Murine Spinal Cord Transcriptome Analysis Following Reduction of Prevalent Myelin cDNA Sequences

From 1,000 randomly selected colonies from cDNA libraries derived from murine spinal cord subtracted against white matter by means of suppression subtractive hybridization, 220 clones were identified as differentially expressed by dot blot analysis. Sequence analysis by the BLAST programming identified 140 unique genes. (1) The percentage of known sequences from myelin and other glial sources was reduced by approximately 75% over previous, similar subtractions employing visual cortex as the driver. (2) Differentially expressed genes tended to reflect existing expectations concerning structure and function of the spinal cord. (3) About 35% of all genes differentially expressed in the spinal cord in this study are also known to be differentially expressed for this structure as tabulated in the UniGene database. (4) About 33% of all genes differentially expressed in the present study are recorded as not present when measured in the spinal cord according to the UniGene database indicating that present techniques are not recording about a third of differentially expressed genes in this structure. (5) About 15% of all differentially expressed genes are for unknown, putative or hypothetical protein products. (6) About 4% of all differentially expressed genes are novel expressed sequence tags for the mouse. The current study demonstrates the importance of reducing the presence of glial associated sequences when comparing brain regions. It is concluded that the persistence of some myelin sequences in the spinal cord when white matter is used as the driver indicates that myelination is more active in this structure than for those areas represented by white matter and corpus callosum.     

ICAM-1–mediated Endothelial Nitric Oxide Synthase Activation via Calcium and AMP-activated Protein Kinase Is Required for Transendothelial Lymphocyte Migration

As a gatekeeper of leukocyte trafficking the vasculature fulfills an essential immune function. We have recently shown that paracellular transendothelial lymphocyte migration is controlled by intercellular adhesion molecule 1 (ICAM-1)-mediated vascular endothelial cadherin (VEC) phosphorylation [Turowski et al., J. Cell Sci. 121, 29–37 (2008)]. Here we show that endothelial nitric oxide synthase (eNOS) is a critical regulator of this pathway. ICAM-1 stimulated eNOS by a mechanism that was clearly distinct from that utilized by insulin. In particular, phosphorylation of eNOS on S1177 in response to ICAM-1 activation was regulated by src family protein kinase, rho GTPase, Ca²⁺, CaMKK, and AMPK, but not Akt/PI3K. Functional neutralization of any component of this pathway or its downstream effector guanylyl cyclase significantly reduced lymphocyte diapedesis across the endothelial monolayer. In turn, activation of NO signaling promoted lymphocyte transmigration. The eNOS signaling pathway was required for T-cell transmigration across primary rat and human microvascular endothelial cells and also when shear flow was applied, suggesting that this pathway is ubiquitously used. These data reveal a novel and essential role of eNOS in basic immune function and provide a key link in the molecular network governing endothelial cell compliance to diapedesis.     

Testing Miller's theory of alleles preventing androgenization as an evolutionary explanation for the genetic predisposition for male homosexuality

The genetic background of male homosexuality presents an evolutionary paradox, since homosexuality could be considered a reproductive disadvantage. We tested E.M. Miller's (2000) balanced polymorphism explanation, which states that alleles partially preventing androgenization in male fetuses during pregnancy would be associated with a homosexual orientation. Having all the alleles produces homosexuality, while heterosexual carriers with only a few of these alleles instead have a reproductive advantage; that is, they have more traits, which, by controlling for excessive aggressiveness and psychopathy, make them more attractive mates. Pairs of brothers were used to test these assumptions. If homosexuality is due to having all the androgenization-preventing alleles, then heterosexual men with homosexual brothers are more likely to also have some of the these alleles compared to heterosexual men with heterosexual brothers. These two categories were compared on variables related both directly and indirectly to reproductive success, with heterosexual men with a homosexual brother hypothesized to have an advantage on the variables. However, no statistically significant findings in support of the theory were detected. The results were discussed together with alternative explanations.     

Lifestyle impacts on the aging‐associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

Cellular aging is characterized by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and activation of DNA damage responses. There is some evidence that DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage, and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging‐associated expression of serum markers of DNA damage (CRAMP, EF‐1α, stathmin, n‐acetyl‐glucosaminidase and chitinase) in comparison with other described markers of cellular aging (p16^INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age‐independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age‐independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16^INK4a expression and negatively with telomere length in peripheral blood T‐lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging.     

Divergent patterns of recent retroviral integrations in the human and chimpanzee genomes: probable transmissions between other primates and chimpanzees

The human genome is littered by endogenous retrovirus sequences (HERVs), which constitute up to 8% of the total genomic sequence. The sequencing of the human (Homo sapiens) and chimpanzee (Pan troglodytes) genomes has facilitated the evolutionary study of ERVs and related sequences. We screened both the human genome (version hg16) and the chimpanzee genome (version PanTro1) for ERVs and conducted a phylogenetic analysis of recent integrations. We found a number of recent integrations within both genomes. They segregated into four groups. Two larger gammaretrovirus-like groups (PtG1 and PtG2) occurred in chimpanzees but not in humans. The PtG sequences were most similar to two baboon ERVs and a macaque sequence but neither to other chimpanzee ERVs nor to any human gammaretrovirus-like ERVs. The pattern was consistent with cross-species transfer via predation. This appears to be an example of horizontal transfer of retroviruses with occasional fixation in the germ line.     

Compensation for unfavorable characteristics of irregular individual shift rotas

Some employees of TV companies, such as those who produce remote TV programs, have to cope with very irregular rotas and many short-term schedule deviations. Many of these employees complain about the negative effects of such on their wellbeing and private life. Therefore, a working group of employers, council representatives, and researchers developed a so-called bonus system. Based on the criteria of the BESIAK system, the following list of criteria for the ergonomic assessment of irregular shift systems was developed: proportion of night hours worked between 22:00 and 01:00 h and between 06:00 and 07:00 h, proportion of night hours worked between 01:00 and 06:00 h, number of successive night shifts, number of successive working days, number of shifts longer than 9 h, proportion of phase advances, off hours on weekends, work hours between 17:00 and 23:00 h from Monday to Friday, number of working days with leisure time at remote places, and sudden deviations from the planned shift rota. Each individual rota was evaluated in retrospect. If pre-defined thresholds of criteria were surpassed, bonus points were added to the worker's account. In general, more bonus points add up to more free time. Only in particular cases was monetary compensation possible for some criteria. The bonus point system, which was implemented in the year 2002 for about 850 employees of the TV company, has the advantages of more transparency concerning the unfavorable characteristics of working-time arrangements, incentive for superiors to design “good” rosters that avoid the bonus point thresholds (to reduce costs), positive short-term effects on the employee social life, and expected positive long-term effects on the employee health. In general, the most promising approach to cope with the problems of shift workers in irregular and flexible shift systems seems to be to increase their influence on the arrangement of working times. If this is not possible, bonus point systems may help to achieve greater transparency and fairness in the distribution of unfavorable working-time arrangements within a team, and even reduce the unnecessary unfavorable aspects of shift systems.     

In silico simulations suggest that Th-cell development is regulated by both selective and instructive mechanisms

Th-cell differentiation is highly influenced by the local cytokine environment. Although cytokines such as IL-12 and IL-4 are known to polarize the Th-cell response towards Th1 or Th2, respectively, it is not known whether these cytokines instruct the developmental fate of uncommitted Th cells or select cells that have already been committed through a stochastic process. We present an individual based model that accommodates both stochastic and deterministic processes to simulate the dynamic behaviour of selective versus instructive Th-cell development. The predictions made by each model show distinct behaviours, which are compared with experimental observations. The simulations show that the instructive model generates an exclusive Th1 or Th2 response in the absence of an external cytokine source, whereas the selective model favours coexistence of the phenotypes. A hybrid model, including both instructive and selective development, shows behaviour similar to either the selective or the instructive model dependent on the strength of activation. The hybrid model shows the closest qualitative agreement with a number of well-established experimental observations. The predictions by each model suggest that neither pure selective nor instructive Th development is likely to be functional as exclusive mechanisms in Th1/Th2 development.     

Rational design and in vitro and in vivo delivery of Dicer substrate siRNA

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.     

Mechanical stress,fracture risk and beak evolution in Darwin's ground finches (Geospiza)

Darwin''s finches have radiated from a common ancestor into 14 descendent species, each specializing on distinct food resources and evolving divergent beak forms. Beak morphology in the ground finches (Geospiza) has been shown to evolve via natural selection in response to variation in food type, food availability and interspecific competition for food. From a mechanical perspective, however, beak size and shape are only indirectly related to birds'' abilities to crack seeds, and beak form is hypothesized to evolve mainly under selection for fracture avoidance. Here, we test the fracture-avoidance hypothesis using finite-element modelling. We find that across species, mechanical loading is similar and approaches reported values of bone strength, thus suggesting pervasive selection on fracture avoidance. Additionally, deep and wide beaks are better suited for dissipating stress than are more elongate beaks when scaled to common sizes and loadings. Our results illustrate that deep and wide beaks in ground finches enable reduction of areas with high stress and peak stress magnitudes, allowing birds to crack hard seeds while limiting the risk of beak failure. These results may explain strong selection on beak depth and width in natural populations of Darwin''s finches.