Biotransformation of Δ1-Progesterone Using Selected Entomopathogenic Filamentous Fungi and Prediction of Its Products' Bioactivity.

This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δ1-progesterone) (2) through microbiological transformation. For the role of catalysts, we used six strains of entomopathogenic filamentous fungi (Beauveria bassiana KCh J1.5, Beauveria caledonica KCh J3.3, Isaria fumosorosea KCh J2, Isaria farinosa KCh KW1.1, Isaria tenuipes MU35, and Metarhizium robertsii MU4). The substrate (2) was obtained by carrying out an enzymatic 1,2-dehydrogenation on an increased scale (3.5 g/L) using a recombinant cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans. All selected strains were characterized by the high biotransformation capacity for the used substrate. As a result of the biotransformation, six steroid derivatives were obtained: 11α-hydroxypregn-1,4-diene-3,20-dione (3), 6ß,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6ß-hydroxypregn-1,4-diene-3,11,20-trione (5), 6ß,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6ß,17ß-dihydroxyandrost-1,4-diene-3-one (7), and 12ß,17α-dihydroxypregn-1,4-diene-3,20-dione (8). The results show evident variability of the biotransformation process between strains of the tested biocatalysts from different species described as entomopathogenic filamentous fungi. The obtained products were tested in silico using cheminformatics tools for their pharmacokinetic and pharmacodynamic properties, proving their potentially high biological activities. This study showed that the obtained compounds may have applications as effective inhibitors of testosterone 17ß-dehydrogenase. Most of the obtained products should, also with a high probability, find potential uses as androgen antagonists, a prostate as well as menopausal disorders treatment. They should also demonstrate immunosuppressive, erythropoiesis-stimulating, and anti-inflammatory properties.

The catalytic activity of mycelial fungi towards 7-oxo-DHEA - an endogenous derivative of steroidal hormone dehydroepiandrosterone.

Seventeen species of fungi belonging to thirteen genera were screened for the ability to carry out the transformation of 7-oxo-DHEA (7-oxo-dehydroepiandrosterone). Some strains expressed new patterns of catalytic activity towards the substrate, namely 16ß-hydroxylation (Laetiporus sulphureus AM498), Baeyer-Villiger oxidation of ketone in D-ring to lactone (Fusicoccum amygdali AM258) and esterification of the 3ß-hydroxy group (Spicaria divaricata AM423). The majority of examined strains were able to reduce the 17-oxo group of the substrate to form 3ß,17ß-dihydroxy-androst-5-en-7-one. The highest activity was reached with Armillaria mellea AM296 and Ascosphaera apis AM496 for which complete conversion of the starting material was achieved, and the resulting 17ß-alcohol was the sole reaction product. Two strains of tested fungi were also capable of stereospecific reduction of the conjugated 7-keto group leading to 7ß-hydroxy-DHEA (Inonotus radiatus AM70) or a mixture of 3ß,7α,17ß-trihydroxy-androst-5-ene and 3ß,7ß,17ß-trihydroxy-androst-5-ene (Piptoporus betulinus AM39). The structures of new metabolites were confirmed by MS and NMR analysis. They were also examined for their cholinesterase inhibitory activity in an enzymatic-based assay in vitro test.

Transcranial Shear Wave Elastography of Neonatal and Infant Brains for Quantitative Evaluation of Increased Intracranial Pressure.

OBJECTIVES: Increased intracranial pressure (ICP) in neonates and infants is a severe disease state that requires adequate diagnosis and, depending on the clinical situation and whether it is increasing, a rapid and efficient therapy. Clinical evaluation, B-mode ultrasound, and Doppler ultrasound give rise to a basic noninvasive diagnosis of increased ICP. The purpose of this prospective study was 2-fold: first, to analyze the technical feasibility of obtaining shear wave elastography (SWE) measurements of an infant's brain, and second, to compare the values of healthy neonates to those who have hydrocephalus and are either suspected of having or invasively shown to have increased ICP. MATERIALS AND METHODS: This was a prospective, institutional review board-approved study of 184 neonates and infants with a mean age of 12 weeks (ranging from 1 day to 12 months). The final, technical evaluable cohort consisted of 166 infants, of whom 110 were healthy asymptomatic infants and 56 were diagnosed with hydrocephalus. Of the latter, 38 showed clinically increased ICP and 18 did not. Invasive ICP measurements were available from 47 of the children. All infants underwent systematic examination using B-mode ultrasound, Doppler ultrasound, and SWE using a high-resolution linear 15-MHz probe (Aixplorer; Supersonic), by 1 of 2 radiologists, each of whom had at least 5 years' experience examining children's brains and applying SWE. Semiquantitative and quantitative SWE measurements were performed.We compared the SWE values to each participant's clinical symptoms and to their invasive ICP measurement results. Correlations were calculated using Pearson and Spearman correlation coefficients. We used Student t test to compare the mean SWE values in healthy children to those of children with increased ICP. RESULTS: Shear wave elastography in the brain was technically feasible, giving reliable SWE measurements in 110 (88.7%) of 124 of healthy children and in 56 (93.3%) of 60 children with hydrocephalus. Shear wave elastography values and, thus, rigidity in the brain's parenchyma were significantly higher in children with hydrocephalus (n = 56) than in healthy children (n = 110; mean, 21.8 kPa vs 14.1 kPa; P = 0.0083). A thorough correlation between invasive ICP measurements and SWE values in a subgroup of patients with hydrocephalus revealed a direct correlation between increased ICP and increased SWE values (r = 0.69, P < 0.001). Mean SWE values were 30.8 kPa (range, 23.9-62.3 kPa) in patients with confirmed increased ICP (n = 35) versus 16.2 kPa (range, 10.2-41.9 kPa) in patients with nonincreased ICP (n = 12). CONCLUSIONS: Shear wave elastography is feasible in neonates with increased ICP and could be a useful additional diagnostic imaging and monitoring method for children verified or suspected to have increased ICP. However, more evidence is necessary to further evaluate the usefulness of SWE measurements in neonates with hydrocephalus. CLINICAL RELEVANCE: Shear wave elastography can be used as a surrogate marker for ICP in neonates and infants.

Metabolic fate of pregnene-based steroids in the lactonization pathway of multifunctional strain Penicillium lanosocoeruleum.

BACKGROUND: Metabolic activities of microorganisms to modify the chemical structures of organic compounds became an effective tool for the production of high-valued steroidal drugs or their precursors. Currently research efforts in production of steroids of pharmaceutical interest are focused on either optimization of existing processes or identification of novel potentially useful bioconversions. Previous studies demonstrated that P. lanosocoeruleum KCH 3012 metabolizes androstanes to the corresponding lactones with high yield. In order to explore more thoroughly the factors determining steroid metabolism by this organism, the current study was initiated to delineate the specificity of this fungus with respect to the cleavage of steroid side chain of progesterone and pregnenolone The effect of substituents at C-16 in 16-dehydropregnenolone, 16α,17α-epoxy-pregnenolone and 16α-methoxy-pregnenolone on the pattern of metabolic processing of these steroids was also investigated. RESULTS AND DISCUSSION: All of the analogues tested (except the last of the listed) in multi-step transformations underwent the Baeyer-Villiger oxidation to their δ-D-lactones. The activity of 3ß-HSD was a factor affecting the composition of the product mixtures. 16α,17α-epoxy-pregnenolone underwent a rare epoxide opening with retention stereochemistry to give four 16α-hydroxy-lactones. Apart from oxidative transformations, a reductive pathway was revealed with the unique hydrogenation of 5-ene double bond leading to the formation of 3ß,16α-dihydroxy-17a-oxa-D-homo-5α-androstan-17-one. 16α-Methoxy-pregnenolone was transformed to the 20(R)-alcohol with no further conversion. CONCLUSIONS: This work clearly demonstrated that P. lanosocoeruleum KCH 3012 has great multi-functional catalytic properties towards the pregnane-type steroids. Studies have highlighted that a slight modification of the D-ring of substrates may control metabolic fate either into the lactonization or reductive and oxidative pathways. Possibility of epoxide opening by enzymes from this microorganism affords a unique opportunity for generation of novel bioactive steroids.

Computational Prediction and Design for Creating Iteratively Larger Heterospecific Coiled Coil Sets.

A major biochemical goal is the ability to mimic nature in engineering highly specific protein-protein interactions (PPIs). We previously devised a computational interactome screen to identify eight peptides that form four heterospecific dimers despite 32 potential off-targets. To expand the speed and utility of our approach and the PPI toolkit, we have developed new software to derive much larger heterospecific sets (≥24 peptides) while directing against antiparallel off-targets. It works by predicting Tm values for every dimer on the basis of core, electrostatic, and helical propensity components. These guide interaction specificity, allowing heterospecific coiled coil (CC) sets to be incrementally assembled. Prediction accuracy is experimentally validated using circular dichroism and size exclusion chromatography. Thermal denaturation data from a 22-CC training set were used to improve software prediction accuracy and verified using a 136-CC test set consisting of eight predicted heterospecific dimers and 128 off-targets. The resulting software, qCIPA, individually now weighs core a-a' (II/NN/NI) and electrostatic g-e'+1 (EE/EK/KK) components. The expanded data set has resulted in emerging sequence context rules for otherwise energetically equivalent CCs; for example, introducing intrahelical electrostatic charge blocks generated increased stability for designed CCs while concomitantly decreasing the stability of off-target CCs. Coupled with increased prediction accuracy and speed, the approach can be applied to a wide range of downstream chemical and synthetic biology applications, in addition more generally to impose specificity on structurally unrelated PPIs.

Insight into the orientational versatility of steroid substrates-a docking and molecular dynamics study of a steroid receptor and steroid monooxygenase.

Numerous steroids are essential plant, animal, and human hormones. The medical and industrial applications of these hormones require the identification of new synthetic routes, including biotransformations. The metabolic fate of a steroid can be complicated; it may be transformed into a variety of substituted derivatives. This may be because a steroid molecule can adopt several possible orientations in the binding pocket of a receptor or an enzyme. The present study, based on docking and molecular dynamics, shows that it is indeed possible for a steroid molecule to bind to a receptor binding site in two or more orientations (normal, head-to-tail reversed, upside down). Three steroids were considered: progesterone, dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. Two proteins were employed as hosts: the human mineralocorticoid receptor and a bacterial Baeyer-Villiger monooxygenase. When the steroids were in nonstandard orientations, the estimated binding strength was found to be only moderately diminished and the network of hydrogen bonds between the steroid and the host was preserved.

Deriving Heterospecific Self-Assembling Protein-Protein Interactions Using a Computational Interactome Screen.

Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein-protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology.

Microbial Baeyer-Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation of D-homo lactones.

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer-Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6ß,11α-dihydroxyprogesterone or 17ß-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5ß-dihydrotestosterone. The experiments have demonstrated that the Baeyer-Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer-Villiger oxidation, also underwent reduction to 17ß-alcohols; activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD) has significant impact on the amount of the formed ring-D δ-lactone.

Plasma HER2 levels are not associated with cardiac function or hypertrophy in control subjects and heart failure patients.

A proper interaction between the endocardial-derived ligand Neuregulin-1 and the myocardial "Human Epidermal growth factor Receptor 2" (HER2) is essential for maintaining heart function. The shed extracellular domain (ECD) of HER2 circulates in blood and serves as a surrogate marker for breast cancer. Altered cardiac loading conditions are accompanied by dysregulation of the myocardial HER2 gene expression. We studied 193 controls with preserved ejection fraction (EF>55%) and 572 patients with different degrees of systolic heart failure: 98 had EF 45-55%, 138 patients EF 35-44%, and 336 patients, EF <35%, respectively. The corresponding mean HER2 levels were 6.44 ± 0.46 ng/mL, 6.07 ± 0.76 ng/mL and 6.57 ± 0.87 ng/mL, and 6.17 ± 0.71 ng/mL, respectively. Furthermore, there was no significant association between plasma HER2 levels and left ventricular filling pressures or the left ventricular wall thickness. The HER2 plasma levels do not reflect the cardiac function and are therefore not useful as a biomarker for heart failure.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca(2+) reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF's profibrotic function in the heart.

Myocarditis in newborn wild-type BALB/c mice infected with the murine gamma herpesvirus MHV-68.

OBJECTIVES: Animal models of human Epstein-Barr virus (EBV) infection include EBV infection of primates and infection of mice with MHV-68, a further gamma herpesvirus (gamma-HV). We aimed at extending the MHV-68 model to study gamma-HV-related cardiac disease. METHODS: Newborn wild-type BALB/c- (n=107), wild-type C57BL/6- (n=17) and immunodeficient B6-(Rag1) mice (n=18) were infected by nasal inoculation and evaluated for histopathological changes as well as tissue viral loads. RESULTS: From day 5 on BALB/c mice showed myocardial viral replication. Whereas focal inflammation occurred simultaneously, necrosis was first observed 9 days post-infection. The maximum rates of necrosis (40%) and of focal inflammation (33%) were found after 10 to 12 and 33 to 35 days, respectively. Some animals developed persistent viral activity and inflammation throughout the observation period of three months. Inflammation was mainly related to T cell infiltrates. Although C57BL/6 mice also showed myocardial inflammation, necrosis was not found suggesting differences in the susceptibility to the virus in distinct mouse strains. In immunodeficient animals higher myocardial viral loads were observed compared to wild-type mice but no cardiac lesions, which suggests that the antiviral immune response contributed to the lesions. CONCLUSIONS: The model system presented here is the first to allow detailed studies on cardiac disease caused by gamma-HV infections and may facilitate the development of more specific treatment options for human cardiac EBV infection.