The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.     

Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.     

Laser capture microdissection of metachromatically stained skeletal muscle allows quantification of fiber type specific gene expression

Molecular and Cellular Biochemistry - Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature, and neurons, thus making it difficult to perform...     

Single-cell responses to ionizing radiation

While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.     

Unintended consequences of delisting routine eye exams on retinopathy screening for people with diabetes in Ontario,Canada

Background:

Routine eye examinations for healthy adults aged 20–64 years were delisted from the Ontario Health Insurance Plan in 2004, but they continue to be insured for people with diabetes regardless of age. We sought to assess whether the delisting of routine eye examinations for healthy adults had the unintended consequence of decreasing retinopathy screening for adults with diabetes.

Methods:

We used administrative data to calculate eye examinations for people with diabetes ages 40–64 years and 65 years and older in each 2-year period from 1998 to 2010. We examined differences by sex, income, rurality and type of health care provider. We used segmented linear regression to assess the change in trend before and after 2004.

Results:

For people with diabetes aged 65 years and older, eye examinations rose gradually from 1998 to 2010, with no substantial change between 2004 and 2006. For people with diabetes aged 40–65 years, there was an 8.7% (95% confidence interval [CI] 6.3%–11.1%) decrease in eye examinations between 2004 and 2006. Results were similar for all population subgroups. Ophthalmologic examinations decreased steadily for both age groups during the study period, and there was a decline in optometry examinations for people ages 40–65 years after 2004.

Interpretation:

The delisting of routine eye examinations for healthy adults in Ontario had the unintended consequence of reducing publicly funded retinopathy screening for people with diabetes. More research is needed to understand whether patients are being charged for an insured service or to what degree misunderstanding has prevented patients from seeking care.Diabetic retinopathy is the leading cause of new cases of blindness in people of working age.¹ In the United States, about 40% of adults with diabetes aged 40 years and older have retinopathy, and 8% have vision-threatening retinopathy.² Studies suggest that, if untreated, 50% of patients with proliferative diabetic retinopathy become legally blind within 5 years, compared with only 5% of patients who receive early treatment.³ Regular dilated eye examinations are effective for early detection and monitoring of asymptomatic retinopathy in people with diabetes⁴ and are recommended by clinical practice guidelines.^5,6In Ontario, Canada’s most populous province, medically necessary services are covered by the Ontario Health Insurance Plan (OHIP) for all permanent residents and Canadian citizens living in the province.⁷ Under OHIP, routine eye examinations were fully insured for all children and adults until November 1, 2004. At that time, routine eye examinations ceased being insured for healthy adults aged 20–64 years, but continued to be insured for children aged 19 years and younger and for adults aged 65 years and older.⁸ Regardless of age, adults with diabetes and some other medical conditions affecting the eye, as well as adults receiving social assistance, continued to have an annual eye examination covered by OHIP. Insured examinations are at no cost to the patient and are reimbursed to the provider at about Can$40. In contrast, healthy adults aged 20–64 years are required to pay out-of-pocket or through private insurance for a routine eye examination, with fees set at the discretion of the optometrist⁹ or physician.¹⁰Health policy experts suggest that delisting services from insurance schemes can have unpredictable effects.¹¹ Understanding the effect of delisting on care is particularly important as governments face fiscal pressures and contemplate further reductions in what is publicly insured.¹² We sought to assess whether delisting routine eye examinations for healthy middle-aged adults in Ontario had the unintended consequence of decreasing retinopathy screening for middle-aged adults with diabetes, even though eye examinations continued to be insured for this population.     

Photoperiodic regulation of gametogenesis in a North Atlantic sea star,Asterias vulgaris

Summary

Individuals of the sea star Asterias vulgaris from New Hampshire were maintained under two contrasting regimes of seasonally changing photoperiod. In one regime the lights turned on and off in phase with local sunrise and sunset; in the other, the daily photoperiod was kept 6 months out of phase with ambient photo- period. When dissected after being maintained for 18 months, the gametogenic condition of the out-of-phase animals was found to be 6 months out of phase with that of the in-phase animals. This experiment demonstrates that photoperiodic regulation of reproduction is important even in marine animals that normally experience marked seasonal changes in sea temperature.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.