A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA

Processing of primary microRNA (pri-miRNA) stem–loops by the Drosha–DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha–DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha–DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.     

Litter quality versus soil microbial community controls over decomposition: a quantitative analysis

The possible effects of soil microbial community structure on organic matter decomposition rates have been widely acknowledged, but are poorly understood. Understanding these relationships is complicated by the fact that microbial community structure and function are likely to both affect and be affected by organic matter quality and chemistry, thus it is difficult to draw mechanistic conclusions from field studies. We conducted a reciprocal soil inoculum × litter transplant laboratory incubation experiment using samples collected from a set of sites that have similar climate and plant species composition but vary significantly in bacterial community structure and litter quality. The results showed that litter quality explained the majority of variation in decomposition rates under controlled laboratory conditions: over the course of the 162-day incubation, litter quality explained nearly two-thirds (64 %) of variation in decomposition rates, and a smaller proportion (25 %) was explained by variation in the inoculum type. In addition, the relative importance of inoculum type on soil respiration increased over the course of the experiment, and was significantly higher in microcosms with lower litter quality relative to those with higher quality litter. We also used molecular phylogenetics to examine the relationships between bacterial community composition and soil respiration in samples through time. Pyrosequencing revealed that bacterial community composition explained 32 % of the variation in respiration rates. However, equal portions (i.e., 16 %) of the variation in bacterial community composition were explained by inoculum type and litter quality, reflecting the importance of both the meta-community and the environment in bacterial assembly. Taken together, these results indicate that the effects of changing microbial community composition on decomposition are likely to be smaller than the potential effects of climate change and/or litter quality changes in response to increasing atmospheric CO₂ concentrations or atmospheric nutrient deposition.     

Mortality risk versus food quality trade-offs in ants: patch use over time

Abstract.

• 1 Foragers of the ant species Lasius pallitarsis (Provancher) and Myrmica incompleta Provancher were given access to two food patches that differed in nutritive quality. In about half of the trials, the higher quality patch also had a mortality risk for the foragers in the form of a large Formica subnuda Emery ant located along the trail. Colonies were given access to the food for 4 or 24 h, every second day.
• 2 Use of the higher quality patch by L.pallitarsis foragers was depressed by the mortality risk. In contrast to the predictions of a food stress hypothesis, foragers used the risky patch more in the longer access trials. This appears due to a decline in the activity level of the F.subnuda, which results in a significantly lower mortality risk per trip.
• 3 M.incompfeta also significantly decreased their use of the higher quality patch when a F.subnuda was present. However, the length of time a colony had access to the patches had no effect on the use of the risky patch. M.incompleta foraging behaviour seems relatively insensitive to short-term food stress or changing levels of mortality risk.
• 4 The differences between species in their exploitation of patches and reactions to F.subnuda are discussed in terms of the species’natural history.

     

Taking babies' temperatures: science versus social taboos in battle over Baby Check.

Baby Check, a scoring system for assessing the severity of illness in babies under 6 months old, has not met with the success its developers expected. The inclusion of rectal temperature in the assessment was strongly opposed by the Royal College of Midwives, which refused to alter its view despite evidence of the safety and accuracy of rectal thermometry. British parents appear not to like rectal thermometers either. Most other medical bodies have supported Baby Check and the reason for midwives'' opposition may have more to do with professional pique at not being consulted than clinical wisdom.     

A Highlights from MBoC Selection: Drosha protein levels are translationally regulated during Xenopus oocyte maturation

MicroRNAs (miRNAs) are ∼21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination–type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing.     

ADAR2 A-->I editing: site selectivity and editing efficiency are separate events

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A→I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.     

Eating Motives and the Controversy over Dieting: Eating Less Than Needed versus Less Than Wanted

Anti‐dieting sentiment has grown in recent years. Critics of restrained eating suggest that it evokes counter‐regulatory responses that render it ineffective or even iatrogenic. However, restrained eaters are not in negative energy balance and overweight individuals show reduced eating problems when losing weight by dieting. A distinction is often drawn between physiological and psychological hunger, and neuroscience research has shown that there is a neurophysiological reality underlying this distinction. The brain has a homeostatic system (activated by energy deficits) and a hedonic system (activated by the presence of palatable food). The omnipresence of highly palatable food in the environment may chronically activate the hedonic appetite system, producing a need to actively restrain eating not just to lose weight but to avoid gaining it. Just as restricting energy intake below homeostatic needs produces physiological deprivation, restricting intake of palatable foods may produce “perceived deprivation” despite a state of energy balance. In summary, the motivation to eat more than one needs appears to be every bit as real, and perhaps every bit as powerful, as the motivation to eat when energy deprived.     

Biotic versus hydrologic control over seasonal nitrate leaching in a floodplain forest

Strong seasonal increases in aquatic (stream, ground and hyporheic water) nitrate have been observed in a variety of ecosystems. In most cases, changes in hydrological and vegetative activity occur contemporaneously, making it difficult to determine whether soil leaching is being driven by increases in the availability of leachable N or is simply due to flushing of N that has accumulated over longer periods. Three studies were conducted to better determine controls on soil nitrate leaching in a near-pristine temperate floodplain ecosystem receiving large N inputs via N-fixation by red alder: 1) an artificial rainfall experiment was conducted to estimate N-leaching potential during the summer, when plant uptake is high and new inputs of organic matter are low; 2) soil solution, groundwater and surface water were sampled during a major autumn storm to document exchanges at the seasonal transition, when plant uptake is low and inputs of senescent organic matter are high; and 3) monthly samples of soil and aquatic nitrogen were collected in 1997 and 1998 to document seasonal patterns of N exchanges. Collectively, these studies demonstrate the importance of hydrologic factors in controlling N flux. Nitrate was rapidly leached from soils during actual and simulated rainstorms. Two pathways of nitrate leaching were identified. Localized flooding and direct leaching of streamside soils into surface waters contributed to high solute concentrations in peak flows. Nitrate that leached into interstitial waters was subject to various factors that could delay or reduce its delivery to surface waters. Greater residence time may increase the influence of this component of stormflow on ecosystem productivity. While soil nitrate pools were rapidly depleted during rainstorms, accumulation of soil nitrate occurred over summer dry periods. Large differences in soil and aquatic nitrate concentrations between two years with contrasting rainfall highlight the potential for inter-annual hydrologic variability to affect ecosystem nutrient cycling.     

Axial versus Appendicular: Constraint versus Selection

SYNOPSIS.Constraint theory has suffered from its many and varyingdefinitions and from repeated confusion with the action of selection.Constraints are difficult to isolate, more difficult to quantify,and their consequences may be simulated by the action of agentssuch as stabilizing selection. This paper presents an alternativemethod of constraint analysis using a simple one-dimensionalmeasurement. A total of 29 skeletal elements from 15 speciesrepresenting 11 orders of mammals were measured for length andnormalized for body mass, after which means, standard deviations,and variances were generated. A coefficient of variation analysiswas performed to normalize for mean element length. The axialskeleton was found to be less variable than the appendicular.The appendicular demonstrated a trend where the more distalelements were the most variable, and variation decreased withmore proximal positions. The three most variable of the 29 elementswere finger V, toe V, and metacarpal V. In summary, the axialskeleton was found to be more conservative in the lengths ofits elements, the more distal appendicular elements were lessconstrained than proximal ones and these constraints were probablythe results of genetic, developmental, and mechanical factors.It is also proposed that stabilizing selection played some rolein maintaining the curb on length variance in these structures,based on mechanical performance. The results of this study areintended to promote discussion of alternative methods of constraintanalysis.     

Pulsed and continuous wave mobile phone exposure over left versus right hemisphere: effects on human cognitive function

The possible effects of continuous wave (CW) and pulse modulated (PM) electromagnetic field (EMF) on human cognition was studied in 36 healthy male subjects. They performed cognitive tasks while exposed to CW, PM, and sham EMF. The subjects performed the same tasks twice during each session; once with left-sided and once with right-sided exposure. The EMF conditions were spread across three testing sessions, each session separated by 1 week. The exposed hemisphere, EMF condition, and test order were counterbalanced over all subjects. We employed a double-blind design: both the subject and the experimenter were unaware of the EMF condition. The EMF was created with a signal generator connected via amplifier to a dummy phone antenna, creating a power output distribution similar to the original commercial mobile phone. The EMF had either a continuous power output of 0.25 W (CW) or pulsed power output with a mean of 0.25 W. An additional control group of 16 healthy male volunteers performed the same tasks without any exposure equipment to see if mere presence of the equipment could have affected the subjects' performance. No effects were found between the different EMF conditions, separate hemisphere exposures, or between the control and experimental group. In conclusion, the current results indicate that normal mobile phones have no discernible effect on human cognitive function as measured by behavioral tests.     

Endogenous versus exogenous nutrient control over decomposition and mineralization in North Carolina peatlands

We examined the relative importance of exogenous (pH, water table, soil nutrient and cation availability) and endogenous (carbon quality, nutrient and cation concentrations of litter) controls on litter decay over both the short term (1 yr) and intermediate term (3 yr) in four freshwater peatland communities that occur along a P and N availability gradient in the Coastal Plain of North Carolina. Four litter types were reciprocally transplanted into each community. Additionally, the effects of exogenous nutrient availability and low pH on decomposition dynamics were examined by fertilizing and liming plots in the most nutrient-deficient community, the short pocosin. Both exogenous and endogenous factors were important in controlling decay rates and nutrient mineralization-immobilization dynamics. The most important site factor controlling decay rates was water table, with greater rates of decomposition in drained sites. High initial soluble phenolic concentrations and a low holocellulose quotient (% holocellulose / % lignocellulose) in litter inhibited decay rates. Despite the extremely low nutrient availability in the pocosins and low soil pH in all three communities, both the cross-community comparison and the amendment experiment in the short pocosin demonstrated that exogenous nutrient availability, endogenous nutrient concentrations in litter, and low soil pH do not inhibit decomposition in these sites. In contrast, immobilization-mineralization dynamics of N and P were largely driven by a source-sink relationship, with greatest immobilization found with high exogenous nutrient availability and low initial endogenous nutrient concentrations. We suggest a conceptual model of nutrient control over decomposition as a function of carbon quality of litter.     

Chromothripsis: Breakage-fusion-bridge over and over again

The acquisition of massive but localized chromosome translocations, a phenomenon termed chromothripsis, has received widespread attention since its discovery over a year ago. Until recently, chromothripsis was believed to originate from a single catastrophic event, but the molecular mechanisms leading to this event are yet to be uncovered. Because a thorough interpretation of the data are missing, the phenomenon itself has wrongly acquired the status of a mechanism used to justify many kinds of complex rearrangements. Although the assumption that all translocations in chromothripsis originate from a single event has met with criticism, satisfactory explanations for the intense but localized nature of this phenomenon are still missing. Here, we show why the data used to describe massive catastrophic rearrangements are incompatible with a model comprising a single event only and propose a molecular mechanism in which a combination of known cellular pathways accounts for chromothripsis. Instead of a single traumatic event, the protection of undamaged chromosomes by telomeres can limit repetitive breakage-fusion-bridge events to a single chromosome arm. Ultimately, common properties of chromosomal instability, such as aneuploidy and centromere fission, might establish the complex genetic pattern observed in this genomic state.     

A comparison of blood pressure measurement over a sleeved arm versus a bare arm

Background

The measurement of blood pressure is a common clinical exam with important health consequences. We sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken on a bare arm.

Methods

We recruited 376 patients between 18 and 85 years of age from a family medicine clinic between September 2004 and November 2006. They all had their blood pressure recorded using the same automatic oscillometric device, with the cuff placed over their bare arms for the first reading. Each patient was then randomly assigned to either the bare-arm group, for which the second blood pressure reading was also taken on a bare arm, or the sleeved-arm group, for which the second reading was taken with the cuff placed over the patient''s sleeve.

Results

The mean age of the 376 participants was 61.6 years (standard deviation 15.0), 61% of the participants were male, 41% had hypertension and 11.7% had diabetes. We found no clinically important differences between the bare-arm group (n = 180) and the sleeved-arm group (n = 196) in age, sex or body mass index. The mean differences between the first and second readings for patients in the bare-arm group were 4.1 mm Hg (95% confidence interval [CI] 2.8 to 5.5) for systolic blood pressure and 0.1 mm Hg (95% CI –0.7 to 0.9) for diastolic blood pressure. The mean differences between the first and second readings for patients in the sleeved-arm group were 3.4 mm Hg (95% CI 2.1 to 4.7) for systolic blood pressure and 0.4 mm Hg (95% CI –0.4 to 1.3) for diastolic blood pressure. The between-group differences in these values was 0.76 mm Hg (95% CI –1.13 to 2.65) for systolic and –0.31 mm Hg (95% CI –1.48 to 0.86) for diastolic blood pressure; neither of these differences was clinically important or statistically significant.

Interpretation

We found that there was no significant difference in blood pressure recorded over a sleeve or on a bare arm. For practical purposes, the decision to measure blood pressure on a bare arm or over a sleeved arm should be left to the judgment of the health care professional taking the blood pressure.The measurement of blood pressure is one of the most common examinations undertaken in family practice and has important health and management consequences for the patient. Accurate assessment of blood pressure, therefore, is very important. Current guidelines outline standards for obtaining accurate and reproducible blood pressure measurements.¹ These standards include guidelines for the sizes of blood pressure cuffs and the position of the patient''s arm, as well as a recommendation that the patient be seated for 5 minutes before his or her blood pressure is taken. In addition, it has been recommended that blood pressure be measured over the patient''s bare arm.¹ However, the current recommended method to determine blood pressure² has several limitations (e.g., high variability of blood pressure at different times of day or if measured only once, loss of proper technique post-training).Three previous studies have assessed whether blood pressure varies significantly when taken over a sleeved arm compared with a bare arm. Details of the literature review are described in Appendix 1, available online at www.cmaj.ca/cgi/content/full/178/5/585/DC2. In one study, involving 36 patients, Holleman et al³ found no significant differences in systolic or diastolic blood pressure readings taken over the sleeved or bare arm of each patient. However, this study was limited because of its small sample and because blood pressure measurements were taken on both arms simultaneously. A study by Kahan et al,⁴ involving 201 patients, compared blood pressure measurements taken over a sleeved arm, a bare arm and below a rolled-up sleeve. They found that the degree of clothing under the sphygmomanometer cuff did not have a clinically important effect on the reading. Although they found no significant difference in the effect of clothing on blood pressure readings, the study was limited by their design of measuring blood pressure below a rolled-up sleeve. The third study, conducted by Liebl et al,⁵ was published while our study was in progress. Their study, which involved 201 patients, compared blood pressure measurements taken over a sleeved arm and a bare arm with both sphygmomanometric and oscillometric devices. They concluded no significant difference in readings between the sleeved and bare-arm groups. In contrast to previous studies, we sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken over a bare arm through the use of a control group in which patients'' blood pressure was taken only over a bare arm.     

Lymphocytes apoptosis: young versus aged and humans versus rats

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.     

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.