Protocol for a randomized controlled multicenter trial assessing the efficacy of leuprorelin for severe polycystic liver disease: the AGAINST-PLD study.

BACKGROUND: In patients with severe polycystic liver disease (PLD), there is a need for new treatments. Estrogens and possibly other female sex hormones stimulate growth in PLD. In some patients, liver volume decreases after menopause. Female sex hormones could therefore be a target for therapy. The AGAINST-PLD study will examine the efficacy of the GnRH agonist leuprorelin, which blocks the production of estrogen and other sex hormones, to reduce liver growth in PLD. METHODS: The AGAINST-PLD study is an investigator-driven, multicenter, randomized controlled trial. Institutional review board (IRB) approval was received at the University Medical Center of Groningen and will be collected in other sites before opening these sites. Thirty-six female, pre-menopausal patients, with a very large liver volume for age (upper 10% of the PLD population) and ongoing liver growth despite current treatment options will be randomized to direct start of leuprorelin or to 18 months standard of care and delayed start of leuprorelin. Leuprorelin is given as 3.75 mg subcutaneously (s.c.) monthly for the first 3 months followed by 3-monthly depots of 11.25 mg s.c. The trial duration is 36 months. MRI scans to measure liver volume will be performed at screening, 6 months, 18 months, 24 months and 36 months. In addition, blood will be drawn, DEXA-scans will be performed and questionnaires will be collected. This design enables comparison between patients on study treatment and standard of care (first 18 months) and within patients before and during treatment (whole trial). Main outcome is annualized liver growth rate compared between standard of care and study treatment. Secondary outcomes are PLD disease severity, change in liver growth within individuals and (serious) adverse events. The study is designed as a prospective open-label study with blinded endpoint assessment (PROBE). DISCUSSION: In this trial, we combined the expertise of hepatologist, nephrologists and gynecologists to study the effect of leuprorelin on liver growth in PLD. In this way, we hope to stop liver growth, reduce symptoms and reduce the need for liver transplantation in severe PLD. Trial registration Eudra CT number 2020-005949-16, registered at 15 Dec 2020. https://www.clinicaltrialsregister.eu/ctr-search/search?query=2020-005949-16 .

Variation in a range of mTOR-related genes associates with intracranial volume and intellectual disability.

De novo mutations in specific mTOR pathway genes cause brain overgrowth in the context of intellectual disability (ID). By analyzing 101 mMTOR-related genes in a large ID patient cohort and two independent population cohorts, we show that these genes modulate brain growth in health and disease. We report the mTOR activator gene RHEB as an ID gene that is associated with megalencephaly when mutated. Functional testing of mutant RHEB in vertebrate animal models indicates pathway hyperactivation with a concomitant increase in cell and head size, aberrant neuronal migration, and induction of seizures, concordant with the human phenotype. This study reveals that tight control of brain volume is exerted through a large community of mTOR-related genes. Human brain volume can be altered, by either rare disruptive events causing hyperactivation of the pathway, or through the collective effects of common alleles.

Spectroscopic and reactivity differences in metal complexes derived from sulfur containing Triphos homologs.

Herein, we report a simplified method for the synthesis of Triphos homologs H3CC(CH2X)n(CH2Y)3-n (X = SPh, Y = PPh2, n = 0-3). The multidentate compounds were tested for their potential to coordinate metals such as Ni, Fe, and Mo under the same experimental conditions. Cyclic voltammetry, spectroelectrochemical IR investigations as well as DFT calculations were used to examine the electronic alterations in a series of [{H3CC(CH2X)n(CH2Y)3-n}Mo(CO)3] complexes and to evaluate their potential to open coordination sites or to release CO upon oxidation or in the presence of different solvents. In addition, we demonstrate that the catalytic hydrosilylation of N,N-dimethylbenzamide to N,N-dimethylbenzylamine is influenced by the applied tripodal ligand. Our investigations show the high potential of such manipulations to selectively alter the dynamics of the binding properties of Triphos-metal complexes and their reactivity.

Modulation of the CO2 fixation in dinickel azacryptands.

While bimetallic azacryptands are known to selectively coordinate CO2, there is little knowledge on how different substitution patterns of the azacryptand cage structure influence CO2 coordination. Stopped-flow UV-vis spectroscopy, electrochemical analysis and DFT calculations were performed on a series of dinickel azacryptands and showed different rates of CO2 coordination to the complexes. We herein present data showing that the different flexibility of the azacryptands is directly responsible for the difference in the CO2 uptake capability of dinickel azacryptand complexes.

Mössbauer and computational investigation of a functional [NiFe] hydrogenase model complex.

Developing biomimetic complexes that model the active site of [NiFe] hydrogenase enzymes in order to catalyze the activation of H2 is a topic of major interest. A functional [NiFe] hydrogenase model complex has recently been described by Ogo et al. (Science, 2013, 339, 682-683). Here, we report a Mössbauer and computational investigation of this model complex. This study affords deeper understanding of the electronic structure, the reactivity and the mechanism of H2 activation by this complex.

B-branch electron transfer in the photosynthetic reaction center of a Rhodobacter sphaeroides quadruple mutant. Q- and W-band electron paramagnetic resonance studies of triplet and radical-pair cofactor states.

The directionality of light-induced charge transfer in bacterial photosynthetic reaction centers (RCs) with respect to their A and B cofactor branches is still poorly understood on the electronic level. A prominent example is primary electron transfer in the RCs from the purple bacterium Rb. sphaeroides. Site-directed mutants with specific alterations of the cofactor binding sites with respect to the native system can deliver useful information toward a better understanding of the directionality enigma. Here we report on electron paramagnetic resonance (EPR) studies of the LDHW quadruple mutant, HL(M182)/GD(M203)/LH(M214)/AW(M260), which contains crucial mutations in the electron-transfer pathway. The directionality of the charge separation process was studied under light- or dark-freezing conditions first directly by 95 GHz (W-band) high-field EPR spectroscopy examining the charge-separated radical pairs (P865•+ Q(B)•−) of the primary donor P865, a bacteriochlorophyll dimer, and the terminal acceptor, QB, a ubiquinone-10. Second, it was studied indirectly by 34 GHz (Q-band) EPR examining the triplet states of the primary donor ((3)P865) that occur as a byproduct of the photoreaction. At 10 K, the triplet state has been found to derive mainly from an intersystem crossing mechanism, indicating the absence of charge-separated radical-pair states with a lifetime longer than 10 ns. B-branch charge separation and formation of the triplet-state (3)P865 via a radical-pair mechanism can be induced with low yield at 10 K by direct excitation of the bacteriopheophytins in the B-branch at 537 nm. At this wavelength, charge separation most probably proceeds via hole transfer from bacteriopheophytin to the primary donor. The triplet state of the primary donor is found to be quenched by the carotenoid cofactor present in the RC. The light-induced transient EPR signal of P•+ Q(B)•− is formed in a minor fraction of RCs (<1%) for RCs frozen in the dark. In contrast, about 70% of RCs illuminated upon freezing are trapped in the long-lived (τ > 104 s) charge-separated-state P•+ Q(B)•−. The temperature dependence of the EPR signals from P•+ Q(B)•− points to two factors responsible for the forward electron transfer to the terminal acceptor QB and for the charge-recombination reaction. The first factor involves a significant protein conformational change to initiate P•+ Q(B)•− charge separation, presumably by moving the quinone from the distal to the proximal position relative to the iron. The second factor includes protein relaxation, which governs the charge-recombination process along the B-branch pathway of the LDHW mutant.

EPR experiments to elucidate the structure of the ready and unready states of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F.

Isolation and purification of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F under aerobic conditions leads to a mixture of two states, Ni-A (unready) and Ni-B (ready). The two states are distinguished by different activation times and different EPR spectra. HYSCORE and ENDOR data and DFT calculations show that both states have an exchangeable proton, albeit with a different (1)H hyperfine coupling. This proton is assigned to the bridging ligand between Ni and Fe. For Ni-B, a hydroxo ligand is found. For Ni-A, either a hydroxo in a different orientation or a hydroperoxo-bridging ligand is present.

An ab initio quantum-chemical study of the blue-copper site of azurin.

The electronic structure of the blue-copper site of Pseudomonas aeruginosa azurin has been investigated by ab initio multireference determinantal configuration interaction (MRD-CI) calculations. A truncated site consisting of copper and its three equatorial ligands has been studied with emphasis on the g tensor and the nitrogen hyperfine tensors of the coordinating histidines. In the ground state the singly occupied molecular orbital (SOMO) involves a copper 3d orbital pi antibonded to the cysteine sulfur and sigma antibonded to the histidine nitrogens. A proper description of the electron-paramagnetic-resonance parameters has been achieved through the use of an effective core potential for copper up to and including the 3s electrons. Both the complete g tensor and the anisotropic hyperfine tensors at the nitrogens are essentially reproduced. Mulliken spin densities of 35 and 59% on copper and sulfur, respectively, and 2.1 and 1.7% on the respective coordinating nitrogens reflect the delocalized character of the SOMO and the inequivalence of the histidines.

Nutritional management during fetal and postnatal life, and the influence on testicular stereology and Sertoli cell numbers in Corriedale ram lambs.

The aim of the experiment was to determine whether supplementation of the lamb-ewe unit during intra-uterine and postnatal life affects testicular stereology, particularly Sertoli cell numbers, in 120 pregnant Corriedale ewes grazed either native pastures (control group) or improved pastures+grain supplement (treated group). Ewes bearing single ram lambs were maintained under the same feeding regime until lambs were castrated (99 days of age). Body weight, testosterone and FSH blood serum levels were recorded at 45, 75 and 99 days of age. Body weight was higher (P<0.01) in the treated group from birth on. Serum testosterone values did not differ between groups. Serum FSH values tended to differ at 45 days of age (P<0.06). Testicular weight and testes histology showed earlier pubertal development and a tendency for higher Sertoli cell numbers in the treated (supplemented) group. This tendency may indicate that extensively reared lambs supplemented during fetal and postnatal life have higher testicular growth and sperm production in later life.

Investigation of contemporary gilded forgeries of ancient coins.

Four contemporary forgeries of ancient gold coins were investigated regarding techniques used for gilding, and the composition of the gold cover and the base metal core. The forged coins are a Daric of the Persian Empire, a Gold Stater in the name of Alexander, and two Solidi of the late Roman Empire. A combination of modern analytical methods such as Scanning Electron Microscopy (SEM), Electron Probe Micro Analysis (EPMA), X-Ray Fluorescence Spectrometry (XRF), and SecondaryIon Mass Spectrometry (SIMS) was used. The results demonstrate that the coins represent the main three technologies of gilding used in antiquity. The core of the Daric is a silver Siglos, plated by leaf gilding. The Gold Stater was forged by foil gilding using a silver core. The Roman Solidi have a core of either silver or copper and were plated by fire gilding. On account of our results it is possible to compare the forgers' profits made by use of the different technologies of forging.

EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus.

The [Cu(I)-Cu(II)] half-met form of the dinuclear copper site of tyrosinase has been probed by continuous wave electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy in the presence and absence of inhibitors. In all cases the EPR spectrum is indicative of a d(x(2)-y(2)) ground state for the unpaired electron. From the cross-peaks observed in the HYSCORE spectra, proton hyperfine coupling constants were obtained that are compatible with a hydroxide ion in an equatorial coordination position of the paramagnetic copper. After changing the water solvent to D(2)O or after addition of the inhibitors p-nitrophenol or L-mimosine, the proton signals disappear. The relevance of these findings for understanding the catalytic cycle is discussed.

Influence of pre- and post-pubertal grazing regimes on adult testicular morphology in extensively reared corriedale rams.

The aim of the present study was to determine whether pre- and post-pubertal young rams on different grazing regimes, resulting in differences in live weight (LW), would show corresponding differences in testicular growth or testicular morphometry that could influence the reproductive traits of these rams upon reaching adulthood. Forty-one spring-born Corriedale rams were reared on either native pasture (low feeding level, Group L, n=22) or improved pasture (higher feeding level, Group H, n=19) from 1 to 7 months of age. Thereafter, half the animals in the native-pasture group were placed on improved pasture and vice versa, thus creating an additional four differential-grazing treatment groups (Groups LL, n=11; LH, n=11; HL, n=10; and HH, n=9). Animals were managed in this way until 18 months of age. Half the animals from each group were then castrated and their testes were subjected to morphometric analysis. The remaining animals (Groups LL, n=6; LH, n=6; HL, n=5; and HH, n=4) were managed together until 30 months of age (from 18 to 27 months on native pastures and from 27 to 30 months of age on improved pastures, at a stocking rate of two to three rams per hectare), whereupon they were also castrated for testicular morphometry. LW and scrotal circumference (SC) were recorded every 60 days. The stereological analysis of testicular parenchyma included counts of elongated spermatids and Sertoli cells. Differences (P<0.001) in LW were observed between feeding levels, even at 30 months of age. Differences (P<0.001) in SC existing at the end of the differential treatment (18 months of age) disappeared (n.s.) soon after. Most differences (P<0.05) in testicular morphometry existing at the end of the differential treatments were no longer significant 1 year later. It is concluded that changes in grazing management during pre- and post-pubertal periods can induce short-lived differences in testicular post-natal growth in Corriedale rams but do not influence testicular morphology or function later in life.

The binding of imidazole in an azurin-like blue-copper site.

Frozen solutions of the azurin mutant His117Gly in the presence of excess of methyl-substituted imidazoles have been investigated by electron spin-echo envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidazole is known to reconstitute a blue-copper site and variation of the non-protein bound ligand [N-methyl-, 2-methyl-, 4(5)-methylimidazole] has allowed the study of the copper-imidazole binding as a model for histidine binding in such sites. Quadrupole and hyperfine tensors of the remote nitrogen of the imidazoles have been determined. The quadrupole tensors indicate that the methyl-substituted imidazoles in the mutant adopt the same orientation relative to copper as the histidine-117 in the wild-type protein. Analysis of the hyperfine tensors in terms of spin densities reveals that the spin density on the remote nitrogen of the substituted imidazole has sigma and a variable pi character, depending on the position of the methyl group. For azurin the corresponding spin density is of virtually pure sigma character. In conclusion, blue-copper sites show subtle variations as regards the histidine/imidazole centred part of the wavefunction of the unpaired electron.

Influence of grazing management on the seasonal change in testicular morphology in Corriedale rams.

The present study was conducted: (a) to determine the degree of seasonal variation in testis stereology in Corriedale rams between autumn and winter; (b) to test the hypothesis that testis stereology of Corriedale rams grazing native pastures during autumn and winter would differ from those of Corriedale rams grazing sown pastures and supplemented with grain during the same period; and (c) to determine whether Sertoli cell numbers differ in adult rams between the breeding season (autumn) and the following non-breeding season (winter). Twenty experimental animals were studied. Six rams (autumn control group, C-A) that had been grazing on native pasture (stocking rate = 2-3 animals ha(-1)) were castrated at the beginning of the experiment (March, early autumn). Seven rams (winter control group, C-W) continued to graze on native pasture at the same stocking rate until the end of the experiment (August, late winter). Another seven rams (treated group, T) grazed on improved pasture (stocking rate = 1-2 animals ha(-1)) and were supplemented with 1 kg grain ram(-1) day(-1) until the end of the experiment. Live weight, scrotal circumference, serum testosterone concentration and selected testicular stereological parameters were measured. The treatment did not impede the winter reduction in testicular activity and reduced its magnitude slightly (group T) compared with controls (group C-W). Sertoli cell numbers were higher in autumn (group C-A) than in winter, both on native (group C-W) and sown pastures (group T). Diminishing Sertoli cell numbers between autumn and the following winter suggest the occurrence of that Sertoli cell death during this period. The results indicate that, although the reproductive activity of Corriedale rams is moderately seasonal, a restricted change in grazing and grain supplementation can only modify it to a limited extent.

Comparative studies of MCs(+)-SIMS and e(-)-beam SNMS for quantitative analysis of bulk materials and layered structures.

For the quantification of heterostructure depth profiles the knowledge of relative sensitivity factors (RSF) and the influence of matrix effects on the measured profiles is necessary. Matrix dependencies of the measured ion intensities have been investigated for sputtered neutral mass spectrometry (SNMS) and MCs(+)-SIMS. The use of Cs as primary ions for SNMS is advantageous compared to Ar because the depth resolution is improved without changing RSFs determined under Ar bombardment. No significant amount of molecules has been found in the SNMS spectra under Cs bombardment. Using MCs(+)-SIMS the RSFs are matrix dependent. An improvement of depth resolution can be achieved by biasing the sample against the primary ion beam for SNMS due to a reduction of the net energy of the primary ions and a resulting more gracing impact angle.

Quantitative determination of element distributions in silicon based thin film solar cells using SNMS.

The determination of elemental distributions in thin film solar cells based on amorphous silicon using electron beam SNMS is possible by quantifying the measured ion intensities. The relative sensitivity factors (RSFs) for all elements measured have to be known. The RSFs have been determined experimentally using implantation and bulk standards with known concentrations of the interesting elements. The measured RSFs have been compared with calculated RSFs. The model used for the calculation of the RSFs takes into account the probability for electron impact ionization and the dwell time of the neutrals inside the postionization region. The comparison between measured and calculated RSF shows, that this model is capable to explain the RSFs for most elements. Differences between calculated and measured values can be explained by the formation of hydride and fluoride molecules (in case of H and F) and influences of the angular distribution of the sputtered neutrals in case of Al. The experimentally determined RSFs have been used for a quantification of depth profiles of the i-, buffer-, p- and front contact layers of a-Si solar cells.