The Circadian Energy Scale (CIRENS): Two Simple Questions for a Reliable Chronotype Measurement Based on Energy

This study presents the Circadian Energy Scale (CIRENS), a very short and simple chronotype measurement tool based on energy. The CIRENS consists of two introspective questions about the usual energy level (very low, low, moderate, high, or very high, scored 1 to 5) in the morning and in the evening. The difference between energy level scores (?4 to 4) felt by respondents in the evening and morning defines the chronotype score and classification. A concurrent validity analysis of the CIRENS with the widely used Horne and Östberg Morningness-Eveningness Questionnaire (MEQ) was conducted using a sample of 225 college students, and with MSFsc, a sleep-based chronotype assessment tool based on the Munich Chronotype Questionnaire (MCTQ), using a sample of 34,530 subjects (18–83 yrs, 27% males). This large sample was collected in a Web survey for behavioral correlates of the CIRENS with variables previously associated with chronotype differences. The correlation of the CIRENS chronotype score was r?=??.70 with the MEQ and r?=?.32 with the MSFsc. CIRENS chronotype scores declined with age and were not affected by sex. Both CIRENS and MSFsc chronotype scores were related to differences in tobacco, caffeine, and cola soft-drink consumption (all higher in evening types). The CIRENS provides a simple chronotype index and a measure of absolute energy throughout the day and seems to be a reliable chronotype assessment tool that may be useful both clinically and for large-scale studies. (Author correspondence: diogorlara@gmail.com)     

Gender differences in chronotype diminish with age: a meta-analysis based on morningness/chronotype questionnaires

Chronotype is an individual difference trait that is closely linked to biological variables. Usually, men and women differ in their chronotype with men being more evening oriented than women. The aim of the study was to assess gender differences in chronotype based on a systematic review and meta-analysis. In addition, many recent studies do not report gender differences any more although researchers collected data on men and women. This motivated us to contact many researchers directly to obtain and use these additional data. We included 164 studies giving 201 effect size measures in a data set, which only included the most commonly used questionnaires (MEQ, rMEQ and CSM) totaling 186,289 participants in total, of which 75,622 were men and 110,667 were women. We calculated an effect size of the standardized mean difference of ?0.072 (95% CI from ?0.062 to ?0.082) in the fixed effects and of ?0.066 (95% CI from ?0.043 to- 0.089) in the random effects model. Men were on average more evening oriented than women. Type of questionnaire did not influence the standardized mean difference in chronotype between genders. Mean age, standard deviation of age and publication year (p ≤ 0.05) were found to have significant effects on the standardized mean difference in chronotype between genders. The standardized mean difference in chronotype between genders is predicted to change from negative to positive with increasing mean age (young women were more morning orientated than young men, but older women were less morning orientated than older men). This suggests that the differences between men and women diminish with time. The standardized mean difference in chronotype between genders is decreasing with increasing publication year, as well as with high variation in age of the samples.

Abbreviations: CSM: Composite Scale of Morningness (Smith et al. 1989); MEQ: Morningness-Eveningness Questionnaire (Horne and Ostberg, 1976); rMEQ: reduced Morningness-Eveningness Questionnaire (Adan & Almiral, 1991); SMD: standardized mean difference (measure of effect size).     

A simple and reliable method for discriminating between Helicoverpa armigera and Helicoverpa assulta (Lepidoptera: Noctuidae)

Abstract The cotton bollworm Helicoverpa armigera and the oriental tobacco budworm H. assulta are sibling species, both being important agricultural pests. Morphologically, the two insects are almost indistinguishable at the egg, larval and pupal stages. One of the big challenges in the study of these insects, in particular in integrated pest management, is a timely and dependable identification of these insects at their early stages of development. Here, we report a H. armigera‐specific nuclear DNA marker, and demonstrate that it can be employed to reliably discriminate between H. armigera and H. assulta by simple polymerase chain reaction amplification experiment.     

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.     

A simple Na(2)SO(3) feeding method for K(L)a measurement in large-scale fermentors

A simple and convenient method for measuring K(L)a in large-scale fermentors was proposed. This method was based on the measurement of the dissolved oxygen concentration under steady state conditions established by an equivalency of the sulfite ion feed and chemical oxidation rates. This method had the following advantages: It was a steady state method, and so it was not necessary to consider the response lag of a dissolved oxygen probe and the response lag due to gas phase mixing in fermentors. The oxygen content of the effluent gas in this measuring system was nearly the same as that of the sparged air. Therefore, it was possible to use the oxygen partial pressure of the sparged air for the calculation of the driving force of oxygen transfer. The detailed information on the kinetics of sulfite oxidation was not necessary, because the dissolved oxygen concentration in steady state was not influenced by sulfite oxidation rates. The K(L)a measurement was finished in as short a period as 150 s, even in a fermentor with a volume of 10 m(3). Since the amount of Na(2)SO(4) accumulation in the test fermentors was very small because of the quick measurement, the K(L)a values obtained by this method were applicable to the electrolyte-free system. Furthermore, we could discharge the used liquid from the fermentors into a drain without any pretreatment due to the low salt concentration.     

Human sleep and circadian rhythms: a simple model based on two coupled oscillators

We propose a model of the human circadian system. The sleep-wake and body temperature rhythms are assumed to be driven by a pair of coupled nonlinear oscillators described by phase variables alone. The novel aspect of the model is that its equations may be solved analytically. Computer simulations are used to test the model against sleep-wake data pooled from 15 studies of subjects living for weeks in unscheduled, time-free environments. On these tests the model performs about as well as the existing models, although its mathematical structure is far simpler.Supported by NIGMS Grant No. 5-R01-GM-30719-03     

Circadian rhythms: a fine c(l)ocktail!

The central pacemaker of circadian behavior in mammals is located in the suprachiasmatic nuclei, each neuron of which has its own circadian rhythm. Recent studies shed light on the integration of these individual cellular rhythms and on novel genetic determinants that have been found to influence circadian behavior.     

Energy and ration requirements of juvenile Pacific halibut (Hippoglossus stenolepis) based on energy consumption and growth rates

Growth of captive juvenile Pacific halibut was linearly related to energy consumption (J g⁻¹ day⁻¹) at 4°C by the following equation: growth (% body weight (b.w.) day⁻¹)=0–007 (consumption J g⁻¹ day⁻¹)– 0.192; r² =0.81. Weight gain was independent of size for fish between 9 and 7000 g when growth was expressed as a function of consumption in J g⁻¹ day⁻¹. Maintenance ration determined in feeding–growth experiments averaged 27.4 J g⁻¹ day⁻¹ at 4–0°C. Small halibut ate significantly more food than large fish. Single meals following 2 day fasts averaged 4.1% b.w. for halibut under 100 g, 1.72% b.w. for 1.2 kg fish and 1.1% B.W. for 6.8 kg fish. Both large and small size categories of halibut tended to evacuate their meal in about 3 days even though small fish ate relatively larger meals. Minimum estimates for daily ration to achieve growth rates observed in the Gulf of Alaska were approximately 0.5 to 2.4% b.w. day⁻¹ depending on fish size and whether northern shrimp or yellowfin sole were their prey.     

A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity

PurposeCancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET.MethodsWe measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines.ResultsWe found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype.ConclusionIndirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.     

The Ecology of Scale: Assessment of Regional Energy Turnover and Comparison with Global Food (5 pp)

Goal, Scope and Background Obviously, people assume that the regional production and distribution of food requires less energy turnover, compared with global transports of food. Politicians claim the term “regionality”, maintaining that regionality is a medicine against wasting so much energy for the global food distribution. Additionally the energy turnover is causing pollution. But remarkably, there is a lack of empirical data to support this idea. At the same time, nobody really requires regional origin for non-food items, e.g. for bicycles, dishwashers, furniture or cars etc. The public mainstream asks for regional origin merely for food. Hence, the first scientific issue worked out in this paper is how the energy turnover of comparable food items can be measured, regarding the lifecycle of food in total. That means to investigate the partial systems of crop or breed, of food production, of packaging including transport and distribution up to the point of sale. Secondly, it has to be checked, if the assumed coincidence of low energy turnover and regional origin can be verified. The specific energy turnover, calculated in the unit [kWh per kg] or [kWh per l] of food, is investigated by comparing regional with global process chains for different food items. Two examples of food – fruit juices and lamb meat – are researched, by personal investigation worldwide. Firstly, fruit juices of high grade quality from Brazil, from European origin and from local German farmers are compared, in terms of energy. Secondly, a comparison of lamb meat from New Zealand and lamb meat from local German farmers is conducted. Lamb meat has been investigated, because it is shipped around the world as frozen natural food, not concentrated like juices. In addition, the business size of the food producers is researched for both examples. Methods As a part of LCA the energy turnover of each process step from the very beginning up to the point of sale is investigated. These primary results are the basic empirical data, in order to allocate the energy turnover at the food items as functional units. The results of regional, European-continental and of global process chains are compared. In addition, the issue is investigated, whether the specific energy turnover depends upon the business size. Results and Discussion Surprisingly, the data in both cases demonstrate a strong degressive relation of the specific energy turnover and the business size. Here it is not important, if the business is regional or not. Merely the efficiency and logistics of the production and the operations determine the specific energy turnover. These findings seem closely connected with the business size, because small companies are not able to invest in energy recovering and saving technology. The regional juices business is worsened by the huge number of smallsized transports of the crop and the nearly bottle-wise distribution. Regarding lamb meat we once more find the disadvantages of comparably small farms again. In addition, the German farmers need daily shepherds, fences by night, stables and usually additional feed during the wintertime. All these efforts are not necessary in New Zealand, where the climatic conditions and the open countryside with low population allow rather easy and low energy breeding of lamb. Conclusion The coincidence of economic and ecological facts is obvious. As a matter of fact, in economics there is a strong degressive relation between the production costs and the number of produced items. This relation is very well known as “Economy of Scale.” Our findings lead to similar conclusions, in terms of ecology. That means, the production ecology depends on the number of produced items. Additionally, our results demonstrate a minimum business size, so that we can claim an “Ecology of Scale” as well. However, on the other hand, the reported data and the conclusions are valid for the investigated food items – fruit juices and lamb meat – only. Nevertheless, one conclusion is already evident: The most popular claims for regional food production and distribution instead of global process chains are not generally valid. Small farmers basically need much more energy to produce and distribute their products, compared with bigger units. Both food items demonstrate clearly, that the ecological quality is mainly influenced by the operational efficiency and not by the marketing distance itself. Recommendation and Outlook Much more detailed data of all the investigated operational units, processing fruit juices and lamb meat, have been published (Fleissner 2001). As a further example, we investigate wine from different countries as a further example. Different from fruit juices, which can be shipped as a concentrate, and different from lamb meat, which is shipped as frozen food, wine of high grade quality is always bottled close to the place of origin. That means, that not only the food, but also the heavy weight packaging is transported around the world. So, we are very curious about the results, which we expect for 2004.     

New substrates for enkephalinase (neutral endopeptidase) based on fluorescence energy transfer

Novel fluorescent substrates for enkephalinase (neutral endopeptidase; EC 3.4.24.11) have been developed. These new assays are based on the disappearance of energy transfer between a tryptophan or a tyrosine residue and the 5-dimethylaminonaphthalene-1-sulfonyl group (dansyl) in the substrates dansyl-Gly-Trp-Gly or dansyl-Gly-Tyr-Gly upon hydrolysis of their Gly-Trp or Gly-Tyr amide bond by enkephalinase. No significant difference in Km or kcat values were found for dansyl-Gly-Trp-Gly and dansyl-Gly-Tyr-Gly, indicating that, in contrast to thermolysin, the active site of enkephalinase easily accommodates tryptophan residues. Both tryptophan and tyrosine-containing substrates can be used for continuous recording of enkephalinase activity and should prove useful for detailed study of the substrate specificity of this enzyme.     

Concanavalin A-perioxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB): a reliable method for dual staining of complex carbohydrates.

A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color alpha-D-glucosyl and alpha-D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.     

Photoperiodic control of diapause in Pseudopidorus fasciata (Lepidoptera: Zygaenidae) based on a qualitative time measurement

In the zygaenid moth, Pseudopidorus fasciata, both larval diapause induction and termination are under photoperiodic control. In this study, we investigated whether photoperiodic time measurement (with a 24-h light-dark cycle) in this moth is qualitative or quantitative. Photoperiodic response curves, at 22, 25, and 28 degrees C indicated that the incidence of diapause depended on whether the scotophases exceeded the critical night length (CNL) or not. All scotophases longer than the CNL-induced diapause; all scotophases shorter than the CNL-inhibited diapause. The CNL was 10.5h at 25 and 28 degrees C, and 10h at 22 degrees C. By transferring from various short photoperiods (LD 8:16, LD 9:15, LD 10:14, LD 11:13, LD 12:12, and LD 13:11) to a long photoperiod (LD 16:8) at different times, the number of light-dark cycles required for 50% diapause induction at 25 degrees C was 7.14 at LD 8:16, 7.2 at LD 9:15, 7.19 at LD 10:14, 7.16 at LD 11:13, and 7.13 at LD 12:12, without showing a significant difference between the treatments. Only at LD 13:11 (near the CNL), the number of light-dark cycles was significantly increased to 7.64. The intensity of diapause induced under different short photoperiods (LD 8:16, LD 9:15, LD 10:14, LD 11:13, and LD 12:12) at 25 degrees C was not significantly different with an average diapause duration of 36 days. The duration of diapause induced under LD 13:11 was significantly reduced to 32 days. All results indicate that the night-lengths are measured as either "long" or "short" compared with some critical value and suggest that photoperiodic time measurement for diapause induction in this moth is based on a qualitative principle.     

The measurement of adenylate energy charge (AEC) in mycobacteria, including Mycobacterium leprae

Abstract The adenylate energy charge (AEC) of Mycobacterium leprae, Mycobacterium lepraemurium and the cultivable Mycobacterium smegmatis were determined following incubation in a variety of culture conditions. The AEC values for M. smegmatis were similar to those reported for other cultivable bacteria. The AEC values for M. leprae and M. lepraemurium purified from host tissue were lower than those of in vitro-grown organisms. The possible use of the AEC in in vitro studies with M. leprae is discussed.     

Towards a reliable technology for antioxidant capacity and oxidative damage evaluation: electrochemical (bio)sensors

To counteract and prevent the deleterious effect of free radicals the living organisms have developed complex endogenous and exogenous antioxidant systems. Several analytical methodologies have been proposed in order to quantify antioxidants in food, beverages and biological fluids. This paper revises the electroanalytical approaches developed for the assessment of the total or individual antioxidant capacity. Four electrochemical sensing approaches have been identified, based on the direct electrochemical detection of antioxidant at bare or chemically modified electrodes, and using enzymatic and DNA-based biosensors.     

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.     

Near-infrared analysis of fennel (Foeniculum vulgare Miller) on different spectrometers--basic considerations for a reliable network

The aim of this study was to investigate the accuracy and transferability of near-infrared (NIR) calibrations for estimating the content and composition of the volatile fraction in fennel fruits (Foeniculum vulgare Miller) as an example of medicinal and spice plants. A master calibration with spectra obtained on a scanning monochromator was generated using 345 samples from three different harvests (1997-1999). A subset of 70 samples from 1999 was also measured on a dispersive grating and a scanning diode array system to gain an insight into the influence of sample presentation and scanning techniques. For all instruments, calibrations with standard errors in the range of the reference method were achieved. Furthermore the influence of storage on NIR spectra and, additionally, the potential of transferring spectra between both scanning monochromators was studied.     

A sensitive and reliable quantification method for mouse interleukin-12 p70 based on fluorometric sandwich ELISA (FS-ELISA)

Interleukin-12 (IL-12) p70 is an important cytokine secreted by antigen-presenting cells in response to antigenic stimulation; it is a heterodimer of p35 and p40 subunits. Here, we report a new, highly sensitive, and reliable method that employs fluorometric sandwich ELISA for quantification of the mouse IL-12 (moIL-12) p70 protein. Our method could quantify moIL-12 p70 in the range of approximately 0.5 to 500 pg/ml. In the assay, no signals were produced by the moIL-12 p40 monomer, homodimer [(p40)2], or mouse IL-23 even up to a concentration of 500 pg/ml; this ensures that our assay has a high specificity for moIL-12 p70. To demonstrate that our method can determine natural moIL-12 in real physiological/pathological samples, we monitored the moIL-12 p70 secretion from peritoneal exudative cells after lipopolysaccharide (LPS) stimulation. IL-12 p70 secretion as early as 3h after LPS stimulation was reliably detected due to the high sensitivity of the method.