MI-Lab - A Laboratory Environment for Medical Informatics Students.

Medical research and health care highly depend on the use of information technology. There is a wide range of application systems (patient administration system, laboratory information system, communication server etc.) and heterogeneous data types (administrative data, clinical data, laboratory data, image data, genomic data etc.). Students and researchers do not often have the possibility to use productive application systems of e.g. hospitals or medical practices to gain practical experiences or examine new components and technologies. Therefore, the aim of this project is to develop a dedicated laboratory environment for patient health care and clinical research. Essential application systems were identified and a suitable architecture was designed for this purpose. It is accompanied by a teaching plan that considers learning modules for bachelor and master degrees in medical informatics. We implemented the laboratory environment called MI-Lab with multiple free and open source software components. All components are installed on virtual machines and/or Docker containers. This modular architecture creates a flexible system which can be deployed in various scenarios. The preliminary evaluation results suggests that laboratory environments like MI-Lab work well in teaching practical aspects of medical informatics and are widely accepted by students.

Stoichiometry and deletion analyses of subunits in the heterotrimeric F-ATP synthase c ring from the acetogenic bacterium Acetobacterium woodii.

The ion-translocating c ring of the Na(+) F1 Fo ATP synthase of the anaerobic bacterium Acetobacterium woodii is the first heteromeric c ring found in nature that contains one V- (c1 ) and two F-type-like c subunits (c2 /c3 ), the latter of identical amino acid sequence. To address whether they are of equal or different importance for function, they were deleted in combination or individually. Deletion of c1 was compensated by incorporation of two c2 /c3 subunits but the enzyme was unstable and largely impaired in Na(+) transport. Deletion of c2 was compensated by incorporation of c3 but also led to a reduction of Na(+) transport. Deletion of c3 had no effect. In contrast, deletion of both c2 and c3 led to a complete loss of ATPase activity at the cytoplasmic membrane. Mass spectrometric analysis of c2 +1 Ala and c2 +2 Ala variants revealed a copy number of 8 : 1 for c2 /c3 which is consistent with the biochemical characteristics of the variants. These data indicate a role of c1 in assembly and a function of c2 as the predominant c ring constituent.

Hybrid rotors in F1F(o) ATP synthases: subunit composition, distribution, and physiological significance.

The c ring of the Na+ F1F(o) ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE1, atpE2 and atpE3. Subunit c1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c2 and c3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid F(o)V(o) c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1F(o) ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one F(o)- and one V(o)-type c subunit in one operon. Their c subunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed.

A low phosphorylation potential in the acetogen Acetobacterium woodii reflects its lifestyle at the thermodynamic edge of life.

The anaerobic, acetogenic bacterium Acetobacterium woodii grows on hydrogen and carbon dioxide and uses the Wood-Ljungdahl pathway to fix carbon but also to synthesize ATP. The free energy change of acetogenesis from H2 + CO2 allows for synthesis of only a fraction of an ATP under environmental conditions, and A. woodii is clearly a paradigm for microbial life under extreme energy limitation. However, it was unknown how much energy is required to make ATP under these conditions. In the present study, we determined the phosphorylation potential in cells metabolizing three different acetogenic substrates. It accounts to 37.9 ± 1.3 kJ/mol ATP during acetogenesis from fructose, 32.1 ± 0.3 kJ/mol ATP during acetogenesis from H2 + CO2 and 30.2 ± 0.9 kJ/mol ATP during acetogenesis from CO, the lowest phosphorylation potential ever described. The physiological consequences in terms of energy conservation under extreme energy limitation are discussed.

High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na⁺-coupled ATP synthase.

All rotary ATPases catalyse the interconversion of ATP and ADP-Pi through a mechanism that is coupled to the transmembrane flow of H(+) or Na(+). Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na(+)-driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na(+) recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring.

Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-ß, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/ß cleft, with bI close to subunit α, whereas bII is proximal to subunit ß. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-ß, α-bI-bII-ß, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.

Functional production of the Na+ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI.

The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its ß subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.

Dissociation of mercaptoacetate's effects on feeding and fat metabolism by dietary medium- and long-chain triacylglycerols in rats.

OBJECTIVE AND METHODS: Mercaptoacetate (MA) inhibits hepatic fatty acid oxidation (FAO) and stimulates feeding in rats fed fat-rich diets. To test whether the feeding stimulation by MA depends on hepatic FAO, we compared the effects of intraperitoneally injected MA (45.6 mg/kg body weight) with saline in rats fed diets containing 18% predominately long-chain triacylglycerols (LCTs; > or =90% 16 C) or 18% medium-chain triacylglycerols (MCTs; 51% 10-12 C). We hypothesized that, because medium-chain fatty acids reach the liver and are oxidized faster than long-chain fatty acids, if MA's feeding-stimulatory effect depends on hepatic FAO, MA should stimulate feeding more in MCT-fed rats than in LCT-fed rats. RESULTS: Although MA injected in mid-light phase stimulated feeding similarly in MCT- and LCT-fed rats, MA injected at light onset initially stimulated food intake (1 h) only in LCT- and not in MCT-fed rats. To investigate MA's metabolic effects during the initial hour, rats were sacrificed 30 min after light-onset injections. At this time plasma beta-hydroxybutyrate appeared to be higher in MCT- than in LCT-fed rats and to be increased by MA. In a final experiment, MA did not affect fatty acid content in liver and duodenum tissues but increased fatty acids in duodenal tissue mitochondria from 12 h-fasted rats fed chow. CONCLUSION: In light-onset tests, adaptation to the MCT diet increased hepatic FAO but not the feeding-stimulatory effect of MA in comparison with adaptation to the LCT diet, suggesting that at this time MA does not act in the liver to stimulate feeding or that this effect is not due to FAO inhibition. Inhibition of duodenal mitochondrial FAO may be another metabolic process through which MA stimulates feeding.

Vagal afferents mediate the feeding response to mercaptoacetate but not to the beta (3) adrenergic receptor agonist CL 316,243.

To evaluate the role of subdiaphragmatic vagal afferents in the anorectic response to peripheral administration of the highly selective beta(3)-adrenergic receptor agonist CL 316,243 (CL), we tested the ability of intraperitoneal (IP) injections of CL to inhibit feeding in rats with subdiaphragmatic vagal deafferentation (SDA, n=13) or sham surgeries (SHAM, n=13). Doses of 10, 100 and 1000 ng/kg CL significantly reduced feeding by statistically similar amounts in SHAM and SDA rats. One hour after IP injection, each dose of CL also significantly increased plasma concentrations of free fatty acids and beta-hydroxybutyrate, an indicator of hepatic fatty acid oxidation (FAO), whereas 6h after injection only the two highest CL doses increased plasma beta-hydroxybutyrate. In contrast, peripheral administration of the FAO inhibitor mercaptoacetate (MA, 45.6 mg/kg IP) stimulated feeding in SHAM but not in SDA rats, extending previous data suggesting a necessary role of vagal afferents in the feeding-stimulatory effect of FAO inhibition. We conclude that subdiaphragmatic vagal afferents are essential for the feeding-stimulatory action of MA but not for the anorectic action of peripheral CL and that CL-induced increase in hepatic FAO is not essential for its feeding-inhibitory effect.

Beta-adrenergic-mediated inhibition of feeding by mercaptoacetate in food-deprived rats.

This study investigated the effect of intraperitoneal (IP) injections of the fatty acid oxidation (FAO) inhibitor mercaptoacetate (MA, 45.6 mg/kg) on feeding in food-deprived rats. As previously, MA significantly stimulated feeding in ad libitum-fed rats. MA, however, reduced feeding in 18 and 36 h-fasted rats despite apparently antagonizing the fasting-induced increase in hepatic FAO. To test whether this anorectic effect involves beta-adrenergic stimulation, 36 h-fasted rats were IP injected with the nonspecific beta-adrenergic receptor antagonist propranolol (PROP, 0.5 mg/kg) just before MA injection. PROP attenuated MA's feeding-inhibitory effect, suggesting that MA anorexia is at least partially mediated by beta-adrenergic stimulation. Finally, we evaluated the role of subdiaphragmatic vagal afferent fibers in MA's feeding-inhibitory effect by testing the ability of MA to inhibit food intake in fasted rats after subdiaphragmatic vagal deafferentation (SDA). MA inhibited feeding similarly in SDA rats and sham-operated rats. These data demonstrate that subdiaphragmatic vagal afferents are not necessary for the feeding-inhibitory effect of peripheral MA. These results suggest that the FAO inhibitor MA elicits a feeding-inhibitory effect in fasted rats that is mediated by a different mechanism than its feeding-stimulatory effect.

Mercaptoacetate fails to block the feeding-inhibitory effect of the beta3-adrenergic receptor agonist CGP 12177A.

Peripherally administered beta3-adrenergic receptor (beta3-AR) agonists stimulate lipolysis and inhibit food intake. To test the hypothesis that this inhibition of feeding is due to a substrate-driven increase in hepatic fatty acid oxidation (FAO), we assessed the ability of the FAO inhibitor mercaptoacetate (MA) to reverse the feeding-inhibitory effect of the beta3-AR agonist CGP 12177A (CGP). Adult male Sprague-Dawley rats received intraperitoneal injections of 1 mg/kg CGP, of 45.6 mg/kg MA, or of both drugs, and the effects on food intake, plasma free fatty acids (FFA), and plasma beta-hydroxybutyrate (BHB), an indicator for hepatic FAO, were assessed. Control rats received saline injections. CGP significantly reduced food intake after 0.5 and 6 h and increased plasma FFA and BHB at 0.5 h, suggesting increased lipolysis and hepatic FAO. MA completely reversed the increase in plasma BHB and thus appeared to effectively abolish CGP's effect on hepatic FAO, but MA failed to affect CGP's feeding-inhibitory action. These findings do not support the hypothesis that the beta3-AR agonist CGP inhibits feeding by enhancing hepatic FAO or ketogenesis. Although the beta3-AR agonist CGP reduced saccharin intake in a one-bottle condition taste aversion test, it seems unlikely that the hypophagic effect of CGP is elicited by malaise.

Beneficial and deleterious effects of hydroxycitrate in rats fed a high-fructose diet.

OBJECTIVE: The present study assessed whether long-term supplementation of a high-fructose diet with hydroxycitrate (HCA), an inhibitor of de novo lipogenesis that is widely used as a non-prescription dietary aid, decreases food intake, visceral fat accumulation, hypertriglyceridemia, and hyperinsulinemia in rats. METHODS: We examined the effects of HCA (1.8% of diet) on food intake, body weight gain, visceral fat accumulation, hypertriglyceridemia, and hyperinsulinemia in rats during a 4-wk period of ad libitum access to a 50% fructose diet after a 3-wk period of food restriction in which they lost about 20% of their body weight. RESULTS: HCA decreased food intake and weight gain throughout the test and reduced visceral fat accumulation compared with control rats fed ad libitum (CON). Rats that were pair-fed (PF) to the HCA rats showed similar decreases in weight gain and visceral fat. HCA did not ameliorate the hypertriglyceridemia induced by high-fructose feeding. HCA improved insulin sensitivity on day 10 in comparison with CON rats, but by day 27 insulin levels were similarly higher and liver glycogen levels were similarly lower in HCA and CON rats in comparison with PF rats. Liver lipid content was elevated in HCA rats compared with CON and PF rats. CONCLUSION: These findings indicate that, although HCA attenuates body weight gain and visceral fat accumulation by reducing food intake under these conditions, it has no lasting beneficial effects on hypertriglyceridemia and hyperinsulinemia and leads to the accumulation of liver lipids.