Experimental evolution of a pheromone signal

Sexual signals are important in speciation, but understanding their evolution is complex as these signals are often composed of multiple, genetically interdependent components. To understand how signals evolve, we thus need to consider selection responses in multiple components and account for the genetic correlations among components. One intriguing possibility is that selection changes the genetic covariance structure of a multicomponent signal in a way that facilitates a response to selection. However, this hypothesis remains largely untested empirically. In this study, we investigate the evolutionary response of the multicomponent female sex pheromone blend of the moth Heliothis subflexa to 10 generations of artificial selection. We observed a selection response of about three‐quarters of a phenotypic standard deviation in the components under selection. Interestingly, other pheromone components that are biochemically and genetically linked to the components under selection did not change. We also found that after the onset of selection, the genetic covariance structure diverged, resulting in the disassociation of components under selection and components not under selection across the first two genetic principle components. Our findings provide rare empirical support for an intriguing mechanism by which a sexual signal can respond to selection without possible constraints from indirect selection responses.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Developing outcome measures for pediatric mitochondrial disorders: Which complaints and limitations are most burdensome to patients and their parents?

Since some drug intervention effects are only experienced by the patient, organizations such as the Food and Drug Administration prefer clinically meaningful outcome measures. Here, we evaluated which symptoms and limitations in daily life are most burdensome to pediatric patients with mitochondrial disorders and their parents, using two questionnaires. In a study of 78 patients, the most burdensome complaints included fatigue, behavior and speech disturbances, epilepsy and muscle weakness and a high degree of limitations in daily activities was found. Importantly, there was a discrepancy between what symptoms metabolic pediatricians estimated would be most burdensome compared to the patients'/caretakers' opinion. To include feasible and relevant outcome measures in intervention studies, the experience and opinions of patients and caretakers should therefore be heard.     

8-Hydroxydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption

8-Hydroxydeoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal and human studies. However, controversial data exist on the relationship between 8-OHdG formation and age, sex and tobacco smoking in humans, while few or no data are available on other exposures such as environmental tobacco smoke, alcohol, coffee and tea consumption. We investigated the level of 8-OHdG in DNA from peripheral leukocytes among 102 healthy adults living in Brescia province, North Italy, aged 25-45 (mean: 35.2 years), of which 51 were males. 8-OHdG levels expressed as a ratio to total deoxyguanosine (8-OHdG/106 dG) in DNA showed wide interindividual variation, the highest value (63.8) being 6. 2-fold greater than the lowest (10.3). Current smokers showed lower mean 8-OHdG values than subjects who never smoked (29.3 and 34.0, respectively, p<0.05), and an inverse relationship was found between 8-OHdG and lifetime smoking, which was independent of age, sex and body mass index. An inverse relationship was also found with coffee drinking while no association was observed with alcohol and tea consumption, exposure to environmental tobacco smoke and use of vitamins in all subjects, and with use of oral contraceptives in females. The inverse relationship between smoking status and 8-OHdG levels could be explained by the presence of efficient repair processes for the oxidative damage induced by smoking. In this study, the smokers were relatively young (77% were less than 40 years) and only 7% smoked 30 or more cigarettes a day. In conclusion, it would appear that 8-OHdG levels in leukocytes may not provide a sensitive marker of exposure to tobacco smoking.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface

Follicular dendritic cells (FDCs) present in lymphoid follicles play a critical role in germinal center reactions. They trap native Ags in the form of immune complexes providing a source for continuous stimulation of specific B lymphocytes. FDCs have been reported to express MHC class II molecules, suggesting an additional role in the presentation of not only native, but also processed Ag in the form of peptide-loaded MHC class II. Adoptive bone marrow transfer experiments have shown that MHC class II molecules are only passively acquired. Up to now the origin of these MHC class II molecules was not clear. Here we show by cryoimmunogold electron microscopy that MHC class II molecules are not present at the plasma membrane of FDCs. In contrast, microvesicles attached to the FDC surface contain MHC class II and other surface proteins not expressed by FDCs themselves. The size and marker profiles of these microvesicles resemble exosomes. Exosomes, which are secreted internal vesicles from multivesicular endosomes, have been shown earlier to stimulate proliferation of specific T lymphocytes in vitro, but their target in vivo remained a matter of speculation. We demonstrate here that isolated exosomes in vitro bind specifically to FDCs and not to other cell types, suggesting that FDCs might be a physiological target for exosomes.     

The fitness of defective interfering murine coronavirus DI-a and its derivatives is decreased by nonsense and frameshift mutations.

The genome of the defective interfering (DI) mouse hepatitis virus DI-a carries a large open reading frame (ORF) consisting of ORF1a, ORF1b, and nucleocapsid sequences. To test whether this fusion ORF is important for DI virus replication, we constructed derivatives of the DI-a genome in which the reading frame was truncated by a nonsense codon or a frameshift mutation. In vitro-transcribed DI RNAs were transfected into mouse hepatitis virus-infected cells followed by undiluted passage of the resulting virus-DI virus stocks. The following observations were made. (i) Truncation of the fusion ORF was not lethal but led to reduced accumulation of DI RNA. (ii) When pairs of nearly identical in-frame and out-of-frame DI RNAs were directly compared by cotransfection, DI viruses containing in-frame genomic RNAs prevailed within three successive passage even when the out-of-frame RNAs were transfected in 10-fold molar excess. (iii) When DI viruses containing out-of-frame genomic RNAs were passaged, mutants emerged and were selected for that had restored the reading frame. We conclude that translation of the fusion ORF is indeed required for efficient propagation of DI-a and its derivatives.     

Glycation induces formation of amyloid cross-beta structure in albumin

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.     

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.