Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the kcat /KM values for two alternative substrates (the ‘discrimination index’). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases’ observed selectivities were remarkably high. In particular, GOT1 reacted ~106-fold less efficiently when the side-chain carboxylate of the ’physiological’ substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using kcat /KM values to derive a global measure of enzyme specificity

A theoretical framework for network monitoring exploiting segment routing counters

Self-driving networks represent the next step of network management techniques in the close future. A fundamental point for such an evolution is the use of Machine Learning based solutions to extract information from data coming from network devices during their activity. In this work we focus on a new type of data, available thanks to the definition of the novel SRv6 paradigm, referred to as SRv6 Traffic Counters (SRTCs). SRTCs provide aggregated measurements related to forwarding operations performed by SRv6 routers. In this work a detailed description of different SRTCs types (SR.INT, PISD, PSID.TM and POL) is provided and their relationships is formalized. The theoretical framework deployed is used to identify, on the basis of network configuration parameters of both SRv6 and IGP protocols, the minimum set of independent SRTCs to characterize the Network Status: we show that about the 80% of counters can be neglected with no information loss. We also apply our framework to two use cases: i) Traffic Matrix (TM) Assessment and ii) Traffic Anomaly Detection. For the TM assessment, we show that in a partially deployed SRv6 scenario a specific type of SRTCs, i.e., PSID, is more reliable than other ones; on the contrary, in a fully deployed scenario POL and PSID.TM counters provide the full TM knowledge. For the Traffic Anomaly Detection case, we show that known solutions based on link load measurements can be improved when integrating SRTCs information

Oxygen diffusion pathways in mutated forms of a LOV photoreceptor from Methylobacterium radiotolerans: a molecular dynamics study.

Mr4511 LOV (Light, Oxygen and Voltage) protein is a blue light sensing photoreceptor from Methylobacterium radiotolerans, binding flavin mononucleotide (FMN) as chromophore. Blue light activation of LOV domains triggers the reversible formation of a FMN-cysteine adduct by a photocycle that goes through the FMN excited triplet state. LOV domains can be engineered as fluorescent sensors and actuators for optogenetics and photomedicine [1]. First experimental data on Mr4511 LOV protein [2] indicate its high potential as a photosensitizer for singlet oxygen (SO) the cytotoxic reactive excited state of molecular oxygen, produced by diffusion limited energy transfer from the FMN triplet state. This feature is obtained after the single mutation of reactive cysteine C71, a change that prevents formation of the photoproduct. In addition, the lack of a tryptophan, conserved in ca. 75% of LOV domains and shown to strongly quench the FMN triplet lifetime (T) in LOV proteins, allows for Mr4511 LOV a longer T than for other LOV domains in C71S and C71G variants [2]. After an homology modeling of Mr4511 LOV, that has lead to a dimeric protein stabilized by the presence of a strong leucine zipper in the C-terminal helices, a mutation of the photocycle substrate cysteine into serine (C71S) has been introduced in silico to make it a SO photosensitizer, and the mutated form stability was tested by MD simulations. Afterwards, both transient and persistent oxygen channels were detected and analysed both in the wt and in the mutated protein. Molecular oxygen was then placed both outer and into the chromophore cavity and potential diffusion pathways were explored with MD simulations, showing a high accessibility of the binding cavity and a high persistence of oxygen inside. Mutations that might favor SO generation were designed based on their position with respect to the FMN and the oxygen channels, taking into account the ability of certain amino acids to quench FMN triplet state and SO. Therefore, C71S/Y61T and C71S/Y61S double mutants were generated in silico and their stability was checked. The analysis of their oxygen diffusion pathways showed an increased diffusion and persistence of oxygen molecules inside the binding cavity, indicating a promising model for SO photosensing and its biomedical and biophysical applications. [1] A. Losi, et al., Chem. Rev. 118 (2018): 10659-10709. [2] E. Consiglieri, et al., Photochem. Photobiol. Sci. 18 (2019): 2657–2660

The adjuvant activity of two urea derivatives on cytokinins: an example of serendipitous dual effect

The aim of this study was to investigate the action spectrum of two urea derivatives, the 1,3-di(benzo[d]oxazol-5-yl)urea (5-BDPU) and the 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU). In order to evaluate a possible adjuvant activity on cytokinins the compounds alone or in the simultaneous presence of different cytokinins were assayed either on in vitro typical cytokinin-related bioassays, or on in planta interaction with cytokinin signal transduction pathway. The compounds ability to activate the cytokinin receptor CRE1/AHK4 was studied either by a heterologous bacterial assay or by a competitive binding assay and docking simulations were performed with the crystal structure of the same receptor. Then, owing to their chemical structure which resembles that of urea-type cytokinins, the ability of 5- and 6-BDPU to inhibit the activity of cytokinin oxidase/dehydrogenase of Zea mays (ZmCKX1) was investigated and docking simulations were performed as well. Accordingly to the experimental results, we speculate that BDPUs could show a dual activity: the blocking of the conformational re-adaption of CRE1/AHK4 receptor maintaining the cytokinin inside its binding pocket, thus possibly enhancing its kinase action; the inhibition of cytokinin oxidase/dehydrogenase activity thus possibly preventing its cleavage of natural cytokinins with isoprenoid side chain. Graphic abstract: [Figure not available: see fulltext.

Dynamic in-network classification for service function chaining ready SDN networks

Service Function Chaining (SFC) paradigm consists in steering traffic flows through an ordered set of Service Functions (SFs) so that to realize complex end to end services. SFC architecture introduces all the logical functions that need to be developed in order to provide the required service. The SFC overlay infrastructure can be built on top of many different underlay network technologies. The high flexibility and centrally controlled feature of Software Defined Networking (SDN), make SDN networks to be a perfect underlay to build the SFC architecture. Due to Ternary Content Address Memory (TCAM) limited size, SDN switches have a limitation in the number of flow rules that can be hosted. This constraint is particularly penalizing in case of the SFC classifier function, since it requires to manage a high number of different flows. The limitation imposed by the TCAM size on the SFC classifier can be a bottleneck for the number of SFC requests that the SDN-based SFC architecture can handle. In this paper we define the Dynamic Chain Request Classification Offloading (D-CRCO) problem, as the one of maximizing the number of accepted SFC requests, having the possibility of: i) implement the SFC classifier also in a node that is internal to the SDN-based SFC domain, and ii) install classification rules in a reactive fashion. Furthermore, we propose the Dynamic Nearest Node (DNN) heuristic to solve the D-CRCO problem. Performance evaluation shows that by using DNN heuristic it is possible to triple the number of accepted requests, with respect to existing solutions

The response of VEGF-stimulated endothelial cells to angiostatic molecules is substrate-dependent

BACKGROUND: The microenvironment surrounding cells can exert multiple effects on their biological responses. In particular the extracellular matrix surrounding cells can profoundly influence their behavior. It has been shown that the extracellular matrix composition in tumors is vastly different than that found in normal tissue with increased amounts of certain matrices such as collagen I. It has been previously demonstrated that VEGF stimulation of endothelial cells growing on type I collagen results in the induction of bcl-2 expression and enhanced endothelial cell survival. We sought to investigate whether this increased endothelial cell survival resulted in the failure of angiostatic molecules to inhibit angiogenesis. RESULTS: We now demonstrate that VEGF-induced survival on collagen I impairs the ability of three known angiostatic molecules, TSP-1, IP-10 and endostatin to inhibit endothelial cell proliferation. Apoptosis of endothelial cells, growing on collagen I, induced by TSP-1 and IP-10 was also inhibited following VEGF stimulation. In contrast, endostatin induced apoptosis in these same cells. Further analysis determined that endostatin did not decrease the expression of bcl-2 nor did it increase activation of caspase-3 in the presence of VEGF. Alternatively, it appeared that in the presence of VEGF, endostatin induced the activation of caspase-8 in endothelial cells grown on collagen I. Furthermore, only endostatin had the ability to inhibit VEGF-induced sprout formation in collagen I gels. CONCLUSION: These data suggest that TSP-1, IP-10 and endostatin inhibit endothelial cells via different mechanisms and that only endostatin is effective in inhibiting angiogenic activities in the presence of collagen I. Our results suggest that the efficacy of angiostatic treatments may be impaired depending on the context of the extracellular matrix within the tumor environment and thus could impede the efficacy of angiostatic therapies

Role of C‐X‐C chemokines as regulators of angiogenesis in lung cancer

Lung cancer is the leading cause of malignancy‐related mortality in the U.S. and is predicted to increase over the remainder of this decade. Despite attempts to advance early diagnosis and use combination therapies, the clinical response of this cancer yields an overall 5‐year survival rate of less than 15%. Clearly, new strategies for therapy are indicated. Although carcinogenesis is complex, tumor growth beyond 1–2 mm3 is dependent on angiogenesis. One of the potential mechanisms that allows for tumorigenesis is dysregulation of the balance of angiogenic and angiostatic factors that favors net neovascularization within the primary tumor. Numerous studies have investigated the role of a variety of molecules in the regulation of angiogenesis. Recently, interleukin‐8 (IL‐8), a member of the C‐X‐C chemokine family, has been found to be an angiogenic factor. In contrast, platelet factor 4 (PF4), another C‐X‐C chemokine, has been shown to have angiostatic properties. It is interesting that the major structural difference between IL‐8 and PF4 is the presence of the NH2‐terminal ELR (Glu‐Leu‐Arg) motif that precedes the first cysteine amino acid residue of IL‐8 and is important in ligand/receptor interactions. We hypothesize that angiogenesis associated with tumorigenesis is dependent on members of the C‐X‐C chemokine family acting as either angiogenic or angiostatic factors. This paradigm predicts that the biological balance in the expression of these C‐X‐C chemokines dictates whether the neoplasm grows and develops metastatic potential or regresses. In this review we discuss our recent laboratory findings that support this contention and suggest that further elucidation of the biology of C‐X‐C chemokines in the context of neovascularization of nonsmall cell lung cancer will permit novel targeted therapy aimed specifically at attenuating tumor growth and metastasis. J. Leukoc. Biol. 57: 752–762; 1995.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141888/1/jlb0752.pd

Integrative and collaborative care models between pediatric oral health and primary care providers: a scoping review of the literature

ObjectivesCollaborative and/or integrative care between oral health and primary care providers can increase access to care to a more expansive population, helping to mitigate oral health related disease. The objective of this review was to present and evaluate different types of care models that exist between oral health and primary care providers in pediatric settings.MethodsA literature search was conducted using five databases: MEDLINE/PubMed, ISI Web of Science, Dentistry and Oral Sciences Source, Cochrane Database, and EMBASE, to identify literature from January 1990 to January 2016. Combinations of controlled terms were utilized. Eligible sources targeted pediatric populations ages 1‐17 and provided descriptions of existing collaborative and/or integrative models.ResultsData related to the practice model, oral care provided, level of integration/collaboration and workflow were extracted. Sixteen articles were included that discussed 24 models of collaboration. These models provided ranges of services, but each offered a minimum of oral health risk assessment, oral health instruction, topical fluoride application and assessment for further treatment. These models included different levels of collaboration based off a ranking system created by the authors with 16.6 percent (4) classified as low, 54.2 percent (13) as medium and 29.2 percent (7) as high.ConclusionsExisting care models offered varying services and levels of integration and/or collaboration, but each offered a baseline of oral care. Most of these collaborations were based within Federally Qualified Health Centers and aimed to ease access to care issues.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145544/1/jphd12267.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145544/2/jphd12267_am.pd

Small Angle X-ray Scattering From Lipid-bound Myelin Basic Protein In Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.861 I455460Altschul, S.F., Madden, T.L., Schäffer, A.A.Z.J., Zhang, Z., Miller, W., Gapped, L.D.J., BLAST and PSI-BLAST: A new generation of protein database search programs (1997) Nucleic Acids Res., 25, pp. 3389-3402Alvord, E.C., Kies, M.W., Suckling, A.L., (1984) Experimental Allergic Encephalomyelitis: A Useful Model for Multiple Sclerosis, , Alan R. Liss Inc., New YorkBates, I.R., Harauz, G., Molecular dynamics exposes α-helices in myelin basic protein (2003) J. Mol. Mod., , published online 24 July 2003Beniac, D.R., Luckevich, M.D., Czamota, G.J., Tompkins, T.A., Ridsdale, R.A., Ottensmeyer, F.P., Moscarello, M.A., Harauz, G., Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles (1997) J. Biol. Chem., 272, pp. 4261-4268Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., Bourne, P.E., The Protein Data Bank (2000) Nucleic Acids Res., 28, pp. 235-242Bobba, A., Munno, I., Greco, B., Pellegrino, N.M., Riccio, P., Jirillo, E., Quagliariello, E., On the spontaneous adherence of myelin basic protein to T-lymphocytes (1991) Biochem. Biophys. Res. Commun., 180, pp. 1125-1129Boggs, J.M., Moscarello, M.A., Papahadiopolos, D., Structural organization of myelin-role of lipid-protein interactions determined in model systems (1982) Lipid-Protein Interactions, 2, pp. 1-51. , P. C. Jost and O. H. Griffith, editors. Wiley & Sons, New YorkDeibler, G., Martenson, R.E., Kies, M.W., Large-scale preparation of myelin basic protein from central nervous tissue of several mammalian species (1972) Prep. Biochem., 2, pp. 139-165Deibler, G.E., Boyd, L.F., Kies, M.W., Proteolytic activity associated with purified myelin basic protein (1984) Experimental Allergic Encephalomyelitis: A Useful Model for Multiple Sclerosis, pp. 249-256. , E. C. Alvord Jr., M. W. Kies, and A. J. Suckling, editors. Liss, New YorkEpand, R.M., Moscarello, M.A., Zierenberg, B., Vail, W.J., The folded conformation of the encephalogenic protein of the human brain (1974) Biochemistry, 13, pp. 1264-1267Garavito, R.M., Ferguson-Miller, S., Detergents as tools in membrane biochemistry (2001) J. Biol. Chem., 276, pp. 32403-32406Glatter, O., Convolution square root of band-limited symmetrical functions and its applications to small-angle scattering data (1981) J. Appl. Crystallogr., 14, pp. 101-108Gow, A., Smith, R., The thermodynamically stable state of myelin basic protein in solution is a flexible coil (1989) Biochem. J., 257, pp. 535-540Guinier, A., Fournet, G., (1955) Small-Angle Scattering of X-Rays, , Wiley & Sons, New YorkHaas, H., Torrielli, M., Steitz, R., Cavatorta, P., Sorbi, R., Riccio, P., Gliozzi, A., Myelin model membranes on solid substrate (1998) Thin Sol. Films, 327-329, pp. 627-631Kellermann, G., Vicentin, E., Tamura, E., Rocha, M., Tolentino, H., Barbosa, A., Craievich, A., Torriani, I., The small-angle x-ray scattering beamline of the Brazilian Synchrotron Light Laboratory (1997) J. Appl. Crystallogr., 30, pp. 880-883Kirschner, D.A., Blaurock, A.E., Organization, phylogenetic variations and dynamic transitions of myelin (1992) Myelin: Biology and Chemistry, pp. 3-78. , R. E. Martenson, editor. CRC Press, Boca Raton, FLKrigbaum, W.R., Hsu, T.S., Molecular conformation of bovine A1 basic protein, a coiling macromolecule in aqueous solution (1975) Biochemistry, 14, pp. 2542-2546Liebes, L.F., Zand, R., Phillips, W.D., Solution behavior, circular dichroism and 22 MHz PMR studies of the bovine myelin basic protein (1975) Biochim. Biophys. Acta, 405, pp. 27-39Liuzzi, G.M., Tamborra, R., Ventola, A., Bisaccia, F., Quagliariello, E., Riccio, P., Different recognition by clostripain of myelin basic protein in the lipid-bound and lipid-free forms (1996) Biochem. Biophys. Res. Commun., 226, pp. 566-571Lolli, F., Liuzzi, G.M., Vergelli, M., Massacesi, L., Ballerini, C., Amaducci, L., Riccio, P., Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis (1993) J. Neuroimmunol., 44, pp. 69-76Massacesi, L., Vergelli, M., Zehetbauer, B., Liuzzi, G.M., Olivotto, J., Ballerini, C., Uccelli, A., Amaducci, L., Induction of the autoimmune encephalomyelitis in rats and immune response to myelin basic protein in lipid bound form (1993) J. Neurol. Sci., 119, pp. 91-98Mazzanti, B., Vergelli, M., Riccio, P., Martin, R., McFarland, H.F., Liuzzi, M.G., Amaducci, L., Massacesi, L., T-cell response to myelin basic protein and lipid-bound myelin basic protein in patients with Multiple Sclerosis and health donors (1998) J. Neuroimmunol., 82, pp. 96-100Moscarello, M.A., Evolving biological concepts and therapeutic approaches (1996) Cell Biology and Pathology of Myelin, , R. M. Devon, R. Doucette, B. H. J. Juurlink, A. J. Nazarali, D. J. Schreyer, and V. M. K. Verge, editors. Plenum Publishing, New YorkOliveira, C.L.P., Dos Santos, D.R., Kellermann, G., Plivelic, T., Torriani, I.L., Data treatment program for the SAXS beamline (1997) LNLS Technical Note, , unpublished, available for beam line usersPerkins, J.P., X-ray and neutron solution scattering (1988) Modern Physical Methods in Biochemistry, Part B, pp. 134-265. , A. Neuberger and L. L. M. van Deenen, editors. Elsevier, Amsterdam, The NetherlandsPolverini, E., Fasano, A., Zito, F., Riccio, P., Cavatorta, P., Conformation of bovine myelin basic protein purified with bound lipids (1999) Eur. Biophys. J., 28, pp. 351-355Readhead, C., Popko, B., Takahashi, N., Shine, H.D., Saavedra, R.A., Sidman, R.L., Hood, L., Expression of a myelin basic protein gene in transgenic mice: Correlation of the dismyelinating phenotype (1987) Cell, 48, pp. 703-712Riccio, P., Rosenbusch, J.P., Quagliarello, E., A new procedure to isolate brain myelin basic protein in a lipid-bound form (1984) FEBS Lett., 177, pp. 236-240Riccio, P., Masotti, L., Cavatorta, P., De Santis, A., Juretic, D., Bobba, A., Pasquali-Ronchetti, I., Quagliariello, E., Myelin basic protein ability to organize lipid bilayers: Structural transitions in bilayers of lysophosphatidylcholine micelles (1986) Biochem. Biophys. Res. Commun., 134, pp. 313-319Riccio, P., Quagliariello, E., Lipid-bound, native-like, myelin basic protein: A well-known protein in a new guise, or an unlikely story? (1993) J. Neurochem., 61, pp. 787-788Riccio, P., Bobba, A., Romito, E., Minetola, M., Quagliariello, E., A new detergent to purify CNS myelin basic protein isoforms in lipid-bound form (1994) Neuroreport, 24, pp. 689-692Riccio, P., Fasano, A., Borenshtein, N., Bleve-Zacheo, T., Kirschner, D.A., Multilamellar packing of myelin modeled by lipid-bound MBP (2000) J. Neurosci. Res., 59, pp. 513-521Ridsdale, R.A., Beniac, D.R., Tompkins, T.A., Moscarello, M.A., Harauz, G., Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis (1997) J. Biol. Chem., 272, pp. 4269-4275Rosenbusch, J.P., Stability of membrane proteins: Relevance for the selection of appropriate methods for high-resolution structure determinations (2001) J. Struct. Biol., 136, pp. 144-157Semenyuk, V., Svergun, D.I., GNOM - A program package for small angle scattering data processing (1991) J. Appl. Crystallogr., 24, pp. 537-540Smith, R., Self-association of myelin basic protein: Enhancement by detergents and lipids (1982) Biochemistry, 12, pp. 2697-2701Smith, R., The basic protein of CNS myelin: Its structure and ligand binding (1992) J. Neurochem., 59, pp. 1589-1608Staugaitis, S.M., Colman, D.R., Pedraza, L., Membrane adhesion and other functions for the myelin basic proteins (1996) Bioessays, 18, pp. 13-18Stoffel, W., The myelin membrane of the central nervous system-essential macromolecular structure and function (1990) Angew. Chem. Int. Ed. Engl., 29, pp. 958-976Svergun, D.I., Petoukhov, M.V., Koch, M.H.J., Determination of domain structure of proteins from x-ray solution scattering (2001) Biophys. J., 80, pp. 2946-2953Vergelli, M., Pinet, V., Vogt, A.B., Kalbus, M., Malnati, M., Riccio, P., Long, E.O., Martin, R., HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing (1997) Eur. J. Immunol., 27, pp. 941-951Vriend, G., WHAT IF: A molecular modeling and drug design program (1990) J. Mol. Graph., 8, pp. 52-5

The complex TIE between macrophages and angiogenesis

Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre-existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro- and anti-angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2-expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin-TIE pathway