to intervention (MR-I) and control (MR-C) groups, respectively. Pretraining and posttraining exercise tests were performed to determine the dynamic balance ability. Dynamic balance ability was measured by means of a balance deck (Lafayette, Lafayette, IN, USA) in 30-, 45-, and 60-second intervals. The MR-I group underwent a 16-week Greek traditional dance training program at a frequency of 3 times per week and for a duration of 45 minutes per season. Posttraining results showed that the individuals with MR in the MR-I group improved during treatment, from their baseline scores on dynamic balance measurements (30 seconds: p < 0.01, 45 seconds: p < 0.05, 60 seconds: p < 0.05). The MR-C group did not show any improvement between the 2 measurements. In conclusion, individuals with MR may be able to improve their dynamic balance when performing a systematic and well-designed Greek traditional dance training program.     

African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ～5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.     

Effect of Linezolid on Clinical Severity and Pulmonary Cytokines in a Murine Model of Influenza A and Staphylococcus aureus Coinfection

Excessive inflammation contributes to the severity of post influenza pneumonia caused by methicillin resistant S.aureus (MRSA). Linezolid, vancomycin, and clindamycin are antibiotics used for MRSA infections. Linezolid has immunomodulatory properties. We report on the effects of the three antibiotics on microbial clearance, pulmonary cytokines and clinical course in a murine model of influenza and MRSA coinfection.

Methods

B6 mice were infected with influenza A virus and 3 days later with MRSA, both intranasally. Treatment with placebo, linezolid, vancomycin or clindamycin started immediately after MRSA infection and continued for 72 hours. Bacterial and viral titers as well as cytokine concentrations in the lungs were assessed 4 and 24 hours after MRSA coinfection. Mice were weighted daily for 13 days.

Results

Coinfected mice had increased pulmonary IL-1β, TNF-α and mKC at 4 and 24 hours, IL-6, IL-10 and IL-12 at 4 hours and IFN-γ at 24 hours after MRSA coinfection (all P<0.05). Compared to placebo, coinfected mice treated with linezolid, vancomycin or clindamycin had decreased pulmonary IL-6 and mKC at 4 hours and IFN-γ at 24 hours after MRSA coinfection (all P<0.05). IL-1β, TNF-α and IL-12 were similar in antibiotic-treated and placebo groups. All antibiotics similarly reduced MRSA without effect on influenza titers. Linezolid-treated mice had less weight loss on days 4–6 after influenza infection compared to placebo (all P<0.05). On all other days weight change was similar among all groups.

Conclusions

This is the first report comparing the effects of antibiotics on cytokines and clinical outcome in a murine model of influenza and MRSA coinfection. Compared to placebo, antibiotic treatment reduced maximum concentration of IL-6, mKC and IFN-γ in the lungs without any difference among antibiotics. During treatment, only linezolid delayed weight loss compared to placebo.     

No impact of SLCO1B1 521T>C, 388A>G and 411G>A polymorphisms on response to statin therapy in the Greek population

Interindividual variability exists in statin lipid-lowering response, partially attributed to genetic factors. Organic anion-transporting polypeptide 1B1 (OATP1B1) encoded by SLCO1B1 gene (solute carrier organic anion transporter family member 1B1) facilitates hepatic uptake of simvastatin and atorvastatin. SLCO1B1 polymorphisms are strongly associated with statin-induced myopathy whereas few studies have assessed their effect on statin differential response. In the present study, we analyzed the association of SLCO1B1 521T>C, 388A>G and 411G>A polymorphisms with response to atorvastatin and simvastatin in 386 adults (201 atorvastatin-treated and 185 simvastatin-treated) with primary hypercholesterolemia, all of Greek origin. Total cholesterol and low-density lipoprotein cholesterol were measured at baseline and on 6 months of treatment. Genetic polymorphisms were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. A novel RFLP protocol was developed for the simultaneous identification of 388A>G and 411G>A polymorphisms. SLCO1B1 521T>C, 388A>G and 411G>A polymorphisms were not associated with lipid-lowering response to atorvastatin or simvastatin. No sex-gene or statin dose-gene interaction was observed on the effect of the analyzed SLCO1B1 polymorphisms in statin lipid lowering response in either statin-treated patient cohort. Further studies in different populations are required to draw firm conclusion on the potential association of SLCO1B1 polymorphisms with statin lipid-lowering response.     

Idiopathic and normal lateral lumbar curves: muscle effects interpreted by 12th rib length asymmetry with pathomechanic implications for lumbar idiopathic scoliosis

Background

The historical view of scoliosis as a primary rotation deformity led to debate about the pathomechanic role of paravertebral muscles; particularly multifidus, thought by some to be scoliogenic, counteracting, uncertain, or unimportant. Here, we address lateral lumbar curves (LLC) and suggest a pathomechanic role for quadrates lumborum, (QL) in the light of a new finding, namely of 12th rib bilateral length asymmetry associated with idiopathic and small non-scoliosis LLC.

Methods

Group 1: The postero-anterior spinal radiographs of 14 children (girls 9, boys 5) aged 9–18, median age 13 years, with right lumbar idiopathic scoliosis (IS) and right LLC less that 10°, were studied. The mean Cobb angle was 12° (range 5–22°). Group 2: In 28 children (girls 17, boys 11) with straight spines, postero-anterior spinal radiographs were evaluated similarly to the children with the LLC, aged 8–17, median age 13 years. The ratio of the right/left 12th rib lengths and it’s reliability was calculated. The difference of the ratio between the two groups was tested; and the correlation between the ratio and the Cobb angle estimated. Statistical analysis was done using the SPSS package.

Results

The ratio’s reliability study showed intra-observer +/−0,036 and the inter-observer error +/−0,042 respectively in terms of 95 % confidence limit of the error of measurements. The 12th rib was longer on the side of the curve convexity in 12 children with LLC and equal in two patients with lumbar scoliosis. The 12th rib ratios of the children with lumbar curve were statistically significantly greater than in those with straight spines. The correlation of the 12th rib ratio with Cobb angle was statistically significant. The 12th thoracic vertebrae show no axial rotation (or minimal) in the LLC and no rotation in the straight spine group.

Conclusions

It is not possible, at present, to determine whether the 12th convex rib lengthening is congenitally lengthened, induced mechanically, or both. Several small muscles are attached to the 12th ribs. We focus attention here on the largest of these muscles namely, QL. It has attachments to the pelvis, 12th ribs and transverse processes of lumbar vertebrae as origins and as insertions. Given increased muscle activity on the lumbar curve convexity and similar to the interpretations of earlier workers outlined above, we suggest two hypotheses, relatively increased activity of the right QL muscle causes the LLCs (first hypothesis); or counteracts the lumbar curvature as part of the body’s attempt to compensate for the curvature (second hypothesis). These hypotheses may be tested by electrical stimulation studies of QL muscles in subjects with lumbar IS by revealing respectively curve worsening or correction. We suggest that one mechanism leading to relatively increased length of the right 12 ribs is mechanotransduction in accordance with Wolff’s and Pauwels Laws.

     

CYP3A5 genotyping for assessing the efficacy of treatment with simvastatin and atorvastatin

In this work, we examined the impact of polymorphism in the cytochrome P450 (CYP) 3A5 gene, CYP3A5*1 (6986A > G, rs 776746), on the reduction in the lipid levels caused by simvastatin and atorvastatin. We studied 350 hyperlipidemic patients who received 10-40 mg of atorvastatin (n = 175) or simvastatin (n = 175) daily. Genotyping for CYP3A5 was done by PCR-RFLP analysis. Differences in the lipid profile before and after treatment were expressed as the % difference. The frequency of CYP3A5polymorphism was 13.4% for heterozygotes and 86.6% for homozygotes. Comparison of the responses to same dose of each drug showed that the highest % difference was associated with total cholesterol (TC) in subjects receiving atorvastatin 40 mg compared with simvastatin 40 mg (p = 0.048). However, comparison of the responses to equivalent doses of atorvastatin vs. simvastatin revealed no difference in the % change in any of the lipid parameters examined. In individuals with the same CYP3A5 genotype, a head to head comparison of the efficacy of the same dose of simvastatin vs. atorvastatin revealed an advantage for atorvastatin. For equivalent doses of atorvastatin vs. simvastatin there was no difference in the % change in any of the lipid parameters examined. Within the same genotype there was a significant difference in the % change related to the drug treatment.     

Improvement of lipase production by addition of catalase during fermentation

A batch fermentation process for lipase production with the recombinant strain Staphylococcus carnosus (pLipMut2) was studied in a bubble column. The rates of growth and lipase production in this type of fermentor were compared with results from shakeflasks. It was seen that cultivation in the bubble column resulted in a prolonged lag time and a reduced lipase activity in comparison to flask cultures. However, by addition of catalase during the fermentation in the bubble column this different behaviour could be avoided. Correspondence to: E. Wenzig