Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.     

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.     

Seeding the future – the issues of supply and demand in restoration in Australia

It has been almost 15 years since concerns about the limited capacity of remnant native vegetation to supply the volumes of seed required to meet increasing restoration demands were first raised. Since that time little progress has been made towards addressing this constraint with the ongoing decline of native vegetation communities, especially since 2000, further challenging seed supply. We provide examples of the size of this demand for seed, as well as major issues associated with seed sourcing. We also discuss how invoking the concept of market forces to drive seed supply and demand is inappropriate and highlight the need for an industry body to oversee seed collection and utilisation standards. We further propose key actions that are required to secure the seed supply chain within the next 20 years to meet existing and future restoration targets. We argue that concerted, coordinated action at Commonwealth, State and regional levels are required to underpin effective future restoration outcomes.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Combined image and genomic analysis of high-grade serous ovarian cancer reveals PTEN loss as a common driver event and prognostic classifier

Background

TP53 and BRCA1/2 mutations are the main drivers in high-grade serous ovarian carcinoma (HGSOC). We hypothesise that combining tissue phenotypes from image analysis of tumour sections with genomic profiles could reveal other significant driver events.

Results

Automatic estimates of stromal content combined with genomic analysis of TCGA HGSOC tumours show that stroma strongly biases estimates of PTEN expression. Tumour-specific PTEN expression was tested in two independent cohorts using tissue microarrays containing 521 cases of HGSOC. PTEN loss or downregulation occurred in 77% of the first cohort by immunofluorescence and 52% of the validation group by immunohistochemistry, and is associated with worse survival in a multivariate Cox-regression model adjusted for study site, age, stage and grade. Reanalysis of TCGA data shows that hemizygous loss of PTEN is common (36%) and expression of PTEN and expression of androgen receptor are positively associated. Low androgen receptor expression was associated with reduced survival in data from TCGA and immunohistochemical analysis of the first cohort.

Conclusion

PTEN loss is a common event in HGSOC and defines a subgroup with significantly worse prognosis, suggesting the rational use of drugs to target PI3K and androgen receptor pathways for HGSOC. This work shows that integrative approaches combining tissue phenotypes from images with genomic analysis can resolve confounding effects of tissue heterogeneity and should be used to identify new drivers in other cancers.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0526-8) contains supplementary material, which is available to authorized users.     

Fitness landscapes for coupled map lattices

Our goal is to match some dynamical aspects of biological systems with that of networks of coupled logistic maps. With these networks we generate sequences of iterates, convert them to symbol sequences by coarse-graining, and count the number of times combinations of symbols occur. Comparison of this with the number of times these combinations occur in experimental data—a sequence of interbeat intervals for example—is a measure of the fitness of each network to describe the target data. The most fit networks provide a cartoon that suggests a decomposition of the experimental data into a component that may be produced by a simple dynamical subsystem, and a residual component, the result of detailed, particular characteristics of the system that generated the target data. In the space of all network parameters, each point corresponds to a particular network. We construct a fitness landscape when we assign a fitness to each point. Because the parameters are distributed continuously over their ranges, and because fitnesses are estimated numerically, any plot of the landscape involves a finite sample of parameter values. We’ll investigate how the local landscape geometry changes when the array of sample parameters is refined, and use the landscape geometry to explore complex relations between local fitness maxima.