Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum

Abstract A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum , from bacteria phylogenetically close to mollicutes ( Clostridium innocuum, C. ramosum and Bacillus subtilis ), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum .     

Temporal variability of top-down forces and their role in host choice evolution of phytophagous arthropods

The role of top-down forces in host choice evolution of phytophagous arthropods is the subject of a vividly animated debate. Empirical evidence for the evolutionary role of top-down forces comes from studies showing that phytophagous arthropods prefer hosts that entail enemy-free space. The aim of this paper is to draw the attention of plant–arthropod researchers to the potentially, temporally variable nature of third trophic level effects. We show that this aspect is largely neglected in studies on enemy-free space, despite the fact that relative enemy impact varies seasonally among plants in at least some studies. We conclude that rigorous testing of the enemy-free space hypothesis can only be performed when within and between season variation in higher trophic level effects is taken into account.     

Isoform-Specific Attachment of Osteoprogenitors to Laminins: Mapping to the Short Arms of Laminin-1

The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.     

How does hydroxyl introduction influence the double helical structure: the stabilization of an altritol nucleic acid:ribonucleic acid duplex

Altritol nucleic acids (ANAs) are a promising new tool in the development of artificial small interfering ribonucleic acids (siRNAs) for therapeutical applications. To mimic the siRNA:messenger RNA (mRNA) interactions, the crystal structure of the ANA:RNA construct a(CCGUAAUGCC-P):r(GGCAUUACGG) was determined to 1.96?? resolution which revealed the hybrid to form an A-type helix. As this A-form is a major requirement in the RNAi process, this crystal structure confirms the potential of altritol-modified siRNAs. Moreover, in the ANA strands, a new type of intrastrand interactions was found between the O2' hydroxyl group of one residue and the sugar ring O4' atom of the next residue. These interactions were further investigated by quantum chemical methods. Besides hydration effects, these intrastrand hydrogen bonds may also contribute to the stability of ANA:RNA duplexes.     

Estimation of Vaccine Efficacy in Non-Randomly Mixing Populations

A deterministic model for the transmission of an acute infectious disease in a heterogeneous, nonrandomly mixing population is developed. This model facilitates the estimation of transmission probabilities from the observed attack rates. If some of the members of the population are vaccinated, then the vaccine efficacy (VE), defined as the relative reduction in the transmission probability due to vaccination, can be estimated. We provide several estimators of VE, depending on the amount of information available on the mixing pattern and on the action of the vaccine. We show that if vaccinated persons increase the frequency of their contacts with infectious persons, then estimators ignoring this change in behavior may substantially underestimate the VE.     

The Effect of Exercise on Visceral Adipose Tissue in Overweight Adults: A Systematic Review and Meta-Analysis

Excessive visceral adipose tissue appears to trigger a cascade of metabolic disturbances that seem to coexist with ectopic fat storage in muscle, liver, heart and the ß-cell. Therefore, the reduction of visceral adipose tissue potentially plays a pivotal role in the treatment of the metabolic syndrome. The purpose of this systematic review and meta-analysis is to describe the overall effect of exercise on visceral adipose tissue and to provide an overview of the effect of different exercise regimes, without caloric restriction, on visceral adipose tissue in obese persons. A systematic literature search was performed according to the PRISMA statement for reporting systematic reviews and meta-analyses. The initial search resulted in 87 articles after removing duplicates. After screening on title, abstract and full-text 15 articles (totalling 852 subjects) fulfilled the a priori inclusion criteria. The quality of each eligible study was assessed in duplicate with “The Critical Review Form for Quantitative Studies”. Using random-effects weights, the standardized mean difference (Hedge''s g) of the change in visceral adipose tissue was −0.497 with a 95% confidence interval of −0.655 to −0.340. The Z-value was −6.183 and the p-value (two tailed) was <0.001. A subgroup analysis was performed based on gender, type of training and intensity. Aerobic training of moderate or high intensity has the highest potential to reduce visceral adipose tissue in overweight males and females. These results suggest that an aerobic exercise program, without hypocaloric diet, can show beneficial effects to reduce visceral adipose tissue with more than 30 cm² (on CT analysis) in women and more than 40 cm² in men, even after 12 weeks.     

Increased Concentrations of Fructose 2,6-Bisphosphate Contribute to the Warburg Effect in Phosphatase and Tensin Homolog (PTEN)-deficient Cells

Unlike normal differentiated cells, tumor cells metabolize glucose via glycolysis under aerobic conditions, a hallmark of cancer known as the Warburg effect. Cells lacking the commonly mutated tumor suppressor PTEN exhibit a glycolytic phenotype reminiscent of the Warburg effect. This has been traditionally attributed to the hyperactivation of PI3K/Akt signaling that results from PTEN loss. Here, we propose a novel mechanism whereby the loss of PTEN negatively affects the activity of the E3 ligase APC/C-Cdh1, resulting in the stabilization of the enzyme PFKFB3 and increased synthesis of its product fructose 2,6-bisphosphate (F2,6P₂). We discovered that when compared with wild-type cells, PTEN knock-out mouse embryonic fibroblasts (PTEN KO MEF) have 2–3-fold higher concentrations of F2,6P₂, the most potent allosteric activator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). Reintroduction of either wild-type or phosphatase mutant PTEN in the PTEN KO cells effectively lowers F2,6P₂ to the wild-type levels and reduces their lactate production. PTEN KO cells were found to have high protein levels of PFKFB3, which directly contribute to the increased concentrations of F2,6P₂. PTEN enhances interaction between PFKFB3 and Cdh1, and overexpression of Cdh1 down-regulates the PFKFB3 protein level in wild-type, but not in PTEN-deficient cells. Importantly, we found that the degradation of endogenous PFKFB3 in PTEN KO cells occurs at a slower rate than in wild-type cells. Our results suggest an important role for F2,6P₂ in the metabolic reprogramming of PTEN-deficient cells that has important consequences for cell proliferation.