First insights into structure-function relationships of alkylglycerol monooxygenase

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O-alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzym

Ultra-wideband 3D image processing for improving landmine detection with GPR, Journal of Telecommunications and Information Technology, 2005, nr 2

This paper describes a new landmine detection algorithm starting from high resolution 3D ground penetrating radar (GPR) images. The algorithm consists of two procedures, object detection and object classification; both strongly depend on the properties of 3D GPR images. The algorithm has been tested on data measured with an ultrawideband (UWB) video impulse radar (VIR) system developed by the International Research Centre for Telecommunications and Radar (IRCTR). It was found that the algorithm is able to detect all landmines (including difficult to detect M14 mines) and classifies almost all landmines correctly with a large reduction in the number of false alarms caused by clutter. It turns out that for clutter removal it is most effective to eliminate detected objects with a small height

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.

Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase

Gamma Knife stereotactic radiosurgery for intraocular retinoblastoma: a 5-year experience

External beam radiotherapy (EBR) remained for a long time the only method of treatment in children with recurrent and resistant retinoblastoma (RB). This method often leads to serious complications, including the occurrence of secondary malignant tumors. Currently, EBR is used as second-line (salvage) therapy. There is no data in the literature of using Gamma Knife stereotactic radiosurgery (GKRS) in RB treatment.Purpose. To present 5-year experience of using GKRS in patients with RB.Material and methods. 16 children (17 eyes) were treated using GKRS in the period from 2015 to 2019. Mean patient age was 34.7 months (range, 12–114 months). The eyes were classified as group B (n=4), C (n=1), D (n=12). 3 children had the last eye. All patients received systemic and local chemotherapy, all types of local treatment modalities before using GKRS. Recurrent and resistant RB was the indication for GKRS. Marginal 50% mean dose was 22 Gу (range, 20–24 Gу), depending on tumour type and location. Radiation doses were evaluated accounting critical eye structures and the orbit bones.Results. Complete regression was achieved in 11 patients, partial in 2. Four patients underwent enucleation after GKRS. Indications for enucleation were retinoblastoma recurrence (n=2) and vitreous hemorrhage with total retinal detachment (n=2). 13 eyes were salvaged with no signs of keratopathy, uveitis or damage of orbital and surrounding tissues during mean follow-up 30.6 months (range, 7–60 months). Сomplications of different severity occurred in 13 patients, including vitreous hemorrhage in 6 patients, which was successfully treated both conservative (n=3) and using pars plana vitrectomy with simultaneous melphalan irrigation (n=3).Conclusion. The first experience of GKRS as an alternative to enucleation in patients with RB was proved to be reasonable and successful

Одномоментная лапароскопическая билатеральная нефруретерэктомия, аллотрансплантация трупной почки и формирование везикостомы у больного с нейрогенным мочевым пузырем

We present a case of simultaneous laparoscopic bilateral nephroureterectomy, cadaveric kidney allotransplantation and performance of vesicostomy. This observation shows that patients with end-stage kidney disease, primarily caused by neurogenic bladder dysfunction, can be successfully treated via surgery. The course of early postoperative period and further rehabilitation did not differ significantly from that obtainable after standard kidney allotransplantation.В статье представлен случай одномоментной лапароскопической билатеральной нефруретерэктомии, аллотрансплантации трупной почки и формирование везикостомы. Данное наблюдение показывает возможность успешного хирургического лечения у пациентов с терминальной стадией почечной недостаточности, первопричиной которой являлась нейрогенная дисфункция мочевого пузыря. Течение раннего послеоперационного периода и дальнейшая реабилитация существенно не отличались от таковых после стандартной аллотрансплантации почки

Global Retinoblastoma Treatment Outcomes Association with National Income Level

Purpose: To compare metastasis-related mortality, local treatment failure, and globe salvage after retinoblastoma in countries with different national income levels. Design: International, multicenter, registry-based retrospective case series. Participants: Two thousand one hundred ninety patients, 18 ophthalmic oncology centers, and 13 countries on 6 continents. Methods: Multicenter registry-based data were pooled from retinoblastoma patients enrolled between January 2001 and December 2013. Adequate data to allow American Joint Committee on Cancer staging, eighth edition, and analysis for the level, as defined by the 2017 United Nations World Population Prospects, and included high-income countries (HICs), upper middle-income countries (UMICs), and lower middle-income countries (LMICs). Patient survival was estimated with the Kaplan-Meier method. Logistic and Cox proportional hazards regression models were used to determine associations between national income and treatment outcomes. Main Outcome Measures: Metastasis-related mortality and local treatment failure (defined as use of secondary enucleation or external beam radiation therapy). Results: Most (60%) study patients resided in UMICs and LMICs. The global median age at diagnosis was 17.0 months and higher in UMICs (20.0 months) and LMICs (20.0 months) than HICs (14.0 months; P < 0.001). Patients in UMICs and LMICs reported higher rates of disease-specific metastasis-related mortality and local treatment failure. As compared with HICs, metastasis-related mortality was 10.3-fold higher for UMICs and 9.3-fold higher for LMICs, and the risk for local treatment failure was 2.2-fold and 1.6-fold higher, respectively (all P < 0.001). Conclusions: This international, multicenter, registry-based analysis of retinoblastoma management revealed that lower national income levels were associated with significantly higher rates of metastasis-related mortality, local treatment failure, and lower globe salvage. (C) 2020 by the American Academy of Ophthalmology.Peer reviewe

Structural model of the open-closed-inactivated cycle of prokaryotic voltage-gated sodium channels

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels

Quantitative Modeling of Currents from a Voltage Gated Ion Channel Undergoing Fast Inactivation

Ion channels play a central role in setting gradients of ion concentration and electrostatic potentials, which in turn regulate sensory systems and other functions. Based on the structure of the open configuration of the Kv1.2 channel and the suggestion that the two ends of the N-terminal inactivating peptide form a bivalent complex that simultaneously blocks the channel pore and binds to the cytoplasmic T1 domain, we propose a six state kinetic model that for the first time reproduces the kinetics of recovery of the Drosophila Shaker over the full range of time scales and hyperpolarization potentials, including tail currents. The model is motivated by a normal mode analysis of the inactivated channel that suggests that a displacement consistent with models of the closed state propagates to the T1 domain via the S1-T1 linker. This motion stretches the bound (inactivating) peptide, hastening the unblocking of the pore. This pulling force is incorporated into the rates of the open to blocked states, capturing the fast recovery phase of the current for repolarization events shorter than 1 ms. If the membrane potential is hyperpolarized, essential dynamics further suggests that the T1 domain returns to a configuration where the peptide is unstretched and the S1-T1 linker is extended. Coupling this novel hyperpolarized substate to the closed, open and blocked pore states is enough to quantitatively estimate the number of open channels as a function of time and membrane potential. A straightforward prediction of the model is that a slow ramping of the potential leads to very small currents

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively