Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations

Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.     

Novel cell-penetrating alpha-keto-amide calpain inhibitors as potential treatment for muscular dystrophy

Dipeptide-derived alpha-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible covalent inhibitors have IC(50) values down to 25nM and exhibit greatly improved activity in muscle cells compared to the reference compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.     

Intracellular recording from a spider vibration receptor

The present study introduces a new preparation of a spider vibration receptor that allows intracellular recording of responses to natural mechanical or electrical stimulation of the associated mechanoreceptor cells. The spider vibration receptor is a lyriform slit sense organ made up of 21 cuticular slits located on the distal end of the metatarsus of each walking leg. The organ is stimulated when the tarsus receives substrate vibrations, which it transmits to the organ’s cuticular structures, reducing the displacement to about one tenth due to geometrical reasons. Current clamp recording was used to record action potentials generated by electrical or mechanical stimuli. Square pulse stimulation identified two groups of sensory cells, the first being single-spike cells which generated only one or two action potentials and the second being multi-spike cells which produced bursts of action potentials. When the more natural mechanical sinusoidal stimulation was applied, differences in adaptation rate between the two cell types remained. In agreement with prior extracellular recordings, both cell types showed a decrease in the threshold tarsus deflection with increasing stimulus frequency. Off-responses to mechanical stimuli have also been seen in the metatarsal organ for the first time.     

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps

Acaulospora alpina sp. nov. forms small (65-85 microm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5-2 microm diam) that are as deep as wide and truncated conical. A "beaded" wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5-6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300-1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.     

Sugar concentrations along and across the Ricinus communis L. hypocotyl measured by single cell sampling analysis

Single cell sap sampling and analysis were used to measure the longitudinal and radial distribution of sucrose, glucose and fructose in the apical cell division zone and in the basal, elongated zone of the Ricinus hypocotyl. Sucrose and hexose increased in concentration from the apex to the base of the seedling axis. In the cell division zone low hexose and sucrose concentrations prevailed in cortex and pith, with a slightly higher hexose concentration in pith cells. The sucrose concentrations in sieve tubes and in phloem were much higher than in the cortex and pith cells. In the basal zone of the hypocotyl high levels of sucrose in phloem, cortex and pith were found, therefore radial, diffusional sucrose flow away from the phloem was considered unlikely. It is proposed that radial flow of growth-water to the hypocotyl periphery together with the down-regulation of a sucrose transporter at the phloem leads to a preferential sucrose flow to the expanding cortex. The pith cells, which do not experience flow of growth-water, are probably insufficiently supplied with sucrose from the phloem resulting eventually in cell death as the plant grows. Shortage of sucrose supply, experimentally achieved by removal of the endosperm, led to sucrose hydrolysis in the pith. The sucrose levels in the other tissues decreased less. It appears that the hydrolysis to hexose was initiated to maintain the osmotic value in the pith cell sap. It is speculated that high hexose levels in the cells are indicative of insufficient sucrose supply via the phloem and that the pith cells are confronted with that situation during early seedling development.     

Flavonoid-rich apples and nitrate-rich spinach augment nitric oxide status and improve endothelial function in healthy men and women: a randomized controlled trial

Flavonoids and nitrates in fruits and vegetables may protect against cardiovascular disease. Dietary flavonoids and nitrates can augment nitric oxide status via distinct pathways, which may improve endothelial function and lower blood pressure. Recent studies suggest that the combination of flavonoids and nitrates can enhance nitric oxide production in the stomach. Their combined effect in the circulation is unclear. Here, our objective was to investigate the independent and additive effects of flavonoid-rich apples and nitrate-rich spinach on nitric oxide status, endothelial function, and blood pressure. A randomized, controlled, crossover trial with healthy men and women (n=30) was conducted. The acute effects of four energy-matched treatments (control, apple, spinach, and apple+spinach), administered in random order, were compared. Measurements included plasma nitric oxide status, assessed by measuring S-nitrosothiols+other nitrosylated species (RXNO) and nitrite, blood pressure, and endothelial function, measured as flow-mediated dilatation of the brachial artery. Results are means and 95% CI. Relative to control, all treatments resulted in higher RXNO (control, 33 nmol/L, 26, 42; apple, 51 nmol/L, 40, 65; spinach, 86 nmol/L, 68, 110; apple+spinach, 69 nmol/L, 54, 88; P<0.01) and higher nitrite (control, 35 nmol/L, 27, 46; apple, 69 nmol/L, 53, 90; spinach, 99 nmol/L, 76, 129; apple+spinach, 80 nmol/L, 61, 104; P<0.01). Compared to control, all treatments resulted in higher flow-mediated dilatation (P<0.05) and lower pulse pressure (P<0.05), and apple and spinach resulted in lower systolic blood pressure (P<0.05). No significant effect was observed on diastolic blood pressure. The combination of apple and spinach did not result in additive effects on nitric oxide status, endothelial function, or blood pressure. In conclusion, flavonoid-rich apples and nitrate-rich spinach can independently augment nitric oxide status, enhance endothelial function, and lower blood pressure acutely, outcomes that may benefit cardiovascular health.     

In vitro metabolic stability of iodinated obestatin peptides

Different iodinated mouse obestatin peptides have been characterized toward their in vitro stability in the main metabolic compartments plasma, liver and kidney. Using HPLC-UV for quantification, significant differences in the degradation kinetics of the iodinated peptides, arising from both enzymatic proteolysis and dehalogenation, were found when compared to the native, unmodified peptide. HPLC-MS/MS analysis demonstrated that the cleavage sites were dependent upon the biological matrix and the location of the amino acid residue incorporating the iodine atom(s). The degrading proteases were found to target peptide bonds further away from the iodine incorporation, while proteolytic cleavages of nearby peptide bonds were more limited. Diiodinated amino acid residue containing peptides were found to be more susceptible to deiodination than the mono-iodinated derivative. In plasma, the percentage of peptide degradation solely attributed to deiodinase activity after 20 min incubation reached up to 25% for 2,5-diiodo-H(19)-obestatin compared to 20% and only 3% for (3,5-diiodo-Y(16))- and (3-iodo-Y(16)) obestatin, respectively. Hence, our results demonstrate that the different iodinated peptides pose significantly different metabolization properties and thus, also different biological activities are expected for peptides upon iodination.     

Decreased skp2 expression is necessary but not sufficient for therapy-induced senescence in prostate cancer

Therapy-induced senescence (TIS), a cytostatic stress response in cancer cells, is induced inefficiently by current anticancer agents and radiation. The mechanisms that mediate TIS in cancer cells are not well defined. Herein, we characterize a robust senescence response both in vitro and in vivo to the quinone diaziquone (AZQ), previously identified in a high-throughput senescence-induction small-molecule screen. Using AZQ and several other agents that induce senescence, we screened a series of cyclin-dependent kinase inhibitors and found that p27(Kip1) was induced in all investigated prostate cancer cell lines. The ubiquitin-ligase Skp2 negatively regulates p27(Kip1) and, during TIS, is translocated to the cytoplasm before its expression is decreased in senescent cells. Overexpression of Skp2 blocks the effects of AZQ on senescence and p27(Kip1) induction. We also find that stable long-term short hairpin RNA knockdown of Skp2 decreases proliferation but does not generate the complete senescence phenotype. We conclude that Skp2 participates in regulating TIS but, alone, is insufficient to induce senescence in cancer cells.