Prediction of various grades of cervical neoplasia on plastic-embedded cytobrush samples. Discriminant analysis with qualitative and quantitative predictors.

The purpose of this study was to investigate whether discrimination into five groups of various grades of cervical preneoplasia and neoplasia is possible using discriminant analysis models. Data were analyzed for 242 cases diagnosed as either slight dysplasia (n = 50), moderate dysplasia (n = 50), severe dysplasia (n = 50), carcinoma in situ (n = 50) or invasive carcinoma (n = 42) and consisted of qualitative and quantitative features of cells derived from a repeat sample taken from the ectocervix as well as the endocervix using Cytobrushes. The samples were embedded in plastic, and thin sections were prepared, resulting in a monolayer of cut nuclei. The percentages of expected correct prediction were obtained by using 10,000 double cross-validation samples; the mean percentage of correct prediction into five groups using cross-validation was 65% (in the original analysis, 72%) and into two groups (dysplasia versus carcinoma in situ and invasive carcinoma) was 91% (93%). The results reflect group discrimination potential; we do not claim reliability of prediction for an individual patient. The patients were not a representative sample of the population; to investigate whether groups of patients could be discriminated on the basis of both qualitative and quantitative features, the data analyzed contain an almost equal number of observations in each of the five groups. The results indicate that features do not classify the cases in the same way; the discriminant analyses suggest that quantitative features play an important role in the discrimination of dysplasia from carcinoma cases, while the majority of the qualitative features are important in discrimination within the three dysplasia groups.     

Microbial community of predatory bugs of the genus <Emphasis Type=Italic>Macrolophus</Emphasis>(Hemiptera: Miridae)

BACKGROUND: The predatory mirids of the genus Macrolophus are key natural enemies of various economically important agricultural pests. Both M. caliginosus and M. pygmaeus are commercially available for the augmentative biological control of arthropod pests in European greenhouses. The latter species is known to be infected with Wolbachia -inducing cytoplasmic incompatibility in its host- but the presence of other endosymbionts has not been demonstrated. In the present study, the microbial diversity was examined in various populations of M. caliginosus and M. pygmaeus by 16S rRNA sequencing and denaturing gradient gel electrophoresis. RESULTS: Besides Wolbachia, a co-infection of 2 Rickettsia species was detected in all M. pygmaeus populations. Based on a concatenated alignment of the 16S rRNA gene, the gltA gene and the coxA gene, the first is phylogenetically related to Rickettsia bellii, whereas the other is closely related to Rickettsia limoniae. All M. caliginosus populations were infected with the same Wolbachia and limoniae-like Rickettsia strain as M. pygmaeus, but did not harbour the bellii-like Rickettsia strain. Interestingly, individuals with a single infection were not found. A PCR assay on the ovaries of M. pygmaeus and M. caliginosus indicated that all endosymbionts are vertically transmitted. The presence of Wolbachia and Rickettsia in oocytes was confirmed by a fluorescence in situ hybridisation. A bio-assay comparing an infected and an uninfected M. pygmaeus population suggested that the endosymbionts had minor effects on nymphal development of their insect host and did not influence its fecundity. CONCLUSION: Two species of the palaearctic mirid genus Macrolophus are infected with multiple endosymbionts, including Wolbachia and Rickettsia. Independent of the origin, all tested populations of both M. pygmaeus and M. caliginosus were infected with three and two endosymbionts, respectively. There was no indication that infection with endosymbiotic bacteria had a fitness cost in terms of development and fecundity of the predators.     

Methane and oxygen dynamics in a shallow floodplain lake: the significance of periodic stratification

Temperature, dissolved oxygen and dissolved methane profiles were measured during autumn and summer, in a shallow floodplain lake in south-eastern Australia to determine the effects of water-column stability on methane and oxygen dynamics. The water column was well mixed in autumn. Strong thermal stratification developed in the late afternoon in summer, with top-to-bottom temperature differences of up to 6 °C. Methane concentrations in surface waters varied over a daily cycle by an 18-fold range in summer, but only by a 2-fold range in autumn. The implication of short-term temporal variation is that static chambers deployed on the water surface for short times (less than a day) in summer will significantly underestimate the diffusive component of methane emissions across the water–atmosphere interface. There was a marked diel variation in dissolved oxygen concentrations in summer, with the highest oxygen values (commonly 5–8 mg l^–1) occurring in the surface waters in late afternoon; the bottom waters were then devoid of oxygen (< 0.2 mg l^–1). Because of high respiratory demands, even the surface water layers could be nearly anoxic by morning in summer. The concentration of dissolved oxygen in the surface waters was always less than the equilibrium value. When the water column became thermally stratified in summer, the dissolved oxygen and methane maxima were spatially separated, and planktonic methanotrophy would be limited to a moving zone, at variable depth, in the water column. In summer the whole-wetland rates of oxygen production and respiration, calculated from long-term (5 h) shifts in dissolved oxygen concentrations over a diel period, were approximately 6–10 and 3–6 mmol m^–3 h^–1, respectively. These values correspond to net and gross primary production rates of 0.7–1.2 and 1.0–1.9 g C m^–3 day^–1, respectively.     

Role of fungi in freshwater ecosystems

There are more than 600 species of freshwater fungi with a greater number known from temperate, as compared to tropical, regions. Three main groups can be considered which include Ingoldian fungi, aquatic ascomycetes and non-Ingoldian hyphomycetes, chytrids and, oomycetes. The fungi occurring in lentic habitats mostly differ from those occurring in lotic habitats. Although there is no comprehensive work dealing with the biogeography of all groups of freshwater fungi, their distribution probably follows that of Ingoldian fungi, which are either cosmopolitan, restricted to pantemperate or pantropical regions, or in a few cases, have a restricted distribution. Freshwater fungi are thought to have evolved from terrestrial ancestors. Many species are clearly adapted to life in freshwater as their propagules have specialised aquatic dispersal abilities. Freshwater fungi are involved in the decay of wood and leafy material and also cause diseases of plants and animals. These areas are briefly reviewed. Gaps in our knowledge of freshwater fungi are discussed and areas in need of research are suggested.     

Numerical study of entrainment of the human circadian system and recovery by light treatment

Background

While the effects of light as a zeitgeber are well known, the way the effects are modulated by features of the sleep-wake system still remains to be studied in detail.

Methods

A mathematical model for disturbance and recovery of the human circadian system is presented. The model combines a circadian oscillator and a sleep-wake switch that includes the effects of orexin. By means of simulations, we characterize the period-locking zone of the model, where a stable 24-hour circadian rhythm exists, and the occurrence of circadian disruption due to both insufficient light and imbalance in orexin. We also investigate how daily bright light treatments of short duration can recover the normal circadian rhythm.

Results

It is found that the system exhibits continuous phase advance/delay at lower/higher orexin levels. Bright light treatment simulations disclose two optimal time windows, corresponding to morning and evening light treatments. Among the two, the morning light treatment is found effective in a wider range of parameter values, with shorter recovery time.

Conclusions

This approach offers a systematic way to determine the conditions under which circadian disruption occurs, and to evaluate the effects of light treatment. In particular, it could potentially offer a way to optimize light treatments for patients with circadian disruption, e.g., sleep and mood disorders, in clinical settings.

     

Leishmania major: nature of immunity induced by immunization with a mutagenized avirulent clone of the parasite in mice

A chemically mutagenized avirulent form of Leishmania major was used to immunize BALB/c and C57B1/6 mice against challenge with virulent L. major. Immunity was elicited when the avirulent parasite was injected intravenously or intraperitoneally, but not subcutaneously. In fact, the latter route of immunization sometimes resulted in exacerbation of a subsequent infection with virulent L. major. Mice immunized with avirulent L. major developed upon challenge with virulent L. major cutaneous lesions which were significantly smaller and contained substantially fewer parasites than lesions on control nonimmune animals. Finally, the protection conferred by immunization with avirulent L. major could be adoptively transferred with T cells of the CD4+ lineage but not the CD8+ lineage.     

Reduced circulating oxidized LDL is associated with hypocholesterolemia and enhanced thiol status in Gilbert syndrome

A protective association between bilirubin and atherosclerosis/ischemic heart disease clearly exists in vivo. However, the relationship between bilirubin and in vivo oxidative stress parameters in a clinical population remains poorly described. The aim of this study was to assess whether persons expressing Gilbert syndrome (GS; i.e., unconjugated hyperbilirubinemia) are protected from thiol oxidation and to determine if this, in addition to their improved lipoprotein profile, could explain reduced oxidized low-density lipoprotein (oxLDL) status in them. Forty-four matched GS and control subjects were recruited and blood was prepared for the analysis of lipid profile and multiple plasma antioxidants and measures of oxidative stress. GS subjects possessed elevated plasma reduced thiol (8.03±1.09 versus 6.75±1.39 nmol/mg protein; P<0.01) and glutathione concentrations (12.7±2.39 versus 9.44±2.45 μM; P<0.001). Oxidative stress status (reduced:oxidized glutathione; GSH:GSSG) was significantly improved in GS (0.49±0.16 versus 0.32±0.12; P<0.001). Protein carbonyl concentrations were negatively associated with bilirubin concentrations and were significantly lower in persons with >40 μM bilirubin versus controls (<17.1 μmol/L; P<0.05). Furthermore, absolute oxLDL concentrations were significantly lower in GS subjects (P<0.05). Forward stepwise regression analysis revealed that bilirubin was associated with increased GSH:GSSG ratio and reduced thiol concentrations, which, in addition to reduced circulating LDL, probably decreased oxLDL concentrations within the cohort. In addition, a marked reduction in total cholesterol concentrations in hyperbilirubinemic Gunn rats is presented (Gunn 0.57±0.09 versus control 1.69±0.40 mmol/L; P<0.001), arguing for a novel role for bilirubin in modulating lipid status in vivo. These findings implicate the physiological importance of bilirubin in protecting from atherosclerosis by reducing thiol and subsequent lipoprotein oxidation, in addition to reducing circulating LDL concentrations.     

Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.