Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601^T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601^T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601^T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601^T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601^T. Genes involved in cyclohexanol degradation were only found in strain K601^T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.     

Multiple RNA interactions position Mrd1 at the site of the small subunit pseudoknot within the 90S pre-ribosome

Ribosomal subunit biogenesis in eukaryotes is a complex multistep process. Mrd1 is an essential and conserved small (40S) ribosomal subunit synthesis factor that is required for early cleavages in the 35S pre-ribosomal RNA (rRNA). Yeast Mrd1 contains five RNA-binding domains (RBDs), all of which are necessary for optimal function of the protein. Proteomic data showed that Mrd1 is part of the early pre-ribosomal complexes, and deletion of individual RBDs perturbs the pre-ribosomal structure. In vivo ultraviolet cross-linking showed that Mrd1 binds to the pre-rRNA at two sites within the 18S region, in helix 27 (h27) and helix 28. The major binding site lies in h27, and mutational analyses shows that this interaction requires the RBD1-3 region of Mrd1. RBD2 plays the dominant role in h27 binding, but other RBDs also contribute directly. h27 and helix 28 are located close to the sequences that form the central pseudoknot, a key structural feature of the mature 40S subunit. We speculate that the modular structure of Mrd1 coordinates pseudoknot formation with pre-rRNA processing and subunit assembly.     

Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion

Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we investigated whether caspase-1 plays a role in triglyceride-(TG)-rich lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1 deficiency reduced the postprandial TG response to an oral lipid load, whereas TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [³H]TG-labeled very low-density lipoprotein (VLDL)-like emulsion particles. An oral gavage of [³H]TG-containing olive oil revealed that caspase-1 deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1 deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1 deficiency did not affect FA oxidation or FA uptake but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.     

Drosophila pupal macrophages – A versatile tool for combined ex vivo and in vivo imaging of actin dynamics at high resolution

Molecular understanding of actin dynamics requires a genetically traceable model system that allows live cell imaging together with high-resolution microscopy techniques. Here, we used Drosophila pupal macrophages that combine many advantages of cultured cells with a genetic in vivo model system. Using structured illumination microscopy together with advanced spinning disk confocal microscopy we show that these cells provide a powerful system for single gene analysis. It allows forward genetic screens to characterize the regulatory network controlling cell shape and directed cell migration in a physiological context. We knocked down components regulating lamellipodia formation, including WAVE, single subunits of Arp2/3 complex and CPA, one of the two capping protein subunits and demonstrate the advantages of this model system by imaging mutant macrophages ex vivo as well as in vivo upon laser-induced wounding.     

The antitumor activity of hydrophobin SC3, a fungal protein

The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.     

STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA+/K+‐ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na⁺/K⁺‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na⁺/K⁺‐ATPase gene and by enzymatic assays with extracted Na⁺/K⁺‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.     

Individual Differences in the Attentional Blink: The Temporal Profile of Blinkers and Non-Blinkers

Background

When two targets are presented in close temporal succession, the majority of people frequently fail to report the second target. This phenomenon, known as the ‘attentional blink’ (AB), has been a major topic in attention research for the past twenty years because it is informative about the rate at which stimuli can be encoded into consciously accessible representations. An aspect of the AB that has long been ignored, however, is individual differences.

Methodology/Principal Findings

Here we compare a group of blinkers (who show an AB) and non-blinkers (who show little or no AB), and investigate the boundary conditions of the non-blinkers'' remarkable ability. Second, we directly test the properties of temporal selection by analysing response errors, allowing us to uncover individual differences in suppression, delay, and diffusion of selective attention across time. Thirdly, we test the hypothesis that information concerning temporal order is compromised when an AB is somehow avoided. Surprisingly, compared to earlier studies, only a modest amount of suppression was found for blinkers. Non-blinkers showed no suppression, were more precise in selecting the second target, and made less order reversals than blinkers did. In contrast, non-blinkers made relatively more intrusions and showed a selection delay when the second target immediately followed the first target (at lag 1).

Conclusion/Significance

The findings shed new light on the mechanisms that may underlie individual differences in selective attention. The notable ability of non-blinkers to accurately perceive targets presented in close temporal succession might be due to a relatively faster and more precise target selection process compared to large blinkers.     

A Rat Model for Muscle Regeneration in the Soft Palate

Background

Children with a cleft in the soft palate have difficulties with speech, swallowing, and sucking. Despite successful surgical repositioning of the muscles, optimal function is often not achieved. Scar formation and defective regeneration may hamper the functional recovery of the muscles after cleft palate repair. Therefore, the aim of this study is to investigate the anatomy and histology of the soft palate in rats, and to establish an in vivo model for muscle regeneration after surgical injury.

Methods

Fourteen adult male Sprague Dawley rats were divided into four groups. Groups 1 (n = 4) and 2 (n = 2) were used to investigate the anatomy and histology of the soft palate, respectively. Group 3 (n = 6) was used for surgical wounding of the soft palate, and group 4 (n = 2) was used as unwounded control group. The wounds (1 mm) were evaluated by (immuno)histochemistry (AZAN staining, Pax7, MyoD, MyoG, MyHC, and ASMA) after 7 days.

Results

The present study shows that the anatomy and histology of the soft palate muscles of the rat is largely comparable with that in humans. All wounds showed clinical evidence of healing after 7 days. AZAN staining demonstrated extensive collagen deposition in the wound area, and initial regeneration of muscle fibers and salivary glands. Proliferating and differentiating satellite cells were identified in the wound area by antibody staining.

Conclusions

This model is the first, suitable for studying muscle regeneration in the rat soft palate, and allows the development of novel adjuvant strategies to promote muscle regeneration after cleft palate surgery.