Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate- zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.      

A methodological study for the quantification and the control of the physico-chemical processes occurring during cell fixation

Summary Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation the fixative is always offered (1) in excess of the cell sample, and the process of fixation is influenced by (2) chemical impurities of the fixative fluid.Both factors influence the succeeding dyeing of cells. In order to avoid these uncontrolled criteria, a new technology for controlled cell fixation has been developed, whereby freshly prepared formaldehyde and methanol gas in an inert gas-flow of helium was applied to thin membranes by aid of a capillary flow-in technique.The instrumental equipment consists of (1) an ultra-high vacuum flowapparatus with a total-pressure measuring unit, (2) a gas-supply device, (3) a mass spectrometer including a pump system, and (4) a Teflon and/or glass-gas chamber for the treatment of synthetic (Hostaphan foils) or biological membranes (mesenterium) with formaldehyde as the fixative gas.The amount of offered, adsorbed, absorbed, diffused, and desorbed fixative gas could be absolutely estimated after the saturation of the membranes with an on-line operating inert mass spectrometer of the Omegatron type.The gas treatment of the Hostaphan foils with formaldehyde showed that nearly all adsorbed gas molecules could be desorbed. In contrast to native membranes the greatest proportion of the gas molecules adhered to the biological surface, and only a small quantity were desorbable. Physisorption or physisorption and chemisorption occured depending on the adsorber surface property.A monolayer of formaldehyde of 5·10¹⁴ to 1·10¹⁵ molecules per 10¹⁶ Ų surface area can be postulated on the basis of these preliminary results. This value corresponds to a mass of about 5·10^–8g CH₂O. It resulted in an area-coverage ratio of CH₂O molecules per cell of 10⁹:1.The membrane surface facing the gas side always amounted to 1 cm². A fixative gas concentration of 10⁶ molecules/cm³, and therefore a degree of coverage of <1/1000 monolayer can be estimated absolutely. For a precise determination of the degree of fixation, further experiments and the evaluation of additional physico-chemical parameters are necessary.     

A methodological study for the quantification and the control of the physico-chemical processes occurring during cell fixation. I. The development of a new technology.

Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation the fixative is always "offered" (1) in excess of the cell sample, and the process of fixation is influenced by (2) chemical impurities of the fixative fluid. Both factors influence the succeeding dyeing of cells. In order to avoid these uncontrolled criteria, a new technology for controlled cell fixation has been developed, whereby freshly prepared formaldehyde and methanol gas in an "inert" gas-flow of helium was applied to thin membranes by aid of a capillary flow-in technique. The instrumental equipment consists of (1) an ultra-high vacuum flow-apparatus with a total-pressure measuring unit, (2) a gas-supply device, (3) a mass spectrometer including a pump system, and (4) a Teflon and/or glass-gas chamber for the treatment of synthetic (Hostaphan foils) or biological membranes (mesenterium) with formaldehyde as the fixative gas. The amount of "offered", adsorbed, absorbed, diffused, and desorbed fixative gas could be absolutely estimated after the saturation of the membranes with an "on-line" operating "inert" mass spectrometer of the Omegatron type. The gas treatment of the Hostaphan foils with formaldehyde showed that nearly all adsorbed gas molecules could be desorbed. In contrast to native membranes the greatest proportion of the gas molecules adhered to the biological surface, and only a small quantity were desorbable. Physisorption or physisorption and chemisorption occured depending on the adsorber surface property. A monolayer of formaldehyde of 5.10(14) to 1.10(15) molecules per 10(16) A2 surface area can be postulated on the basis of these preliminary results. This value corresponds to a mass of about 5.10(-8) g CH2O. It resulted in an area-coverage ratio of CH2O molecules per cell of 10(9):1. The membrane surface facing the gas side always amounted to 1 cm2. A fixative gas concentration of 10(6) molecules/cm3, and therefore a degree of coverage of less than 1/1000 monolayer can be estimated absolutely. For a precise determination of the degree of fixation, further experiments and the evaluation of additional physico-chemical parameters are necessary.     

An influence of hormones on hoarding behavior in the Mongolian gerbil (Meriones unguiculatus)

Three experiments on food hoarding behavior in the Mongolian gerbil (Meriones unguiculatus) demonstrated (1) that females hoarded more than males, (2) that castration increased food hoarding in males, and (3) that administration of testosterone propionate to castrate males inhibited hoarding. The adaptive value of these relationships and their implications for a physiological model of food hoarding in the rodent are discussed.     

Secular trends in acute dialysis after elective major surgery �� 1995 to 2009

Background:

Acute kidney injury is a serious complication of elective major surgery. Acute dialysis is used to support life in the most severe cases. We examined whether rates and outcomes of acute dialysis after elective major surgery have changed over time.

Methods:

We used data from Ontario’s universal health care databases to study all consecutive patients who had elective major surgery at 118 hospitals between 1995 and 2009. Our primary outcomes were acute dialysis within 14 days of surgery, death within 90 days of surgery and chronic dialysis for patients who did not recover kidney function.

Results:

A total of 552 672 patients underwent elective major surgery during the study period, 2231 of whom received acute dialysis. The incidence of acute dialysis increased steadily from 0.2% in 1995 (95% confidence interval [CI] 0.15–0.2) to 0.6% in 2009 (95% CI 0.6–0.7). This increase was primarily in cardiac and vascular surgeries. Among patients who received acute dialysis, 937 died within 90 days of surgery (42.0%, 95% CI 40.0–44.1), with no change in 90-day survival over time. Among the 1294 patients who received acute dialysis and survived beyond 90 days, 352 required chronic dialysis (27.2%, 95% CI 24.8–29.7), with no change over time.

Interpretation:

The use of acute dialysis after cardiac and vascular surgery has increased substantially since 1995. Studies focusing on interventions to better prevent and treat perioperative acute kidney injury are needed.More than 230 million elective major surgeries are done annually worldwide.¹ Acute kidney injury is a serious complication of major surgery. It represents a sudden loss of kidney function that affects morbidity, mortality and health care costs.² Dialysis is used for the most severe forms of acute kidney injury. In the nonsurgical setting, the incidence of acute dialysis has steadily increased over the last 15 years, and patients are now more likely to survive to discharge from hospital.^3–5 Similarly, in the surgical setting, the incidence of acute dialysis appears to be increasing over time,^6–10 with declining inhospital mortality.^8,10,11Although previous studies have improved our understanding of the epidemiology of acute dialysis in the surgical setting, several questions remain. Many previous studies were conducted at a single centre, thereby limiting their generalizability.^6,12–14 Most multicentre studies were conducted in the nonsurgical setting and used diagnostic codes for acute kidney injury not requiring dialysis; however, these codes can be inaccurate.^15,16 In contrast, a procedure such as dialysis is easily determined. The incidence of acute dialysis after elective surgery is of particular interest given the need for surgical consent, the severe nature of the event and the potential for mitigation. The need for chronic dialysis among patients who do not recover renal function after surgery has been poorly studied, yet this condition has a major affect on patient survival and quality of life.¹⁷ For these reasons, we studied secular trends in acute dialysis after elective major surgery, focusing on incidence, 90-day mortality and need for chronic dialysis.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.