Loss of function in the Drosophila clock gene period results in altered intermediary lipid metabolism and increased susceptibility to starvation.

The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period01 (per01) clock mutants and Canton-S wildtype (WTCS) flies in an isogenic and non-isogenic background using LC-MS. In the non-isogenic background, metabolites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per01 mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WTCS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per01 mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per01 did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per01 mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants.

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography-tandem mass spectrometry-based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.

Fully automated registration of vibrational microspectroscopic images in histologically stained tissue sections.

BACKGROUND: In recent years, hyperspectral microscopy techniques such as infrared or Raman microscopy have been applied successfully for diagnostic purposes. In many of the corresponding studies, it is common practice to measure one and the same sample under different types of microscopes. Any joint analysis of the two image modalities requires to overlay the images, so that identical positions in the sample are located at the same coordinate in both images. This step, commonly referred to as image registration, has typically been performed manually in the lack of established automated computational registration tools. RESULTS: We propose a corresponding registration algorithm that addresses this registration problem, and demonstrate the robustness of our approach in different constellations of microscopes. First, we deal with subregion registration of Fourier Transform Infrared (FTIR) microscopic images in whole-slide histopathological staining images. Second, we register FTIR imaged cores of tissue microarrays in their histopathologically stained counterparts, and finally perform registration of Coherent anti-Stokes Raman spectroscopic (CARS) images within histopathological staining images. CONCLUSIONS: Our validation involves a large variety of samples obtained from colon, bladder, and lung tissue on three different types of microscopes, and demonstrates that our proposed method works fully automated and highly robust in different constellations of microscopes involving diverse types of tissue samples.

Cutting to the chase: Participation factors, behavioral effects, and cultural perspectives of participants in an adult circumcision campaign.

Following the adoption of key national policy, several campaigns aimed at increasing the number of adult males receiving circumcisions have been implemented across South Africa. Evidence as to the likely effectiveness of such interventions comes predominantly from three large randomized-controlled trials. However, little has been written about how these campaigns are perceived by the participants. This is significant given the importance of the social issues that are implicit in determining both the ethical acceptability, and effectiveness of these campaigns. We report on a study aimed at identifying and exploring motivating factors for participation, behavioral effects, and cultural attitudes of participants towards a circumcision campaign undertaken in the Northern Cape Province. For this interpretive sociological research project, semi-structured interviews were conducted with 29 participants. These were recorded, transcribed, and qualitatively analyzed. The main reasons given for participation included that of reducing the risk of acquiring HIV and other sexually transmitted infections (STIs), as well as the enhancement of sexual experience. Participants insisted that they would continue to use condoms after the circumcision, although felt that other community members receiving circumcisions would not do so. Several advantages were described when receiving a circumcision at a public health facility, as opposed to the manner more traditional to the participant's culture. Whilst they did not report intentions for risk compensation, the reasons given for participation and their willingness to attribute this problem to other community members casts doubt on the veracity of their reported intentions. Furthermore, participants did not appear to have a complete understanding as to how circumcision is protective. Participants shared the belief that circumcisions as performed in the context of this campaign were safer than the traditional circumcision occurring in the area, which represents an important area for further research.

Natural Zeitgebers Under Temperate Conditions Cannot Compensate for the Loss of a Functional Circadian Clock in Timing of a Vital Behavior in Drosophila.

The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling (Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling.

C/EBPß is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPß. C/EBPß-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPß broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPß, and co-activator p300, and is down-regulated by C/EBPß-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPß-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPß-inhibitory STLs. Overall, our data identify C/EBPß as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPß may have therapeutic potential against AML.

The Circadian Clock Improves Fitness in the Fruit Fly, Drosophila melanogaster.

It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant's natural cycle, i.e., a 19-h day in the case of per s mutants and a 29-h day for per l mutants. The arrhythmic per 0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but per l mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, per s and per 0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and - as revealed for per l mutants - this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that per l and per s persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.

Non-Canonical Control of Neuronal Energy Status by the Na+ Pump.

Neurons have limited intracellular energy stores but experience acute and unpredictable increases in energy demand. To better understand how these cells repeatedly transit from a resting to active state without undergoing metabolic stress, we monitored their early metabolic response to neurotransmission using ion-sensitive probes and FRET sensors in vitro and in vivo. A short theta burst triggered immediate Na+ entry, followed by a delayed stimulation of the Na+/K+ ATPase pump. Unexpectedly, cytosolic ATP and ADP levels were unperturbed across a wide range of physiological workloads, revealing strict flux coupling between the Na+ pump and mitochondria. Metabolic flux measurements revealed a "priming" phase of mitochondrial energization by pyruvate, whereas glucose consumption coincided with delayed Na+ pump stimulation. Experiments revealed that the Na+ pump plays a permissive role for mitochondrial ATP production and glycolysis. We conclude that neuronal energy homeostasis is not mediated by adenine nucleotides or by Ca2+, but by a mechanism commanded by the Na+ pump.