The role of the ecological scaffold in the origin and maintenance of whole-group trait altruism in microbial populations.

BACKGROUND: Kin and multilevel selection provide explanations for the existence of altruism based on traits or processes that enhance the inclusive fitness of an altruist individual. Kin selection is often based on individual-level traits, such as the ability to recognize other altruists, whereas multilevel selection requires a metapopulation structure and dispersal process. These theories are unified by the general principle that altruism can be fixed by positive selection provided the benefit of altruism is preferentially conferred to other altruists. Here we take a different explanatory approach based on the recently proposed concept of an "ecological scaffold". We demonstrate that ecological conditions consisting of a patchy nutrient supply that generates a metapopulation structure, episodic mixing of groups, and severe nutrient limitation, can support or "scaffold" the evolution of altruism in a population of microbes by amplifying drift. This contrasts with recent papers in which the ecological scaffold was shown to support selective processes and demonstrates the power of scaffolding even in the absence of selection. RESULTS: Using computer simulations motivated by a simple theoretical model, we show that, although an altruistic mutant can be fixed within a single population of non-altruists by drift when nutrients are severely limited, the resulting altruistic population remains vulnerable to non-altruistic mutants. We then show how the imposition of the "ecological scaffold" onto a population of non-altruists alters the balance between selection and drift in a way that supports the fixation and subsequent persistence of altruism despite the possibility of invasion by non-altruists. CONCLUSIONS: The fixation of an altruistic mutant by drift is possible when supported by ecological conditions that impose a metapopulation structure, episodic mixing of groups, and severe nutrient limitation. This is significant because it offers an alternative explanation for the evolution of altruism based on drift rather than selection. Given the ubiquity of low-nutrient "oligotrophic" environments in which microbes exist (e.g., the open ocean, deep subsurface soils, or under the polar ice caps) our results suggest that altruistic and cooperative behaviors may be highly prevalent among microbial populations.

Evolution of the connectivity and indispensability of a transferable gene: the simplicity hypothesis.

BACKGROUND: The number of interactions between a transferable gene or its protein product and genes or gene products native to its microbial host is referred to as connectivity. Such interactions impact the tendency of the gene to be retained by evolution following horizontal gene transfer (HGT) into a microbial population. The complexity hypothesis posits that the protein product of a transferable gene with lower connectivity is more likely to function in a way that is beneficial to a new microbial host compared to the protein product of a transferable gene with higher connectivity. A gene with lower connectivity is consequently more likely to be fixed in any microbial population it enters by HGT. The more recently proposed simplicity hypothesis posits that the connectivity of a transferable gene might increase over time within any single microbial population due to gene-host coevolution, but that differential rates of colonization of microbial populations by HGT in accordance with differences in connectivity might act to counter this and even reduce connectivity over time, comprising an evolutionary trade-off. RESULTS: We present a theoretical model that can be used to predict the conditions under which gene-host coevolution might increase or decrease the connectivity of a transferable gene over time. We show that the opportunity to enter new microbial populations by HGT can cause the connectivity of a transferable gene to evolve toward lower values, particularly in an environment that is unstable with respect to the function of the gene's protein product. We also show that a lack of such opportunity in a stable environment can cause the connectivity of a transferable gene to evolve toward higher values. CONCLUSION: Our theoretical model suggests that the connectivity of a transferable gene can change over time toward higher values corresponding to a more sessile state of lower transferability or lower values corresponding to a more itinerant state of higher transferability, depending on the ecological milieu in which the gene exists. We note, however, that a better understanding of gene-host coevolutionary dynamics in natural microbial systems is required before any further conclusions about the veracity of the simplicity hypothesis can be drawn.

Fatty acids differentially regulate insulin resistance through endoplasm reticulum stress-mediated induction of tribbles homologue 3: a potential link between dietary fat composition and the pathophysiological outcomes of obesity.

AIMS/HYPOTHESIS: Previous studies have shown that saturated fatty acids cause insulin resistance (IR) that is prevented by unsaturated fatty acids. Tribbles homologue 3 (TRIB3) is a putative endogenous inhibitor of insulin signalling, but its role in insulin signalling is controversial. This study aimed to determine whether fatty acids regulate IR via TRIB3. METHODS: We treated HepG2 cells with saturated and unsaturated fatty acids and evaluated TRIB3 expression. We then tested whether regulation of TRIB3 occurred through endoplasmic reticulum (ER) stress, and whether modulating TRIB3 and ER stress marker genes was necessary and/or sufficient for regulation of insulin signalling. To test the in vivo significance of this mechanism, we fed mice obesogenic diets with different fatty acid profiles and assessed physiological variables of diabetes, ER stress markers and Trib3 expression in the liver. RESULTS: Our data show that fatty acids differentially regulate IR through ER stress-mediated induction of TRIB3. Intriguingly, a standard and widely used obesogenic diet high in unsaturated fats failed to induce ER stress, TRIB3 or IR. However, an alternative obesogenic diet with lower unsaturated fat recapitulated the cell studies by causing ER stress, TRIB3 induction and IR. CONCLUSIONS/INTERPRETATION: This study revealed a novel mechanism linking dietary fat composition to IR. Given the emerging roles for ER stress in non-alcoholic liver disease, we conclude that dietary fat composition rather than total amount may mediate hepatic pathology associated with obesity.

Enhanced tumor cures after Foscan photodynamic therapy combined with the ceramide analog LCL29. Evidence from mouse squamous cell carcinomas for sphingolipids as biomarkers of treatment response.

To improve anticancer therapeutic success of photodynamic therapy (PDT), combination treatments represent a viable strategy. Sphingolipid analogs combined with anticancer drugs can enhance tumor response. We have shown that LCL29, a C6-pyridinium ceramide, promotes therapeutic efficacy of Photofrin-PDT in mouse SCCVII squamous cell carcinoma tumors. The long-term effect of the combination PDT + LCL29 is unknown. In this study we used the same model to test the long-term curative potential of Foscan-PDT + LCL29. We show that treatment of SCCVII tumors with the combination led to enhanced long-term tumor cure compared to PDT alone. LCL29 itself did not prevent tumor growth. All treatments triggered early increases in tumor-associated C16-ceramide, C18-ceramide, dihydrosphingosine, and global levels of dihydroceramides. PDT-evoked increases in tumor-associated sphingosine-1-phosphate and dihydrosphingosine-1-phosphate remained elevated or were attenuated after the combination, respectively; in contrast, LCL29 had no effect on these two sphingolipids. Our data demonstrate that adjuvant LCL29 improves PDT long-term therapeutic efficacy, implying translational potential of the combination. Furthermore, our findings indicate that changes in the sphingolipid profile might serve as predictive biomarkers of tumor response to treatments.

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas.

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination.

Mitochondrially targeted ceramide LCL-30 inhibits colorectal cancer in mice.

The sphingolipid ceramide is intimately involved in the growth, differentiation, senescence, and death of normal and cancerous cells. Mitochondria are increasingly appreciated to play a key role in ceramide-induced cell death. Recent work showed the C16-pyridinium ceramide analogue LCL-30 to induce cell death in vitro by mitochondrial targeting. The aim of the current study was to translate these results to an in vivo model. We found that LCL-30 accumulated in mitochondria in the murine colorectal cancer cell line CT-26 and reduced cellular ATP content, leading to dose- and time-dependent cytotoxicity. Although the mitochondrial levels of sphingosine-1-phosphate (S1P) became elevated, transcription levels of ceramide-metabolising enzymes were not affected. In mice, LCL-30 was rapidly absorbed from the peritoneal cavity and cleared from the circulation within 24 h, but local peritoneal toxicity was dose-limiting. In a model of subcutaneous tumour inoculation, LCL-30 significantly reduced the proliferative activity and the growth rate of established tumours. Sphingolipid profiles in tumour tissue also showed increased levels of S1P. In summary, we present the first in vivo application of a long-chain pyridinium ceramide for the treatment of experimental metastatic colorectal cancer, together with its pharmacokinetic parameters. LCL-30 was an efficacious and safe agent. Future studies should identify an improved application route and effective partners for combination treatment.

Distribution and dynamic changes of sphingolipids in blood in response to platelet activation.

BACKGROUND: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking. OBJECTIVES: To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets. METHODS: Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo. RESULTS: Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P. CONCLUSIONS: Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.

Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.

Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.

The origin and evolution of geminivirus-related DNA sequences in Nicotiana.

A horizontal transmission of a geminiviral DNA sequence, into the germ line of an ancestral Nicotiana, gave rise to multiple repeats of geminivirus-related DNA, GRD, in the genome. We follow GRD evolution in Nicotiana tabacum (tobacco), an allotetraploid, and its diploid relatives, and show GRDs are derived from begomoviruses. GRDs occur in two families: the GRD5 family's ancestor integrated into the common ancestor of three diploid species, Nicotiana kawakamii, Nicotiana tomentosa and Nicotiana tomentosiformis, on homeologous group 4 chromosomes. The GRD3 family was acquired more recently on chromosome 2 in a lineage of N. tomentosiformis, the paternal ancestor of tobacco. Both GRD families include individual members that are methylated and diverged. Using relative rates of synonymous and nonsynonymous nucleotide substitutions, we tested for evidence of selection on GRD units and found none within the GRD3 and GRD5 families. However, the substitutions between GRD3 and GRD5 do show a significant excess of synonymous changes, suggesting purifying selection and hence a period of autonomous evolution between GRD3 and GRD5 integration. We observe in the GRD3 family, features of Helitrons, a major new class of putative rolling-circle replicating eukaryotic transposon, not found in the GRD5 family or geminiviruses. We speculate that the second integration event, resulting in the GRD3 family, involved a free-living geminivirus, a Helitron and perhaps also GRD5. Thus our data point towards recurrent dynamic interplay between geminivirus and plant DNA in evolution.

The mechanism of bile salt-induced hemolysis.

The hemolytic activities of sodium deoxycholate (DChol) and its tauro-conjugate (TDChol) and glyco-conjugate (GDChol) were analysed. 50 % hemolysis occurred in 30 min at pH 7.3, at the concentrations of these detergents equal to 0.044, 0.042 and 0.040 % respectively. These values are below their critical micellar concentrations. Based on its kinetics, this hemolysis is classified as being of permeability type. The detergents increase the permeability of erythrocyte membranes to KCl, and colloid osmotic hemolysis occurs. The minimum of hemolytic activity of the three cholates is at about pH 7.5. A very high increase in hemolytic activity occurs at pHs below 6.8, 6.5 and 6.2 for DChol, TDChol, and GDChol, respectively. These values are close to the pK(a) for DChol (6.2), but much higher than the pK(a) for TDChol (1.9) and GDChol (4.8). It is therefore suggested that the increase in hemolytic activity is not a result of the protonation of the anionic groups of the cholates. At acidification below pH 6, the kinetics of DChol induced hemolysis change to the damage type characterised by nonselective membrane permeability. Such a transition is not observed in TDChol and GDChol induced hemolysis. It is therefore suggested that the change in the type of hemolysis depends on protonation of the anionic group of cholates.

Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution.

The selective pressure at the protein level is usually measured by the nonsynonymous/synonymous rate ratio (omega = dN/dS), with omega < 1, omega = 1, and omega > 1 indicating purifying (or negative) selection, neutral evolution, and diversifying (or positive) selection, respectively. The omega ratio is commonly calculated as an average over sites. As every functional protein has some amino acid sites under selective constraints, averaging rates across sites leads to low power to detect positive selection. Recently developed models of codon substitution allow the omega ratio to vary among sites and appear to be powerful in detecting positive selection in empirical data analysis. In this study, we used computer simulation to investigate the accuracy and power of the likelihood ratio test (LRT) in detecting positive selection at amino acid sites. The test compares two nested models: one that allows for sites under positive selection (with omega > 1), and another that does not, with the chi2 distribution used for significance testing. We found that use of the chi(2) distribution makes the test conservative, especially when the data contain very short and highly similar sequences. Nevertheless, the LRT is powerful. Although the power can be low with only 5 or 6 sequences in the data, it was nearly 100% in data sets of 17 sequences. Sequence length, sequence divergence, and the strength of positive selection also were found to affect the power of the LRT. The exact distribution assumed for the omega ratio over sites was found not to affect the effectiveness of the LRT.

Positive and negative selection in the DAZ gene family.

Because a microdeletion containing the DAZ gene is the most frequently observed deletion in infertile men, the DAZ gene was considered a strong candidate for the azoospermia factor. A recent evolutionary analysis, however, suggested that DAZ was free from functional constraints and consequently played little or no role in human spermatogenesis. The major evidence for this surprising conclusion is that the nonsynonymous substitution rate is similar to the synonymous rate and to the rate in introns. In this study, we reexamined the evolution of the DAZ gene family by using maximum-likelihood methods, which accommodate variable selective pressures among sites or among branches. The results suggest that DAZ is not free from functional constraints. Most amino acids in DAZ are under strong selective constraint, while a few sites are under diversifying selection with nonsynonymous/ synonymous rate ratios (d(N)/d(S)) well above 1. As a result, the average d(N)/d(S) ratio over sites is not a sensible measure of selective pressure on the protein. Lineage-specific analysis indicated that human members of this gene family were evolving by positive Darwinian selection, although the evidence was not strong.

Substitution rates in Drosophila nuclear genes: implications for translational selection.

The relationships between synonymous and nonsynonymous substitution rates and between synonymous rate and codon usage bias are important to our understanding of the roles of mutation and selection in the evolution of Drosophila genes. Previous studies used approximate estimation methods that ignore codon bias. In this study we reexamine those relationships using maximum-likelihood methods to estimate substitution rates, which accommodate the transition/transversion rate bias and codon usage bias. We compiled a sample of homologous DNA sequences at 83 nuclear loci from Drosophila melanogaster and at least one other species of Drosophila. Our analysis was consistent with previous studies in finding that synonymous rates were positively correlated with nonsynonymous rates. Our analysis differed from previous studies, however, in that synonymous rates were unrelated to codon bias. We therefore conducted a simulation study to investigate the differences between approaches. The results suggested that failure to properly account for multiple substitutions at the same site and for biased codon usage by approximate methods can lead to an artifactual correlation between synonymous rate and codon bias. Implications of the results for translational selection are discussed.

Rates of nucleotide substitution and mammalian nuclear gene evolution. Approximate and maximum-likelihood methods lead to different conclusions.

Rates and patterns of synonymous and nonsynonymous substitutions have important implications for the origin and maintenance of mammalian isochores and the effectiveness of selection at synonymous sites. Previous studies of mammalian nuclear genes largely employed approximate methods to estimate rates of nonsynonymous and synonymous substitutions. Because these methods did not account for major features of DNA sequence evolution such as transition/transversion rate bias and unequal codon usage, they might not have produced reliable results. To evaluate the impact of the estimation method, we analyzed a sample of 82 nuclear genes from the mammalian orders Artiodactyla, Primates, and Rodentia using both approximate and maximum-likelihood methods. Maximum-likelihood analysis indicated that synonymous substitution rates were positively correlated with GC content at the third codon positions, but independent of nonsynonymous substitution rates. Approximate methods, however, indicated that synonymous substitution rates were independent of GC content at the third codon positions, but were positively correlated with nonsynonymous rates. Failure to properly account for transition/transversion rate bias and unequal codon usage appears to have caused substantial biases in approximate estimates of substitution rates.

[Choice of correct stabilization in long bone fractures]. / W sprawie wyboru systemu stabilizacji zlaman kosci dlugich.

The paper presents an algorithm to facilitate the choice of the correct stabilization system in fractures of long bones. The algorithm is based on fracture morphology. The indications and contractions for surgical treatment are discussed.

[Is posttraumatic osteomyelitis of iatrogenic nature?]. / Czy pourazowe zapalenie kosci jest choroba jatrogenna?

A series of 112 patients with posttraumatic osteomyelitis has been reviewed. Analysis of the management revealed, that malpractice contributed to the onset of osteomyelitis in many cases. Since politrauma patients with extensive bone and soft tissue injury are prone to infection errors in management of these patients lead to serious complications. Neglecting of primary immobilization of the open fracture in politrauma patients, incorrect methods of fracture fixation or technical errors at surgery, erroneous soft tissue injury management (complete closure of the wound, neglecting compartment syndrome) and disregarding local antibiotics are among most frequently committed errors. The authors conclude, that in majority of cases posttraumatic osteomyelitis is of iatrogenic nature.

[Chronic bone abscess]. / Przewlekly ropien kostny.

The review of 68 patients with chronic bone abscess as a long-term sequel to ostomyelitis is presented. Antibiotic instillation within abscess cavity is recommended by the authors. Once the drainage is concluded immunotherapy should be introduced to secure long-lasting remission as in 90% of presented cases.