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Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cryb mutant. Several features of this variant's light responsiveness imply either that CRYb retains circadian-photoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry0's) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry0 flies frequently exhibited two separate circadian components in LL, not observed in most previous cryb analyses. Temperature-dependent circadian phenotypes exhibited by cry0 flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry0 with an externally blinding mutation (norpAP24), which caused the most severe decrements of circadian photoreception observed so far. cryb cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry0 to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry0 and cryb eclosed rhythmically in LL. These findings indicate that the novel cry0 type causes more profound defects than does the cryb mutation, implying that CRYb retains residual activity. Because some norpAP24 cry0 individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei , the newly designed inhibitors can be further improved and explored to identify their actual target(s).

A soluble form of endoglin (sEng) released into the circulation was suggested to be a direct inducer of endothelial dysfunction, inflammation and contributed to the development of hypertension by interfering with TGF-β signaling in cardiovascular pathologies. In the present study, we assessed the hypothesis that high sEng level-induced hypertension via a possible sEng interference with TGF-β signaling pathways may result in inflammatory, structural or fibrotic changes in hearts of Sol - Eng + mice (mice with high levels of soluble endoglin) fed either chow or high-fat diet. Female Sol - Eng + mice and their age matched littermates with low plasma levels of sEng were fed either chow or high-fat diet (HFD). Heart samples were subsequently analyzed by histology, qRT-PCR and Western blot analysis. In this study, no differences in myocardial morphology/hypertrophy and possible fibrotic changes between Sol - Eng + mice and control mice were detected on both chow and HFD. The presence of sEng did not significantly affect the expression of selected members of TGF-β signaling (membrane endoglin, TGFβRII, ALK-5, ALK-1, Id-1, PAI-1 and activated Smad proteins-pSmad 1,5 and pSmad 2,3), inflammation, heart remodeling (PDGFb, Col1A1) and endothelial dysfunction (VCAM-1, ICAM-1) in the hearts of Sol - Eng + mice compared to control mice on both chow and high-fat diet. High levels of soluble endoglin did not affect microscopic structure (profibrotic and degenerative cardiomyocyte changes), and specific parts of TGF-β signaling, endothelial function and inflammation in the heart of Sol - Eng + mice fed both chow diet or HFD. However, we cannot rule out a possibility that a long-term chronic exposure (9 months and more) to soluble endoglin alone or combined with other cardiovascular risk factors may contribute to alterations of heart function and structure in Sol - Eng + mice, which is the topic in our lab in ongoing experiments.

In the present paper potato plants were cultivated in the presence of ammonium sulphate or elemental sulphur supplementation into the soil to reveal the effects of different sulphur forms on content of nitrogen, phosphorus, potassium, calcium, magnesium and sulphur, and yield of tubers. During the investigation of the influence of different sulphur forms on yield of potato tubers we did not observe significant changes. Average weight of tubers of control plants per one experimental pot was 355 g. Application of sulphur in both forms resulted in moderate potato tubers weight reduction per one experimental pot compared to control group; average value ranged from 320 to 350 g per one experimental pot. Further we treated the plants with two different supplementation of sulphur with cadmium(II) ions (4 mg of cadmium(II) acetate per kilogram of the soil). The significantly lowest cadmium content (p < 0.05) was determined in tissues of plants treated with the highest dosage of elemental sulphur (0.64 mg Cd/kg) compared to control plants (0.82 mg Cd/kg). We also aimed our attention on the cadmium content in proteins, lipids or soluble carbohydrates and ash. Application of sulphate as well as elemental sulphur resulted in significant cadmium content reduction in lipid fraction compared to control plants. In addition to this we quantified content of low molecular mass thiols in potatoes tissues. To determine the thiols content we employed differential pulse voltammetry Brdicka reaction. After twelve days of the treatment enhancing of thiols level was observed in all experimental groups regardless to applied sulphur form and its concentration. Finally we evaluated the effect of sulphur supplementation on Phytophora infestans infection of potato plants.

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites' viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis with inflammation and fibrosis. Membrane endoglin (Eng) expression is shown to participate in fibrosis, and plasma concentrations of soluble endoglin (sEng) are increased in patients with hypercholesterolemia and type 2 diabetes mellitus. We hypothesize that NASH increases both hepatic Eng expression and sEng in blood and that high levels of sEng modulate cholesterol and bile acid (BA) metabolism and affect NASH progression. Three-month-old transgenic male mice overexpressing human sEng and their wild type littermates are fed for six months with either a high-saturated fat, high-fructose high-cholesterol (FFC) diet or a chow diet. Evaluation of NASH, Liquid chromatography–mass spectrometry (LC/MS) analysis of BA, hepatic expression of Eng, inflammation, fibrosis markers, enzymes and transporters involved in hepatic cholesterol and BA metabolism are assessed using Real-Time Quantitative Reverse Transcription Polymerase Chain reaction (qRT-PCR) and Western blot. The FFC diet significantly increases mouse sEng levels and increases hepatic expression of Eng. High levels of human sEng results in increased hepatic deposition of cholesterol due to reduced conversion into BA, as well as redirects the metabolism of triglycerides (TAG) to its accumulation in the liver, via reduced TAG elimination by β-oxidation combined with reduced hepatic efflux. We propose that sEng might be a biomarker of NASH development, and the presence of high levels of sEng might support NASH aggravation by impairing the essential defensive mechanism protecting NASH liver against excessive TAG and cholesterol accumulation, suggesting the importance of high sEng levels in patients prone to develop NASH.

Estrogen‐induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase‐1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol‐induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down‐regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up‐regulated Mrp3 (348%, P ≤ 0.05). Heme pre‐treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up‐regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol‐treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme‐induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up‐regulation (Mrp2/Mrp4) or down‐regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2‐dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol‐induced cholestasis.