Genotype-Specific Incidence and Clearance of Human Papillomavirus in Oral Mucosa of Women: A Six-Year Follow-Up Study

Background

There are no previous longitudinal studies on genotype-specific natural history of human papillomavirus (HPV) infections in oral mucosa of women.

Methods

In the Finnish Family HPV Study, 329 pregnant women were enrolled and followed up. HPV-genotyping of oral scrapings was performed with nested PCR and Multimetrix® test (Progen, Heidelberg, Germany). Incidence and clearance times and rates for each HPV-genotype identified in oral mucosa were determined. Predictors for incident and cleared HPV infections for species 7/9 genotypes were analyzed using Poisson regression model.

Results

Altogether, 115 baseline HPV-negative women acquired incident oral HPV infection, and 79 women cleared their infection. HPV16 and multiple HPVs most frequently caused incident infections (65% and 12%) in 13.3 and 17.1 months respectively, followed by HPV58, HPV18 and HPV6 (close to 5% each) in 11–24 months. HPV58, HPV18 and HPV66 were the most common to clear. HPV6 and HPV11 had the shortest clearance times, 4.6 months and 2.5 months, and the highest clearance rates, 225.5/1000 wmr and 400/1000 wmr, respectively. The protective factors for incident oral HPV-species 7/9 infections were 1) new pregnancy during follow-up and 2) having the same sexual partner during FU. Increased clearance was related with older age and a history of atopic reactions, whereas previous sexually transmitted disease and new pregnancy were associated with decreased clearance.

Conclusions

HPV16 was the most frequent genotype to cause an incident oral HPV-infection. Low risk HPV genotypes cleared from oral mucosa more quickly than high risk HPV genotypes. Pregnancy affected the outcome of oral HPV infection.     

Unveiling Distribution Patterns of Freshwater Phytoplankton by a Next Generation Sequencing Based Approach

The recognition and discrimination of phytoplankton species is one of the foundations of freshwater biodiversity research and environmental monitoring. This step is frequently a bottleneck in the analytical chain from sampling to data analysis and subsequent environmental status evaluation. Here we present phytoplankton diversity data from 49 lakes including three seasonal surveys assessed by next generation sequencing (NGS) of 16S ribosomal RNA chloroplast and cyanobacterial gene amplicons and also compare part of these datasets with identification based on morphology. Direct comparison of NGS to microscopic data from three time-series showed that NGS was able to capture the seasonality in phytoplankton succession as observed by microscopy. Still, the PCR-based approach was only semi-quantitative, and detailed NGS and microscopy taxa lists had only low taxonomic correspondence. This is probably due to, both, methodological constraints and current discrepancies in taxonomic frameworks. Discrepancies included Euglenophyta and Heterokonta that were scarce in the NGS but frequently detected by microscopy and Cyanobacteria that were in general more abundant and classified with high resolution by NGS. A deep-branching taxonomically unclassified cluster was frequently detected by NGS but could not be linked to any group identified by microscopy. NGS derived phytoplankton composition differed significantly among lakes with different trophic status, showing that our approach can resolve phytoplankton communities at a level relevant for ecosystem management. The high reproducibility and potential for standardization and parallelization makes our NGS approach an excellent candidate for simultaneous monitoring of prokaryotic and eukaryotic phytoplankton in inland waters.     

Phytoplankton indicator taxa for reference conditions in Northern and Central European lowland lakes

Phytoplankton data from 606 lakes were used to characterize indicator taxa of near-pristine reference conditions in clearwater and humic lowland lakes of Northern and Central Europe. Reference lakes were selected based on low pressure from catchment land-use, low population density and the absence of point sources. Reference lakes had low phytoplankton biomass and taxa richness compared to non-reference lakes. In low alkalinity lakes of Northern Europe, the reference communities had high biomass proportions of chrysophytes and low proportions of cyanobacteria; in the Central European high alkalinity lakes, the biomass was distributed more evenly among algal groups. Indicator species analysis and similarity analysis listed 5–29 taxa indicating reference conditions. Indicator taxa differed especially between the low alkalinity and the high alkalinity lakes, but there were also country-specific differences. Most common indicator taxa for the northern reference lakes were chrysophytes (e.g. Bitrichia, Dinobryon). In the Central European reference lakes, diatoms (e.g. Cyclotella) were more characteristic. Despite the differences, there was a general finding that taxa present in reference lakes were often also present in non-reference lakes, but typically in lower biomass proportions; another characteristic of the reference communities is the absence of many taxa typically found in non-reference lakes.     

Spermine prevents cytochrome c release in glucocorticoid-induced apoptosis in mouse thymocytes

Apoptosis can be induced in primary cultures of mouse thymocytes using the glucocorticoid dexamethasone. Addition of the polyamine spermine simultaneously with dexamethasone reduces the induction of apoptosis compared to treatment with dexamethasone alone. We investigated the signal transduction pathway at the mitochondrial level in order to elucidate spermine's protective effect. Mitochondrial involvement is evident due to the loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol and activation of caspase-9 in dexamethasone-treated thymocytes. The addition of spermine inhibited the release of cytochrome c from the mitochondria into the cytosol, and also the activation of caspase-9. When the mitogen concanavalin A (Con A) was added to dexamethasone- plus spermine-treated thymocytes, the number of apoptotic cells in the pre-G(1)peak was reduced compared to thymocytes treated with only dexamethasone plus spermine. Comparing concanavalin A added to dexamethasone-treated or to dexamethasone plus spermine-treated thymocytes, showed a markedly reduced pre-G(1)peak in the latter. Thus, the spermine-induced inhibition of cytochrome c release confers a survival advantage on thymocytes.     

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.     

Increased toxicity of a trinuclear Pt-compound in a human squamous carcinoma cell line by polyamine depletion

ABSTRACT: BACKGROUND: Mononuclear platinum anticancer agents hold a pivotal place in the treatment of many forms of cancers, however, there is a potential to improve response to evade resistance development and toxic side effects. BBR3464 is a promising trinuclear platinum anticancer agent, which is a polyamine mimic. The aim was to investigate the influence of polyamine pool reduction on the cytotoxic effects of the trinuclear platinum complex BBR3464 and cisplatin. Polyamine pool reduction was achieved by treating cells with either the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) or the polyamine analogue N1,N11-diethylnorspermine (DENSPM). METHODS: A human squamous cell carcinoma cell line, LU-HNSCC-4, established from a primary head and neck tumour was used to evaluate cellular effects of each drug alone or combinations thereof. High-performance liquid-chromatography was used to quantify intracellular polyamine contents. Inductively coupled mass spectroscopy was used to quantify intracellular platinum uptake. Cells were exposed to DFMO or DENSPM during 48 h at concentrations ranging from 0 to 5 mM or 0 to 10 muM, respectively. Thereafter, non-treated and treated cells were exposed to cisplatin or BBR3464 during 1 h at concentrations ranging from 0 to 100 muM. A 96-well assay was used to determine cytotoxicity after five days after treatment. RESULTS: The cytotoxic effect of BBR3464 on LU-HNSCC-4 cells was increased after cells were pre-treated with DENSPM or DFMO, and the interaction was found to be synergistic. In contrast, the interaction between cisplatin and DFMO or DENSPM was near-additive to antagonistic. The intracellular levels of the polyamines putrescine and spermidine were decreased after treatment with DFMO, and treatment with DENSPM resulted in an increase in putrescine level and concomitant decrease in spermidine and spermine levels. The uptake of BBR3464 was significantly increased after pre-treatment of the cells with DFMO, and varied dependent on the concentration of DENSPM. The uptake of cisplatin was unchanged. Conclusions: Taken together, these results demonstrate that combinations of polyamine synthesis inhibitors with BBR3464 appear to be a promising approach to enhance the anticancer activity against HSCC.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

beta1-Integrins induce phosphorylation of Akt on serine 473 independently of focal adhesion kinase and Src family kinases

Adhesion by means of beta1-integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3-kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion-induced activation of class Ia PI3K. Here we show that PI3K-dependent phosphorylation of Akt in response to ligation of beta1-integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant beta1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion-dependent activation of PI3K/Akt is triggered by the membrane proximal part of the beta1 subunit in a FAK and Src-independent manner.     

Reactivity of Trametes laccases with fatty and resin acids

Lipophilic extractives commonly referred to as wood pitch or wood resin can have a negative impact on paper machine runnability and product quality. The lipophilic extractives are composed mainly of fatty acids, resin acids, sterols, steryl esters and triglycerides. In this work, the suitability of laccases for the modification of fatty and resin acids was studied, using two model fractions. In the treatments, resin and fatty acid dispersions were treated with two different laccases, i.e. laccases from Trametes hirsuta and T. villosa. Different chromatographic methods were used to elucidate the effects of laccase treatments on the chemistry of the fatty and resin acids. Both laccases were able to modify the fatty and resin acids to some extent. In the case of fatty acids, a decrease in the amount of linoleic, oleic and pinolenic acids was observed, whereas the modification of resin acids resulted in a reduced amount of conjugated resin acids.     

Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases

beta-Alanine synthase (beta AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, beta ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of beta AS from Saccharomyces kluyveri to a resolution of 2.7 A. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of beta AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed alpha/beta-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by beta AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.