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.     

Use of Psoralen Monoadducts to Compare The Structure of 16S rRNA in Active and Inactive 30S Ribosomal Subunits

Abstract

E. coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4′ -aminomethyl-4,5′,8-trimethylpsoralen (AMT). This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region. After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation. Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks. The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles. However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3′ end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different.     

Human Core Temperature Responses during Exercise and Subsequent Recovery: An Important Interaction between Diurnal Variation and Measurement Site

Chronobiological investigations into core temperature during and after exercise can involve ambulatory measurements of intestinal temperature during actual competitions, esophageal temperature measurements in laboratory simulations, or rectal temperature, which can be measured in both the field and laboratory. These sites have yet to be compared during both morning and afternoon exercise and subsequent recovery. At 08∶00 and 17∶00 h, seven recreationally active males exercised at 70% peak oxygen uptake for 30 min and then recovered passively for 30 min. During the experiment, esophageal, rectal, intestinal, and skin temperatures, plus sweat loss, heart rate, and ratings of perceived exertion (RPE), were monitored. We found that the diurnal variation in intestinal temperature responses (0.45±0.32°C; mean±SD) was significantly larger compared with rectal (0.33±0.24°C) and, particularly, esophageal temperature responses (0.21±0.20°C; p= 0.019). This reflected a greater difference of 0.25–0.40°C between the esophagus and the other two sites in the afternoon, compared to inter‐site differences of only 0.13–0.16°C in the morning. Diurnal variation was small for skin temperature, heart rate, sweat loss, and RPE responses during exercise (p>0.05). Our data suggest that the relative differences between intestinal, rectal, and esophageal temperature during exercise and subsequent recovery depend on time of day to the extent that inferences from studies on experimental and applied chronobiology will be affected.     

The Endocytosis of Cellulose Synthase in Arabidopsis Is Dependent on μ2, a Clathrin-Mediated Endocytosis Adaptin

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.Cellulose microfibrils, as the major load-bearing polymers in plant cell walls, are the predominant component that enforces asymmetric cell expansion (Green, 1962). In higher plants, cellulose is synthesized by multimeric rosettes, which are also referred to as cellulose synthase complexes (CSCs; Kimura et al., 1999). Genetic and coimmunoprecipitation studies have indicated that CELLULOSE SYNTHASE1 (CESA1), CESA3, and CESA6-like (CESA6, CESA2, CESA5, and CESA9) isoforms are constituents of CSCs during primary cell wall synthesis (Persson et al., 2005; Desprez et al., 2007; Persson et al., 2007; Wang et al., 2008), whereas CESA4, CESA7, and CESA8 are implicated in the cellulose synthesis of secondary cell walls (Taylor et al., 1999, 2003; Brown et al., 2005). Knowledge about cellulose synthesis has recently been enhanced by the development of a system whereby the dynamics of CESA can be imaged in living cells (Paredez et al., 2006; Desprez et al., 2007). In agreement with earlier transmission electron microscopy studies in which rosettes were visualized in Golgi cisternae, vesicles, and at the plasma membrane (Haigler and Brown, 1986), fluorescent protein tagging of CESA has identified CESA localization at the plasma membrane, in Golgi bodies, and in small intracellular compartments (Paredez et al., 2006; Desprez et al., 2007; Crowell et al., 2009; Gutierrez et al., 2009; Gu et al., 2010; Lei et al., 2012; Li et al., 2012b).Assuming that cellulose synthesis occurs solely at the plasma membrane, the trafficking of CSCs to and from the plasma membrane may act as a significant regulatory mechanism. Although the mechanistic details of CESA trafficking are lacking, live cell imaging has shown that CESA localizes to various subcellular compartments. A subset of CESAs colocalize with markers of the trans-Golgi network (TGN)/early endosome (EE), an organelle that is part of both the secretory and endocytic pathways in Arabidopsis (Arabidopsis thaliana; Dettmer et al., 2006; Lam et al., 2007; Crowell et al., 2009, 2010; Viotti et al., 2010). CESAs also localize to microtubule-associated cellulose synthase compartments (MASCs) and small CESA-containing compartments (SmaCCs). The exact function of SmaCCs/MASCs is unknown, but it has been proposed that SmaCCs/MASCs might result from the internalization of CSCs or might act in the delivery of CSCs to the plasma membrane (Crowell et al., 2009, 2010; Gutierrez et al., 2009).Clathrin-mediated endocytosis (CME) has been shown to be a major endocytic pathway in Arabidopsis (Holstein, 2002; Samaj et al., 2005; Dhonukshe et al., 2007; Kleine-Vehn and Friml, 2008; Chen et al., 2011; Beck et al., 2012; Wang et al., 2013), although there is also evidence of clathrin-independent endocytosis mechanisms (Bandmann and Homann, 2012). The function of many CME proteins has been extensively characterized in mammals (McMahon and Boucrot, 2011), and homologs of many CME components are encoded by the Arabidopsis genome, including multiple copies of clathrin H chain and clathrin light chain (CLC), all four subunits of the heterotetrameric ADAPTOR PROTEIN COMPLEX2 (AP2) complex, dynamin-related proteins, and accessory proteins such as AP180 (Holstein, 2002; Chen et al., 2011); however, many CME components have yet to be characterized in plants.It has been suggested that CME might also function in controlling cell wall metabolism. For example, dividing and growing cells internalize cross-linked cell wall pectins, which might allow for cell wall remodeling (Baluska et al., 2002, 2005; Samaj et al., 2004). Moreover, the importance of endocytosis for cell wall morphogenesis is apparent from the functional characterization of proteins involved in CME. A dynamin-related protein, DRP1A, plays a significant role in endocytosis and colocalizes with CLC (Collings et al., 2008; Konopka and Bednarek, 2008). Defective endocytosis in RADIAL SWELLING9 (rsw9) plants, which contain a mutation in DRP1A, results in cellulose deficiency and defects in cell elongation (Collings et al., 2008). A mutation in rice, brittle culm3 (bc3), was mapped to the dynamin-related gene OsDRP2A, which has been proposed to function in CME. The brittle-culm phenotype in this mutant was attributed to cellulose deficiency (Xiong et al., 2010). Although the abundance of OsCESA4 was also altered in bc3, it remains unclear whether the cellulose deficiency of either bc3 or rsw9 results directly from perturbations in CESA trafficking.To identify proteins involved in the regulation of cellulose biosynthesis, a yeast two-hybrid (Y2H) screen was performed in which the central domain of CESA6 (CESA6CD) was used as bait to screen an Arabidopsis complementary DNA library for potential interaction partners of CESA6 (Gu et al., 2010; Gu and Somerville, 2010). The Y2H screen identified μ2 as a putative interaction partner of CESA6CD. The mammalian homolog of μ2 is the medium subunit of the tetrameric AP2, which acts as the core of the CME machinery by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis (Jackson et al., 2010; McMahon and Boucrot, 2011; Cocucci et al., 2012). In this study, we provide evidence that μ2 plays a role in CME in Arabidopsis, that CESAs are a new set of CME cargo proteins, and that plant cells might regulate cellulose synthesis by controlling the abundance of CSCs at the plasma membrane through CME. To our knowledge, this study is the first to show the affect of an AP2 complex component on endocytosis in plants and the first to visualize an AP2 complex component in living plant cells. Furthermore, our data suggest that the role of AP2 in plants may differ from what has been shown in animals.     

Fecal Influenza in Mammals: Selection of Novel Variants

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2^E627K, HA^F128V, HA^F454L, or HA^H300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.     

The Mature Virion of Ectromelia Virus,a Pathogenic Poxvirus,Is Capable of Intrahepatic Spread and Can Serve as a Target for Delayed Therapy

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.