The majority of microRNAs detectable in serum and saliva is concentrated in exosomes

There is an increasing interest in using microRNAs (miRNA) as biomarkers in autoimmune diseases. They are easily accessible in many body fluids but it is controversial if they are circulating freely or are encapsulated in microvesicles, particularly exosomes. We investigated if the majority of miRNas in serum and saliva are free-circulating or concentrated in exosomes. Exosomes were isolated by ultracentrifugation from fresh and frozen human serum and saliva. The amount of selected miRNAs extracted from the exosomal pellet and the exosome-depleted serum and saliva was compared by quantitative RT-PCR. Some miRNAs tested are ubiquitously expressed, others were previously reported as biomarkers. We included miRNAs previously reported to be free circulating and some thought to be exosome specific. The purity of exosome fraction was confirmed by electronmicroscopy and western blot. The concentration of miRNAs was consistently higher in the exosome pellet compared to the exosome-depleted supernatant. We obtained the same results using an equal volume or equal amount of total RNA as input of the RT-qPCR. The concentration of miRNA in whole, unfractionated serum, was between the exosomal pellet and the exosome-depleted supernatant. Selected miRNAs, which were detectable in exosomes, were undetectable in whole serum and the exosome-depleted supernantant. Exosome isolation improves the sensitivity of miRNA amplification from human biologic fluids. Exosomal miRNA should be the starting point for early biomarker studies to reduce the probability of false negative results involving low abundance miRNAs that may be missed by using unfractionated serum or saliva.     

Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression

The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.     

Reactive oxygen species and mitochondrial diseases

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.     

Prenatal development of cutaneous afferent connections in the spinal cord of fetal sheep

In this study we have examined the physiological and neurochemical development of the cutaneous afferent pathways from the hindlimb to the spinal cord in fetal sheep. We have shown that somatosensory input from the hindlimb evokes activity in DRG neurons at 87d gestation and in cells in the dorsal horn at 92d (term, 146d). There is evidence of immunoreactivity for substance P, calcitonin gene-related peptide and glutamine several days prior to this at 77-80 days. The implication of these findings are discussed.     

Genetic and cytogenetic analysis of the fruit fly Rhagoletis cerasi (Diptera: Tephritidae).

The European cherry fruit fly, Rhagoletis cerasi, is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the "weak points", and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.     

Respiratory fluoroquinolones for the treatment of community-acquired pneumonia: a meta-analysis of randomized controlled trials

Background

We investigated whether the use of respiratory fluoroquinolones was associated with better clinical outcomes compared with the use of macrolides and β-lactams among adults with pneumonia.

Methods

We searched PubMed, Current Contents, Scopus, EMBASE, ClinicalTrials.gov and Cochrane with no language restrictions. Two reviewers independently extracted data from published trials that compared fluoroquinolones (levofloxacin, moxifloxacin, gemifloxacin) with macrolides or β-lactams or both. A meta-analysis was performed with the clinical outcomes of mortality, treatment success and adverse outcomes.

Results

We included 23 trials in our meta-analysis. There was no difference in mortality among patients who received fluoroquinolones or the comparator antibiotics (OR 0.85, 95% CI 0.65–1.12). Pneumonia resolved in more patients who received fluoroquinolones compared with the comparator antibiotics for the included outcomes in the intention-to-treat population (OR 1.17, 95% CI 1.00–1.36), clinically evaluable population (OR 1.26, 95% CI 1.06–1.50) and the microbiologically assessed population (OR 1.67, 95% CI 1.28–2.20). Fluoroquinolones were more effective than a combination of β-lactam and macrolide (OR 1.39, 95% CI 1.02–1.90). They were also more effective for patients with severe pneumonia (OR 1.84, 95% CI 1.02–3.29), those who required admission to hospital (OR = 1.30, 95% CI 1.04–1.61) and those who required intravenous therapy (OR = 1.44, 15% CI 1.13–1.85). Fluoroquinolones were more effective than β-lactam and macrolide in open-label trials (OR = 1.35, 95% CI 1.08–1.69) but not in blinded randomized controlled trials (OR = 1.13, 95% CI 0.85–1.50).

Interpretation

Fluoroquinolones were associated with higher success of treatment for severe forms of pneumonia; however, a benefit in mortality was not evident. A randomized controlled trial that includes patients with severe pneumonia with or without bacteremia is needed.Community-acquired pneumonia is among the leading reasons for hospital admission¹ and resource consumption.^2,3 It is the most frequent cause of community-acquired infections among patients admitted to intensive care units.⁴ In addition, it is among the leading causes of death worldwide.Physicians must choose an optimal therapeutic regimen that eliminates the infection effectively, minimizes the risk of developing drug resistance and does not compromise the safety of the patient. The combination of β-lactam and macrolide covers the most common possible pathogens involved in the pathogenesis of pneumonia.⁵ More recently, fluoroquinolones with enhanced activity against Streptococcus pneumoniae were introduced in clinical practice. The favourable pharmacokinetic profile of fluoroquinolones allows for once daily administration, often eliminating the need for parenteral treatment. Furthermore, initial treatment with fluoroquinolones was among the predictors of lower treatment failure among patients with pneumonia.⁶In 2007, the Infectious Diseases Society of America and the American Thoracic Society released new guidelines for the management of care for adult patients with community-acquired pneumonia.⁷ In these guidelines, levofloxacin, gemifloxacin and moxifloxacin were reported to be equally effective as the combination of β-lactam and macrolide, and were proposed to be the preferred treatment option for patients who require admission to hospital, as well as for patients with comorbidity who receive treatment as outpatients. In addition to being safe, these fluoroquinolones are more effective against the most common types of bacteria responsible for the development of community-acquired pneumonia.⁷ For example, S. pneumoniae strains are not fully susceptible to ciprofloxacin. On the other hand, trovafloxacin, clinafloxacin, gatifloxacin and other quinolones are not used because of safety concerns or because they are not widely available. The trials that compared fluoroquinolones with other antibiotics regimens for the treatment of pneumonia were designed on the basis of noninferiority (i.e., an antibiotic is equally effective to a comparator), and several were conducted in order to receive approval from the relevant agencies.We sought to examine whether the use of fluoroquinolones was associated with more advantages or disadvantages than the use of macrolides or β-lactams in terms of mortality, resolution of pneumonia and adverse effects.     

Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels

The objective of the study was the comparison of anti-Müllerian hormone (AMH) levels among obese or overweight and normal-weight women with the four different polycystic ovary syndrome (PCOS) phenotypes and healthy control subjects. AMH levels were evaluated in four age- and body mass index (BMI)-matched groups of 25 normal-weight and 25 obese or overweight women each, belonging to the four main subsets of the syndrome resulting from combinations of the three diagnostic criteria [group 1: oligo- and/or anovulation (ANOV), hyperandrogenemia (HA), and polycystic ovaries (PCO) on ultrasonographic evaluation; group 2: ANOV and HA; group 3: HA and PCO, group 4: ANOV and PCO], and in 50 (25 obese or overweight and 25 normal weight) age- and BMI-matched healthy control subjects. Age, BMI, waist circumference, FSH, LH, prolactin, testosterone, Delta(4)-androstenedione, dehydroepiandrosterone-sulfate, 17alpha-OH-progesterone, fasting insulin, glucose, AMH, free androgen index, and homeostasis model assessment for insulin resistance index were analyzed. AMH levels were significantly higher in PCOS groups 1 and 2 compared with groups 3 and 4 and the control group and higher in PCOS groups 3 and 4 compared with the control group. AMH levels were significantly increased in normal-weight compared with obese and overweight women. AMH concentrations were independently predicted, in order of significance, by LH and testosterone levels, BMI (negatively), and the total number of follicles 2-9 mm in diameter. The differences in circulating AMH levels between the main phenotypic groups of PCOS women appear to reflect the severity of the syndrome, but are negatively affected by obesity. Increased LH levels might be the most significant independent link between PCOS-associated disorders of ovulation and the observed increase in circulating AMH concentration.