Reproducibility of fluctuating asymmetry measurements in plants: Sources of variation and implications for study design

Fluctuating asymmetry (FA), i.e. small, non-directional deviations from perfect symmetry in morphological characters, increases under genetic and/or environmental stress. Ecological and evolutionary studies addressing FA became popular in past decades; however, their outcomes remain controversial. The discrepancies might be at least partly explained by inconsistent and non-standardised methodology. Our aim was to improve the methodology of these studies by identifying factors that affect the reproducibility of FA measurements in plant leaves. Six observers used a highly standardised measurement protocol to measure FA using the width, area and weight of the same set of leaves of 10 plant species that differed in leaf size, shape of the leaf margin and other leaf traits. On average, 24% of the total variation in the data was due to measurement error. Reproducibility of measurements varied with the shape of leaf margin, leaf size, the measured character and the experience of the observer. The lowest reproducibility of the width of leaf halves was found for simple leaves with serrate margins and the highest for simple leaves with entire margins and for compound pinnate leaves. The reproducibility was significantly lower for the weight of leaf halves than for either their width or area, especially for plants with small leaves. The reproducibility was also lower for measurements made by experienced observers than by naïve observers. The size of press-dried leaves decreased slightly but significantly relative to fresh leaves, but the FA of press-dried leaves adequately reflected the FA of fresh leaves. In contrast, preservation in 60% ethanol did not affect leaf size, but it decreased the width-based values of FA to 89.3% of the values measured from fresh leaves. We suggest that although reproducibility of leaf FA measurements depends upon many factors, the shape of the leaf margin is the most important source of variation. We recommend, whenever possible, choosing large-leaved plants with entire leaf margins as model objects for studies involving measurements of FA using the width of leaf halves. These measurements should be conducted with high accuracy from images of fresh or press-dried leaves.     

Mechanisms underlying the age-related decrement in the human sweating response

To examine the mechanisms underlying the age-related decrement in the ability to sweat, seven older (64-76 years) and seven younger (20-24 years) men participated in a 60-min sweating test. The test consisted of placing the subject's lower legs in a water bath at 42 degrees C while sitting in a controlled environment of 35 degrees C ambient temperature and 45% relative humidity. The rectal (Trc) and skin temperatures, local sweating rates (m(sw): on the forehead, chest, back, forearm and thigh) and the frequency of sweat expulsion (f(sw)) were measured during the test. No group difference was observed in the mean body temperature (Tb) throughout the passive heating, although the older men had a higher Tre and a lower mean skin temperature during the last half of the 60-min test. There were no group differences in the Tb threshold for sweating, although the time to the onset of sweating tended to be longer for the older men regardless of body site. The m(sw) increased gradually for approximately 35 min after the start of heat exposure in the older men and for 30 min in the younger men and then reached a steady state. During the first half of the test, the older men had a significantly lower m(sw) at all sites. During the last half of the test, only m(sw) on the thigh was significantly lower in the older men than in the younger men. There was no group difference in the slope of f(sw) versus Tb (an indicator of the change in the central sudomotor response to thermal input). The slope of m(sw) versus f(sw) (an indicator of the change in peripheral activity in response to central sudomotor changes) was significantly lower on the thigh in the older men, but there were no differences for the other sites. These results suggest that in older men the lower thigh m(sw) observed during the last half of the heat test was possibly due to age-related modifications of peripheral mechanisms involving the sweat glands and surrounding tissues. It was not due to a change in the central drive to sudomotor function. Furthermore, the sluggish m(sw) responses in the older men appear to have been related to age-related modifications of the sensitivity of thermoreceptors in various body regions to thermal stimuli. They may also involve lower sweat glands' sensitivity to cholinergic stimulus or sluggish vasodilatation, and do not reflect age-related changes in the central drive.     

Some implications of chaos theory for the genetic analysis of human development and variation.

Non-linear epigenetic processes are a potential underlying source of phenotypic differences in development. Simulation studies of twin pairs using simple non-linear development models characterised by chaotic or near-chaotic behavior are presented. The effect of chaotic processes on correlations is to lower them from their initial values, but high initial correlations are affected much less by chaotic and near-chaotic processes than intermediate correlations. Therefore, we would predict that traits affected by chaotic processes would have high MZ and low DZ twin correlations and this is reminiscent of certain traits such as EEG spectra. However the much more frequent observation of MZ correlations close to twice their DZ counterparts would suggest that the role of chaos in development is quite limited.     

Structural analysis of heme proteins: implications for design and prediction

Background  

Heme is an essential molecule and plays vital roles in many biological processes. The structural determination of a large number of heme proteins has made it possible to study the detailed chemical and structural properties of heme binding environment. Knowledge of these characteristics can provide valuable guidelines in the design of novel heme proteins and help us predict unknown heme binding proteins.     

Animal temperament and human disturbance: implications for the response of wildlife to tourism

Studies on the response of wildlife to human disturbance generally focus on demographic changes or on physiological and behavioural modifications directly related to stress response. Yet fewer studies have explored whether the distribution of individual animals in response to human disturbance is influenced by temperament. Temperament represents the consistency of responses of individuals in reaction to novel or challenging situations. Individuals are thus assumed to express highly consistent behaviour-hormonal response under specific stress conditions. In this study, we investigate the relations between exploration, grooming-scanning continuum, emotionality, and docility of individual Eastern chipmunks (Tamias striatus) and location of their burrow respective to frequentation by humans. We then assess the relationship between cortisol accumulated in the hair and both temperament and frequentation by humans. Explorative or docile chipmunks were more common in frequented areas. Hair cortisol increased with docility, but was not related to human frequentation. These results indicate that temperament may cause animals to distribute themselves in a non-random way in response to human disturbance. Integrating temperament into studies of the stress response of wildlife to humans could therefore help us understand the impact of tourism on wildlife.     

Environmental exposure and mitochondrial epigenetics: study design and analytical challenges

The environment can influence human health and disease in many harmful ways. Many epidemiological studies have been conducted with the aim of elucidating the association between environmental exposure and human disease at the molecular and pathological levels, and such associations can often be through induced epigenetic changes. One such mechanism for this is through environmental factors increasing oxidative stress in the cell, and this stress can subsequently lead to alterations in DNA molecules. The two cellular organelles that contain DNA are the nucleus and mitochondria, and the latter are particularly sensitive to oxidative stress, with mitochondrial functions often disrupted by increased stress. There has been a substantial increase over the past decade in the number of epigenetic studies investigating the impact of environmental exposures upon genomic DNA, but to date there has been insufficient attention paid to the impact upon mitochondrial epigenetics in studying human disease with exposure to environment. Here, in this review, we will discuss mitochondrial epigenetics with regard to epidemiological studies, with particular consideration given to study design and analytical challenges. Furthermore, we suggest future directions and perspectives in the field of mitochondrial epigenetic epidemiological studies.     

Biological implications of isochore boundaries in the human genome

The human genome is composed of large sequence segments with fairly homogeneous GC content, namely isochores, which have been linked to many important functions; biological implications of most isochore boundaries, however, remain elusive, partly due to the difficulty in determining these boundaries at high resolution. Using the segmentation algorithm based on the quadratic divergence, we re-determined all 79 boundaries of previously identified human isochores at single-nucleotide resolution, and then compared the boundary coordinates with other genome features. We found that 55.7% of isochore boundaries coincide with termini of repeat elements; 45.6% of isochore boundaries coincide with termini of highly conserved sequences based on alignment of 17 vertebrate genomes, i.e., the highly conserved genome sequence switches to a less or non-conserved one at the isochore boundary; some isochore boundaries coincide with abrupt change of CpG island distribution (note that one boundary can associate with more than one genome feature). In addition, sequences around isochore boundaries are highly conserved. It seems reasonable to deduce that the boundaries of all the isochores studied here would be replication timing sites in the human genome. These results suggest possible key roles of the isochore boundaries and may further our understanding of the human genome organization.     

Homotypic variation of canine flexor tendons: implications for the design of experimental studies in animal models

Water, collagen and glycosamimoglycan contents, cross-sectional area, stiffness and elastic modulus were carefully quantitated in flexor digitorum superficialis tendons from mature canines. From these data the within- and between-animal variability was estimated and used to demonstrate sample size calculations for both two-group and paired (within-animal) study designs. The estimated between-dog variance was typically 50% or less of the total variance for the parameters investigated. In other words, the correlation among the tendons within an animal for most measures was not strong. Therefore, for some variables (e.g., elastic modulus) in this animal and tendon model, there is no appreciable gain in statistical power by using a paired study design. A two-group design could be used, but any within-animal correlation must be accounted for in the analysis. For other variables such as collagen content, a paired design would gain substantial power.     

Characterization of a human peptide deformylase: implications for antibacterial drug design

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.     

Biological rhythm experimentation: a longitudinal design and analysis.

Certain experiments exploring the relationship between biological rhythms and external factors such as pharmacologic agents are typified by the particular importance of interaction and conditional effect parameters. The category of biological rhythm experiments presented here also necessarily involves a deliberate confounding of periodicity rooms and biological rhythm stages (time points). These features, together with the longitudinal nature of the observations, require special precautions in the experimental design and analysis.     

Genotypic variation in plant response to low boron and implications for plant breeding

Plant response to low B in the soil varies widely among species, and among genotypes within a species. Boron efficient genotypes are those that are able to grow well in soils in which other genotypes are adversely affected by B deficiency. This review considers the extent of variation in B efficiency in plant species and genotypes, the physiological nature of the efficiency mechanisms, what is known of the genetic basis for inheritance, screening techniques and the practical implications of the genotypic variations.Frequently, B efficiency is the sole reason for a difference between an average yield and complete crop failure. Severe yield losses can be effectively prevented by the inclusion of B efficiency as a selection criterion in crop breeding and improvement programmes for regions with low B soils. In addition, the expression of B deficiency primarily through male sterility, which is common in many species, creates opportunities for outcrossing in normally self-fertilised species. This, in turn, leads to two possibilities. Firstly, self fertilisation, and therefore maintenance of pure lines, cannot always be assumed in self pollinated species where B efficient and inefficient genotypes are grown side by side on low B soils. Secondly, B deficiency, in soil or artificial media, may be used as a fertility selective medium in which the male sterile B inefficient genotypes and the male fertile B efficient genotypes could hybridise naturally. This would be useful as a simple and economical method for creating heterozygous populations in breeding programmes as well as for producing hybrid seeds. Now that the roles of B in plant growth and development are beginning to be clarified, the efficiency mechanisms as well as the governing genetics can be explained. Practical benefits from the genetic diversity of B efficiency will be enhanced by a better understanding of B efficiency mechanisms and the molecular bases for their genetic control.     

Conditional analysis of mixed Poisson processes with baseline counts: implications for trial design and analysis

The design of clinical trials is typically based on marginal comparisons of a primary response under two or more treatments. The considerable gains in efficiency afforded by models conditional on one or more baseline responses has been extensively studied for Gaussian models. The purpose of this article is to present methods for the design and analysis of clinical trials in which the response is a count or a point process, and a corresponding baseline count is available prior to randomization. The methods are based on a conditional negative binomial model for the response given the baseline count and can be used to examine the effect of introducing selection criteria on power and sample size requirements. We show that designs based on this approach are more efficient than those proposed by McMahon et al. (1994).     

Interpopulation variation in life-history traits of Poeciliopsis prolifica: implications for the study of placental evolution

Placental reproduction is widespread across vertebrate taxa, but little is known about its life-history correlates and putative adaptive value. We studied variation in life-history traits in two populations of the placental poeciliid fish Poeciliopsis prolifica to determine whether differences in post-fertilization maternal provisioning to embryos have a genetic basis and how food availability affects reproduction. Life histories were characterized for wild-caught females and for second-generation lab-born females raised under two levels of food availability. We found that the two populations did not differ significantly in the wild for any life-history traits except for the lipid dry weight in females and in embryos at an advanced stage of development. When environmental effects were experimentally controlled, however, populations exhibited significant differences in several traits, including the degree of maternal provisioning to embryos. Food availability significantly affected female size at first parturition, brood size and offspring dry weight at birth. Altogether, these results demonstrate that population differences in maternal provisioning and other life-history traits have a genetic basis and show a plastic response to food availability. We infer that phenotypic plasticity may mask population differences in the field. In addition, when comparing life-history patterns in these two populations with known patterns in placental and non-placental poeciliids, our results support the hypotheses that placentation is an adaptive reproductive strategy under high-resource conditions but that it may represent a cost under low-food conditions. Finally, our results highlight that age at maturity and reproductive allotment may be key life-history traits accompanying placental evolution.     

Tawny Owl Strix aluco response to call-broadcasting and implications for survey design

Capsule: The use of call-broadcasting significantly increases the number of Tawny Owls Strix aluco detected in winter point counts, but requires careful survey design to avoid introducing potential sources of bias into population estimates.

Aims: To examine Tawny Owl response to call-broadcasting to aid survey design in national monitoring efforts.

Methods: A nocturnal survey was undertaken at 36 survey points over three nights in winter in Thetford Forest, England. Each survey consisted of four consecutive five-minute segments: a passive count, followed by three counts with the use of call-broadcasting.

Results: Few (4%) Tawny Owls were recorded during passive surveys, whereas the greatest response was during the first and second call-broadcast segments (49% and 36%, respectively). New detections declined to 11% in the final segment. Response was fastest at dusk, although time of night did not significantly affect the number of individuals detected. Male owls accounted for 79% of detections.

Conclusion: Our results show that ten minutes of call-broadcast surveying will detect 85% of responsive Tawny Owls, thus vastly improving detection compared to passive listening alone. However, simultaneous counts of geographically separated detections should be used to provide a minimum count and reduce potential double-counting of mobile individuals.     

The human ankle during walking: implications for design of biomimetic ankle prostheses

The non-disabled human ankle joint was examined during walking in an attempt to determine overall system characteristics for use in the design of ankle prostheses. The hypothesis of the study was that the quasi-stiffness of the ankle changes when walking at different walking speeds. The hypothesis was examined using sagittal plane ankle moment versus ankle angle curves from 24 able-bodied subjects walking over a range of speeds. The slopes of the moment versus ankle angle curves (quasi-stiffness) during loading appeared to change as speed was increased and the relationship between the moment and angle during loading became increasingly non-linear. The loading and unloading portions of the moment versus angle curves showed clockwise loops (hysteresis) at self-selected slow speeds that reduced essentially to zero as the speed increased to self-selected normal speeds. Above self-selected normal speeds, the loops started to traverse a counter-clockwise path that increased in area as the speed was increased. These characteristics imply that the human ankle joint could be effectively replaced with a rotational spring and damper for slow to normal walking speeds. However, to mimic the characteristics of the human ankle during walking at fast speeds, an augmented system would be necessary. This notion is supported by the sign of the ankle power at the time of opposite heel contact, which was negative for slow speeds, was near zero at normal speeds, and was positive for fast walking speeds.     

Control of sweating during the human menstrual cycle

Thermoregulatory responses were studied in seven women during two separate experimental protocols in the follicular (F, days 4-7) phase and during the luteal (L, days 19-22) phase of the menstrual cycle. Continuous measurements of esophageal temperature (Tes), mean skin temperature (Tsk), oxygen uptake and forearm sweating (ms) were made during all experiments. Protocol I involved both passive heat exposure (3 h) and cycle exercise at approximately 80% VO2 peak during which the environmental chamber was controlled at Ta = 50.0 degrees C, rh = 14% (Pw = 1.7 kPa). In protocol II subjects were tested during thirty-five minutes of exercise at approximately 85% VO2 peak at Ta = 35 degrees C and rh = 25% (Pw = 1.4 kPa). The normal L increase in resting Tes (approximately 0.3 degrees C) occurred in all seven subjects. Tsk was higher during L than F in all experiments conducted at 50 degrees C. During exercise and passive heat exposure, the Tes threshold for sweating was higher in L, with no change in the thermosensitivity (slope) of ms to Tes between menstrual cycle phases. This rightward or upward shift in Tes threshold for initiation of sweating averaged 0.5 degrees C for all experiments. The data indicate the luteal phase modulation in the control of sweating in healthy women is also apparent during severe exercise and/or heat stress.