Longitudinal qPCR Study of the Dynamics of L. crispatus,L. iners,A. vaginae, (Sialidase Positive) G. vaginalis,and P. bivia in the Vagina

Background

To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles.

Methods

Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar.

Results

Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D).VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H₂O₂-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF.

Conclusions

This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies.     

An evaluation of small-molecule p53 activators as chemoprotectants ameliorating adverse effects of anticancer drugs in normal cells

Pharmacological activation of wild-type p53 has been found to protect normal cells in culture from cytotoxicity and nuclear aberrations caused by conventional cancer therapeutics. Hence, small-molecule p53 activators could have clinical benefits as chemoprotectants for cancer patients bearing p53-mutant tumors. We have evaluated 16 p53-based cyclotherapy regimes combining p53 activators tenovin-6, leptomycin B, nutlin-3 and low dose actinomycin D, with clinically utilized chemotherapeutic agents (S- and M-phase poisons), vinblastine, vinorelbine, cytosine arabinoside and gemcitabine. All the p53 activators induce reversible cell-cycle arrest in primary human fibroblasts and protect them from both S- and M-phase poisons. Furthermore, studies with p53-mutant cancer cell lines show that nutlin-3 and low dose actinomycin D do not affect the sensitivity of these cells to any of the chemotherapeutics tested. Thus, these two small molecules could be suitable choices for cyclotherapy regimes involving S- or M-phase poisons. In contrast, pre-incubation of p53-mutant cells with tenovin-6 or leptomycin B reduces the efficacy of vinca alkaloids, suggesting that these p53 activators could be effective as chemoprotectants if combined with S- but not M-phase poisons. Discrepancies were observed between the levels of protection detected immediately after treatment and following recovery in fresh medium. This highlights the need to assess both short- and long-term effects when evaluating compounds as potential chemoprotectants for cancer therapy.     

Geographic variation in Thomas langur (Presbytis thomasi) loud calls

Geographic variation in primate vocalizations has been described at two levels. First, at the level of acoustic variation within the same call type between populations and, second, at the level of presence or absence of certain call types in different populations. Acoustic variation is of interest because there are several factors that can explain this variation, such as gene flow, ecological factors and population density. Here we focus on the first level in a Southeast Asian primate, the Thomas langur. We recorded male loud calls in four populations that differed in their geographic distances from each other and had varying geographic barriers in between them, such as rivers and mountain ranges. The presence of these barriers leads to expectations of loud call variation under the gene flow model, which are examined here. We conducted a principal components analysis to condense the number of acoustic variables. With a subsequent discriminant function analysis on the six principal component scores, we found that the percentage of loud calls that were correctly assigned to a population was relatively high (50.0-76.2%) when three randomly selected loud calls from each male were used. Using the discriminant functions from this analysis to predict population membership of the remainder of the loud calls yielded lower, but still relatively high correct assignment percentages (26.2-66.7%). Analyses to examine the influence of barriers on similarities between populations confirm our expectations. We discuss that differences in loud calls are probably most parsimoniously explained by gene flow (or the lack thereof) between the populations and that future studies of genetic differences are crucial to test this hypothesis.     

Regulation of thermogenesis in flowering Araceae: the role of the alternative oxidase

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.     

The Arabidopsis Somatic Embryogenesis Receptor Kinase 1 Gene Is Expressed in Developing Ovules and Embryos and Enhances Embryogenic Competence in Culture

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.     

The ligase chain reaction distinguishes hepatitis B virus S-gene variants

Abstract The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.     

Calcium,a pivotal player in photodynamic therapy?

Photodynamic therapy combines three non-toxic components: light, oxygen and a photosensitizer to generate singlet oxygen and/or other ROS molecules in order to target destruction of cancer cells. The damage induced in the targeted cells can furthermore propagate to non-exposed bystander cells thereby exacerbating the damage. Ca²⁺ signaling is strongly intertwined with ROS signaling and both play crucial roles in cell death. In this review we aimed to review current knowledge on the role of Ca²⁺ and ROS signaling, their effect on cell-cell propagation via connexin-linked mechanisms and the outcome in terms of cell death. In general, photodynamic therapy results in an increased cytosolic Ca²⁺ concentration originating from Ca²⁺ entry or Ca²⁺ release from internal stores. While photodynamic therapy can certainly induce cell death, the outcome depends on the cell type and the photosensitizer used. Connexin channels propagating the Ca²⁺ signal, and presumably regenerating ROS at distance, may play a role in spreading the effect to neighboring non-exposed bystander cells. Given the various cell types and photosensitizers used, there is currently no unified signaling scheme to explain the role of Ca²⁺ and connexins in the responses following photodynamic therapy. This article is part of a Special Issue entitled: Calcium signaling in health, disease and therapy edited by Geert Bultynck and Jan Parys.