Model reduction of genome-scale metabolic models as a basis for targeted kinetic models.

Constraint-based, genome-scale metabolic models are an essential tool to guide metabolic engineering. However, they lack the detail and time dimension that kinetic models with enzyme dynamics offer. Model reduction can be used to bridge the gap between the two methods and allow for the integration of kinetic models into the Design-Built-Test-Learn cycle. Here we show that these reduced size models can be representative of the dynamics of the original model and demonstrate the automated generation and parameterisation of such models. Using these minimal models of metabolism could allow for further exploration of dynamic responses in metabolic networks.

Safety and efficacy of low-dose PI3K inhibitor taselisib in adult patients with CLOVES and Klippel-Trenaunay syndrome (KTS): the TOTEM trial, a phase 1/2 multicenter, open-label, single-arm study.

PURPOSE: PIK3CA pathogenic variants in the PIK3CA-related overgrowth spectrum (PROS) activate phosphoinositide 3-kinase signaling, providing a rationale for targeted therapy, but no drug has proven efficacy and safety in this population. Our aim was to establish the six-month tolerability and efficacy of low-dose taselisib, a selective class I PI3K inhibitor, in PROS patients. METHODS: Patients over 16 years with PROS and PIK3CA pathogenic variants were included in a phase IB/IIA multicenter, open-label single-arm trial (six patients at 1 mg/day of taselisib, then 24 at 2 mg/day). The primary outcome was the occurrence of dose limiting toxicity (DLT). Efficacy outcomes were the relative changes after treatment of (1) tissue volume at affected and unaffected sites, both clinically and on imaging; (2) cutaneous vascular outcomes when relevant; (3) biologic parameters; (4) quality of life; and (5) patient-reported outcomes. RESULTS: Among 19 enrolled patients, 2 experienced a DLT (enteritis and pachymeningitis) leading to early trial termination (17 treated, 10 completed the study). No serious adverse reaction occurred in the 1 mg cohort (n = 6). No significant reduction in affected tissue volume was observed (mean -4.2%; p = 0.81; SD 14.01). Thirteen (76.4%) participants reported clinical improvement (pain reduction, chronic bleeding resolution, functional improvement). CONCLUSION: Despite functional improvement, the safety profile of low-dose taselisib precludes its long-term use.

Mechanism of plasma electrolytic oxidation in Mg3ZnCa implants: a study of double-layer formation and properties through nanoindentation.

Plasma electrolytic oxidation (PEO), applied to light metals such as titanium, aluminum, and magnesium, creates a two-layer coating and has become increasingly important in metal coatings. However, due to the high voltage and temperature of the process, no online instrument could monitor the underlying mechanism. This paper presents a new image proving that the surface of PEO-coated Mg3ZnCa boiled during the process and argues that three hypotheses are involved in the PEO mechanism based on boiling caused by tolerating high voltage during the PEO process, which could explain the current‒voltage diagram of the process. Finally, nanoindentation was used to measure the elastic module and hardness of the PEO layers. The nanoindentation test results revealed the similarity of the elastic module of the outer porous layer and the primary alloy, with values of 40.25 GPa and 41.47 GPa, respectively, confirming that the outer porous layer corresponds to the cold plasma-gas phase formed during the PEO process.

Slow magnetic order-order transition in the spin chain antiferromagnet Ca3Co2O6.

Using powder neutron diffraction, we have discovered an unusual magnetic order-order transition in the Ising spin chain compound Ca3Co2O6. On lowering the temperature, an antiferromagnetic phase with a propagation vector k=(0.5,-0.5,1) emerges from a higher temperature spin density wave structure with k=(0,0,1.01). This transition occurs over an unprecedented time scale of several hours and is never complete.

Mesocarp of Brazil nut (Bertholletia excelsa) as inspiration for new impact resistant materials.

Aiming to produce bioinspired impact and puncture resistant materials, the mesocarp of the Brazil nut (Bertholletia excelsa) was characterized. The mesocarp composition was investigated by chemical extraction and its microstructure was analyzed by optical microscopy and microtomography (microCT). A compression test evaluated the force needed to open the mesocarp shell. Shore D hardness testing and nanoindentation measured the local mechanical properties at different length scales. Brazil nut mesocarp has a higher content of lignin (56%) than other nutshells and is mainly composed of sclereids and fibers cells arranged together and not in separated layers as usually found in nature. The mesocarp has an internal and external layer with fibers oriented from peduncle to opercular opening and a middle layer where entangled fibers are latitudinally oriented. To open a Brazil nut mesocarp, compression forces of 10 079 ± 1460 N (parallel to latitudinal section) and 14 785 ± 4050 N (perpendicular to latitudinal section) are needed. Such forces are higher than the forces needed to open most nutshells, if fracture force is normalized by shell thickness. The Shore D hardness test showed that hardness is uniform in the mesocarp, although it is higher in the center of the thickness than close to the inner or outer surface. The cell wall of fibers has a higher reduced modulus than the cell wall of sclereids although they have a similar hardness. These microstructural and mechanical results indicate that Brazil nutshell has great potential as a source for bioinspiration and motivates further studies.

In vitro investigations on the differential pro-oxidant and/or antioxidant properties of cyclosporin A and tacrolimus in human and rat liver microsomes.

OBJECTIVE: The use of cyclosporin A (CSA) and tacrolimus (TAC) in organ transplantation and in the therapy of immune disorders is often hampered by adverse effects, mainly nephro-, hepato- and neurotoxicity. For the development of these side effects, among others, an increased formation of reactive oxygen species, probably generated by the cytochrome P450 (CYP) system, has been accused. Since in this respect literature data are inconsistent, in the present study possible pro- and/or antioxidant effects of CSA and TAC and the involvement of the CYP system were re-evaluated in vitro. METHODS: Effects of CSA and TAC were examined on CYP mediated oxidase functions by stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) or luminol (LM) amplified chemiluminescence (CL) in liver microsomes of either untreated rats or of rats treated with beta-naphthoflavone (BNF), phenobarbital (PB) or dexamethasone (DEX) and in human liver microsomes. RESULTS: In rat liver microsomes, CSA displayed pro-oxidant properties (though only very slightly), whereas in human liver microsomes small antioxidant effects were seen. With TAC in both species the antioxidant capacity prevailed. Treatment of rats with BNF or DEX caused an increase in the pro-oxidant effects of CSA with respect to LPO or LM-CL, whereas in liver microsomes of DEX-treated rats H2O2 production and LC-CL were diminished. CONCLUSIONS: CSA seems to have both pro-oxidant and antioxidant properties, whereas with TAC mainly an antioxidant capacity was seen. The CYP system seems to be involved in the pro-oxidant influence of CSA. Whether pro-oxidant or antioxidant effects predominate may depend on the antioxidant capacity of a tissue and on the CYP isoforms mainly present.

[In vitro investigations of phase I metabolism of the fomocaine derivative Oe 9000 with pig liver homogenates]. / In-vitro-Untersuchungen zum Phase-1-Metabolismus des Fomocain-Derivats Oe 9000 mit Schweineleberhomogenaten.

2,2'-[4-(4-Phenoxymethylphenyl)butylimino]diethanol (Oe 9000) is a new, highly potent local anaesthetic related to fomocaine. It displays a long duration of action, low toxicity and is superior to fomocaine with regard to aqueous solubility and efficacy. In view of the development of new application forms, e.g. for the treatment of postoperative pain, the elucidation of the biotransformation of the drug is required. Therefore, experiments with 10000 x g supernatants and microsomes from pig liver homogenates were conducted. Using specifically synthesized reference compounds six phase I metabolites could be identified by LC-MS. Apart from the predominating oxidative desamination of the compound, that led after redox reactions to the corresponding butyric acid and butanol derivatives, oxygenation of the exocycle, oxidative N-desalkylation, and N-oxidation were observed. Thus, with the exception of one compound only metabolites are generated, that are expected to have no local anaesthetic activity due to their reduced basicity.

Biomimetic cellular metals-using hierarchical structuring for energy absorption.

Fruit walls as well as nut and seed shells typically perform a multitude of functions. One of the biologically most important functions consists in the direct or indirect protection of the seeds from mechanical damage or other negative environmental influences. This qualifies such biological structures as role models for the development of new materials and components that protect commodities and/or persons from damage caused for example by impacts due to rough handling or crashes. We were able to show how the mechanical properties of metal foam based components can be improved by altering their structure on various hierarchical levels inspired by features and principles important for the impact and/or puncture resistance of the biological role models, rather than by tuning the properties of the bulk material. For this various investigation methods have been established which combine mechanical testing with different imaging methods, as well as with in situ and ex situ mechanical testing methods. Different structural hierarchies especially important for the mechanical deformation and failure behaviour of the biological role models, pomelo fruit (Citrus maxima) and Macadamia integrifolia, were identified. They were abstracted and transferred into corresponding structural principles and thus hierarchically structured bio-inspired metal foams have been designed. A production route for metal based bio-inspired structures by investment casting was successfully established. This allows the production of complex and reliable structures, by implementing and combining different hierarchical structural elements found in the biological concept generators, such as strut design and integration of fibres, as well as by minimising casting defects. To evaluate the structural effects, similar investigation methods and mechanical tests were applied to both the biological role models and the metallic foams. As a result an even deeper quantitative understanding of the form-structure-function relationship of the biological concept generators as well as the bio-inspired metal foams was achieved, on deeper hierarchical levels and overarching different levels.

Renal handling of drugs and amino acids after impairment of kidney or liver function--influences of maturity and protective treatment.

Renal tubular cells are involved both in secretion and in reabsorption processes within the kidney. Normally, most xenobiotics are secreted into the urine at the basolateral membrane of the tubular cell, whereas amino acids are reabsorbed quantitatively at the luminal side. Under different pathological or experimental circumstances, these transport steps may be changed, e.g., they may be reduced by renal impairment (reduction of kidney mass, renal ischemia, administration of nephrotoxins) or they may be enhanced after stimulation of transport carriers. Furthermore, a distinct interrelationship exists between excretory functions of the kidney and the liver. That means liver injury can influence renal transport systems also (hepato-renal syndrome). In this review, the following aspects were included: based upon general information concerning different transport pathways for xenobiotics and amino acids within kidney cells and upon a brief characterization of methods for testing impairment of kidney function, the maturation of renal transport and its stimulation are described. Similarities and differences between the postnatal development of kidney function and the increase of renal transport capacity after suitable stimulatory treatment by, for example, various hormones or xenobiotics are reviewed. Especially, renal transport in acute renal failure is described for individuals of different ages. Depending upon the maturity of kidney function, age differences in susceptibility to kidney injury occur: if energy-requiring processes are involved in the transport of the respective substance, then adults, in general, are more susceptible to renal failure than young individuals, because in immature organisms, anaerobic energy production predominates within the kidney. On the other hand, adult animals can better compensate for the loss of renal tissue (partial nephrectomy). With respect to stimulation of renal transport capacity after repeated pretreatment with suitable substances, age differences also exist: most stimulatory schedules are more effective in young, developing individuals than in mature animals. Therefore, the consequences of the stimulation of renal transport can be different in animals of different ages and are discussed in detail. Furthermore, the extent of stimulation is different for the transporters located at the basolateral and at the luminal membranes: obviously the tubular secretion at the contraluminal membrane can be stimulated more effectively than reabsorption processes at the luminal side.

Methods in testing interrelationships between excretion of drugs via urine and bile.

The liver and kidney are largely responsible for inactivating and eliminating drugs and other chemicals. As the excretory capabilities of the two organs overlap, a damage of one system might be compensated by the other. Because of the specificity of both renal and hepatic elimination mechanisms such an alternative excretion route is not possible generally. Several interferences are possible to characterize the relation between hepatic and renal excretion of drugs and xenobiotics. Firstly, the simultaneous assay of excreted drug amounts in urine and bile can give some information concerning the main transport routes of this drug. Thereafter the total interruption of liver or kidney function elucidates the general possibility of alternative excretion routes. But it is important for clinical practice to distinguish between different localizations of organ damages. Today some experimental possibilities exist to exclude partial functions of both kidney and liver separately. Thus it can be clarified why a compound might be excreted via liver or kidney. Moreover it can be characterized whether or not a compensation for the loss of one main excretion organ is possible or not. Such investigations are of some practical importance. Dosing guidelines for drug therapy must be completed for cases of renal or hepatic failure. Moreover the developmental pattern of both elimination routes has consequences for drug use in paediatrics as well as geriatrics. Beside this point of view such investigations are necessary for the prediction of changes in the toxicity of drugs after renal or hepatic insufficiency.

Effect of stress ratio on the fatigue behaviour of compact bone.

During the fatigue process of bone, cracks generally initiate from the inherent defects existing in the bone. The fatigue lives of bone specimens at different stress levels as well as at different stress ratios R were evaluated using a computer simulation in which the crack propagation behaviour initiated from the inherent defects in the bone are herein considered. The S-N curves as well as the distributions of fatigue lives obtained by the simulations accurately conform with the experimental results. With the strain threshold epsilon(max) representing fatigue failure of the bone specimen, the values of 1500 microepsilon for R = -1, 2500 microepsilon for R = 0.1 and 4000 microepsilon for R = 10 were extrapolated from the simulations. These values conform with experimental values reported in the literature. Such conformity indicates that the strain threshold for fatigue failure is associated with the threshold value for crack propagation.

Homocysteine, its metabolites, and B-group vitamins in renal transplant patients.

BACKGROUND: Increased plasma total homocysteine (tHcy) levels are an independent risk factor for cardiovascular morbidity in patients with normal and impaired renal function, including stable renal transplant recipients (RTRs). Plasma concentrations of the metabolites of Hcy, such as cystathionine (Cys), methylmalonic acid (MMA), 2-methylcitric acid (MC), and its diastereomers MCI and MCII have been reported in only a few articles. We therefore looked for the serum concentration of these metabolites and their relationship to renal function, cardiovascular diseases, the immunosuppressive treatment, and serum concentrations of cobalamin and folate. METHODS: Fifty RTRs (mean age 50.4 +/- 11.8 years, 35.9 +/- 44.4 months after kidney transplantation) and 35 controls (NP; mean age 43.5 +/- 14.4 years) were studied. Total Hcy and its metabolites were measured by gas chromatography-mass spectrometry (GC-MS). RESULTS: Total Hcy, MMA, Cys, and MC were elevated twofold to sixfold as compared with NP, with a significant interrelationship between these compounds. With the exception of Cys, they were significantly correlated with serum creatinine. Serum folate levels were inversely correlated with tHcy, Cys and cobalamin with MMA and the ratio of MCI/MCII. There was no correlation between tHcy concentration and its metabolites with immunosuppressive treatment (CsA vs. FK506), clinical history, or current findings of cardiovascular complications and blood pressure profile. CONCLUSION: Prospective studies are needed to find out whether the lowering or normalization of serum concentrations of tHcy and its metabolites due to treatment with B vitamins should be achieved to reduce the cardiovascular risk and improve the long-term outcome of allografts and of patients.

Serum concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine in renal failure patients.

BACKGROUND: Nitric oxide (NO) synthesis is inhibited by the ADMA that accumulates in the plasma of patients with renal failure; however, the concentration of SDMA also is enhanced. Therefore, it has been hypothesized that ADMA and SDMA may contribute to hypertension in these patients. METHODS: We measured the concentrations of ADMA, SDMA and 21 endogenous amino acids in 257 persons by high pressure liquid chromatography (HPLC). RESULTS: The plasma concentrations of both ADMA and SDMA were significantly elevated in patients with chronic renal failure (CRF). The increase was more pronounced for SDMA (2.05 +/- 0.1 micromol/L vs. 0.5 +/- 0.04 micromol/L), whereas it was only moderate for ADMA (0.85 +/- 0.03 micromol/L vs. 0.73 +/- 0.06 micromol/L). In dialysis patients, the concentrations were further increased (ADMA, 1.05 +/- 0.04 micromol/L; SDMA, 2.68 +/- 0.13 micromol/L). After kidney transplantation, the concentration of SDMA returned to the baseline value (1.15 +/- 0.11 micromol/L), but that of ADMA remained enhanced (0.99 +/- 0.06 micromol/L). CONCLUSIONS: In CRF, especially the concentration of SDMA is significantly increased. Not only ADMA, but also SDMA are likely to be responsible for hypertension. Competition for reabsorption between SDMA and arginine within the kidney has to be considered for the interpretation of changes in the ratio between dimethylarginines and arginine in renal failure. Hemodialysis is not suitable for a long-lasting removal of methylarginines. Whether the administration of arginine could have promising effects on hypertension and complications of CRF needs to be studied in prospective trials.

Measurement of ultrasonic-induced chlorhexidine liberation: correlation of the activity of chlorhexidine-silver-sulfadiazine-impregnated catheters to agar roll technique and broth culture.

The diagnosis of intravascular catheter-related infections continues to be a challenge to both the clinician and the microbiologist. To assess the antiseptic effects of silver-sulfadiazine-chlorhexidine-impregnated central venous catheters (SSC) on catheter culture systems, segments of fresh antiseptic- and non antiseptic-impregnated catheters as well as extracted catheters following five days of immersion in PBS were sonicated. The chlorhexidine liberated from the catheter material by ultrasonication was measured by HPLC. Fresh antiseptic-impregnated catheter segments rolled on seeded agar plates produced inhibition zones unlike catheters which had been extracted for >five days in phosphate buffered saline (PBS). Scanning electron microscopy (SEM) revealed that chlorhexidine-silversulfadiazine crystals were located in the superficial catheter matrix. Direct contact of superficially located drug particles with seeded agar plates probably caused the inhibition of bacterial growth. The study suggests that antiseptic compounds readily elute from fresh catheters during solid medium-based culturing processes and ultrasonication. The addition of inhibitors of silversulfadiazine-chlorhexidine to media may be prudent especially when culturing antimicrobial loaded catheters removed after short inwelling times.

Failure of physostigmine in intoxications with tricyclic antidepressants in rats.

In experiments on rats there is a moderate antagonistic effect of physostigmine against intoxications with the tricyclic antidepressants (TAD) clomipramine, desipramine and imipramine, respectively. During the first hours after TAD intoxication the survival rate is higher in physostigmine treated rats. Especially after relatively low doses of TAD the lethality seems to be reduced by physostigmine treatment. However, at the end of the observation period (96 h) the lethality after TAD is equal with and without physostigmine treatment. The effectivity of physostigmine does not depend on the mode of administration: repeated administration and intravenous infusion are not more effective than a single injection of physostigmine. The influence of TAD on heart rate and respiratory rate was not abolished by physostigmine salicylate. Intoxications with high doses of physostigmine were antagonized by atropine; on the other hand there are no signs for an antagonistic effect of desipramine against physostigmine intoxication, that means their anticipated anticholinergic properties could not be proved.

Renal amino acid transport in immature and adult rats during thallium-induced nephrotoxicity.

The effect of Tl2SO4 (Tl, 2 mg/100 g b.wt.) on renal amino acid excretion and plasma amino acid composition was investigated in 10- and 55-day-old rats. Tl decreased glomerular filtration rate only in adult rats. On the other hand, the renal fractional excretion (FE) of amino acids was distinctly higher in adult rats as a sign of lower amino acid reabsorption capacity after Tl. In immature animals FE was increased only for a few amino acids. However, in both age groups Tl administration significantly decreased plasma amino acid concentrations, and was more pronounced in immature rats. The investigation of renal amino acid handling (1) confirms that Tl was more nephrotoxic in 55-day-old animals as demonstrated before using other parameters for nephrotoxicity testing and (2) showed that determination of renal amino acid handling is a suitable marker for nephrotoxicity in adult rats.

Functional and morphological aspects of thallium-induced nephrotoxicity in rats.

Until now the effect of thallium (Tl) on renal function has not been investigated systematically. Therefore, the dose (5, 10, 15, 20 mg Tl2SO4/kg body wt., intraperitoneally) and time-dependence of renal damage was investigated in diuresis experiments on conscious rats. Morphology was evaluated after perfusion fixation in situ. Morphologic changes were localized in the thick ascending limb of the loop of Henle, mostly expressed at the 2nd day after Tl administration, which were completely normalized again at the 10th day. Other parameters such as Tl concentration, changes in water content and the activity of Na+/K(+)-ATPase as well as the diuretic effect of furosemide confirmed the Tl effect to be localized in the renal medulla. One single Tl administration is followed by a decrease in glomerular filtration rate (GFR) and urine volume and an increase of proteinuria. Electrolyte excretion was only slightly changed. All changes were reversible within the 10-day investigation period.

LLU-alpha, an endogenous metabolite of gamma-tocopherol, is more effective against metal nephrotoxicity in rats than gamma-tocopherol.

Antioxidants of the vitamin E family have protective effects against metal toxicity. We examined the protective effect of racemic LLU-alpha [2,7,8-trimethyl-2-(carboxyethyl)-6-hydroxychroman] a metabolite of gamma-tocopherol, in comparison to the effect of alpha- and gamma-tocopherol in rats treated with sodium dichromate (Cr) or thallium sulfate (Tl). We measured metal nephrotoxicity based on urinary protein excretion and discussed it with respect to the metal concentration in renal tissue. The ranking of antioxidant activity (iron stimulated lipid peroxidation, luminol and lucigenin amplified chemiluminescence) was determined in the following order: alpha-tocopherol

Counterion evaporation.

We study the adsorption behavior of a highly charged rodlike polyelectrolyte approaching an oppositely charged planar wall in an unbounded electrolyte solution. The grand potential, the entropy, and the total number of screening particles are calculated as functions of the rod-wall distance, using input parameters that are typical of a DNA-molecule and charged lipid bilayers. It is found that counterions which are bound to the polyelectrolyte at infinite rod-wall distances will be released, or "evaporated," as the DNA molecule moves closer to the charged wall. This effect can be regarded as the opposite of the ion-condensation process. The transition of ions from the system of screening ions into the reservoir of bulk ions can lead to an increase of the enthalpy. This gain of enthalpy for the whole system manifests itself as an attractive contribution to the effective interaction between the wall and the polyelectrolyte.

Biliary amino acid excretion in rats before and after bilateral nephrectomy.

In previous studies it could be shown that after bilateral nephrectomy (NX) the excretory function of the liver is disturbed. To further clarify whether or not this "renohepatic syndrome" is caused by toxic effects of uremia or by competition phenomena between various uraemic toxins an additional aspect was investigated: the biliary excretion of endogenous amino acids. Furthermore, previously it could be shown that renal and hepatic excretory functions overlap. Therefore, the renal excretion of effectively biliary eliminated amino acids (glutamic acid, alanine, tyrosine, isoleucine) is very low and vice versa. That means, that the renal excretion of amino acids with low hepatic elimination (tryptophan, citrulline, lysine, taurine) dominates. The hepatic excretion of amino acids is hardly altered after NX. Remarkably, the removal of both kidneys is followed by a distinct reduction in amino acid plasma concentrations, especially if these concentrations are relatively high in the controls. Interestingly, there is no correlation between plasma concentrations and biliary excretion of amino acids. But the calculation of the bile to plasma concentration ratios of amino acids makes it possible to differentiate three groups of amino acids: Amino acids excreted actively into bile (ratio > or = 1), amino acids with ratios below 1, indicating effective retention, and amino acids with ratios of about 1, whose hepatic handling is passive. After NX these ratios tended to approach 1; low ratios increased and high ratios decreased. That means, active processes involved in excretion or retention are obviously disturbed. These changes could indicate uraemic liver damage as proved regarding influence of NX on hepatic excretion of other endogenous substances and xenobiotics.