Academic literature on the topic 'C-type inactivation'
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Journal articles on the topic "C-type inactivation"
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Villalba-Galea, Carlos A., Takeharu Kawano, and Diomedes E. Logothetis. "C-Type Inactivation in KV2.1 Channels." Biophysical Journal 116, no. 3 (February 2019): 15a. http://dx.doi.org/10.1016/j.bpj.2018.11.122.
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Yan, Jiusheng, Qin Li, and Richard W. Aldrich. "Closed state-coupled C-type inactivation in BK channels." Proceedings of the National Academy of Sciences 113, no. 25 (June 13, 2016): 6991–96. http://dx.doi.org/10.1073/pnas.1607584113.
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Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.
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Li, Xiaoyan, Glenna C. L. Bett, Xuejun Jiang, Vladimir E. Bondarenko, Michael J. Morales, and Randall L. Rasmusson. "Regulation of N- and C-type inactivation of Kv1.4 by pHo and K+: evidence for transmembrane communication." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 1 (January 1, 2003): H71—H80. http://dx.doi.org/10.1152/ajpheart.00392.2002.
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Kv1.4 encodes a slowly recovering transient outward current ( I to), which inactivates by a fast N-type (intracellular ball and chain) mechanism but has slow recovery due to C-type inactivation. C-type inactivation of the NH2-terminal deletion mutant (fKv1.4ΔN) was inhibited by 98 mM extracellular K+concentration ([K+]o), whereas N-type was unaffected. In 98 mM [K+]o, removal of intracellular K+ concentration ([K+]i) speeded C-type inactivation but had no effect on N-type inactivation, suggesting that C-type inactivation is sensitive to K+ binding to intracellular sites. C-type inactivation is thought to involve closure of the extracellular pore mouth. However, a valine to alanine mutation on the intracellular side of S6 (V561A) of fKv1.4ΔN alters recovery and results in anomalous speeding of C-type inactivation with increasing [K+]o. Extracellular pH (pHo) modulated both N- and C-type inactivation through an S5-H5 linker histidine (H508) with acidosis speeding both N- and C-type inactivation. Mutation of an extracellular lysine to a tyrosine (K532Y) slowed C-type inactivation and inhibited the pH dependence of both N- and C-type inactivation. These results suggest that mutations, [K+], and pH modulate inactivation through membrane-spanning mechanisms involving S6.
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Kurata, Harley T., Zhuren Wang, and David Fedida. "NH2-terminal Inactivation Peptide Binding to C-type–inactivated Kv Channels." Journal of General Physiology 123, no. 5 (April 12, 2004): 505–20. http://dx.doi.org/10.1085/jgp.200308956.
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In many voltage-gated K+ channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na+ permeability of C-type–inactivated K+ channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type–inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na+ tail currents normally observed through C-type–inactivated channels, an effective blockade of the peak Na+ tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type–inactivated channels. In C-type–deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type–inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na+ tail of C-type–inactivated channels. Stable binding between the inactivation peptide and the C-type–inactivated state results in slower current decay, and a reduction of the Na+ tail current magnitude, due to slower transition of channels through the Na+-permeable states traversed during recovery from inactivation.
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Claydon, T. W., M. R. Boyett, A. Sivaprasadarao, and C. H. Orchard. "Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K+ channel." American Journal of Physiology-Cell Physiology 283, no. 4 (October 1, 2002): C1114—C1121. http://dx.doi.org/10.1152/ajpcell.00542.2001.
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Acidosis inhibits current through the Kv1.4 K+ channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Δ2–146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Δ2–146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Δ2–146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.
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Starkus, John G., Lioba Kuschel, Martin D. Rayner, and Stefan H. Heinemann. "Ion Conduction through C-Type Inactivated Shaker Channels." Journal of General Physiology 110, no. 5 (November 1, 1997): 539–50. http://dx.doi.org/10.1085/jgp.110.5.539.
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C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.
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Trefilov, B. B., N. V. Nikitina, and I. K. Leonov. "THE KINETICS OF THE INACTIVATION OF THE HEPATITIS VIRUS TYPE I (AVIHEPATOVIRUS, PICORNAVIRIDAE)." Problems of Virology, Russian journal 63, no. 3 (June 20, 2018): 135–38. http://dx.doi.org/10.18821/0507-4088-2018-63-3-135-138.
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Experimental data on the kinetics of the inactivation of the vaccine strain of the duckling hepatitis virus of the type I with increased temperature and aminoethyl ethylenimine are presented. It was shown that the vaccine strain 3M-UNIIP of the hepatitis virus of ducklings of type I was comparatively thermostable at 56°C and sensitive to the action of aminoethyl ethylenimine; the time of complete inactivation of the virus at a final concentration of 0.1% at 37°C was 24 h. The obtained results suggest that aminoethyl ethylenimine can be used as an inactivator in manufacturing inactivated vaccine against viral hepatitis of ducklings of type I.
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Mathes, Chris, Joshua J. C. Rosenthal, Clay M. Armstrong, and William F. Gilly. "Fast Inactivation of Delayed Rectifier K Conductance in Squid Giant Axon and Its Cell Bodies." Journal of General Physiology 109, no. 4 (April 1, 1997): 435–48. http://dx.doi.org/10.1085/jgp.109.4.435.
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Inactivation of delayed rectifier K conductance (gK) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (∼−10 mV in 50–70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12–18°C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at −10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12°C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kv1 channels studied in heterologous expression systems.
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Biagi, B. A., and J. J. Enyeart. "Multiple calcium currents in a thyroid C-cell line: biophysical properties and pharmacology." American Journal of Physiology-Cell Physiology 260, no. 6 (June 1, 1991): C1253—C1263. http://dx.doi.org/10.1152/ajpcell.1991.260.6.c1253.
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The whole cell version of the patch-clamp technique was used to characterize voltage-gated Ca2+ channels in the calcitonin-secreting rat thyroid C-cell line 6-23 (clone 6). Three types of Ca2+ channels could be distinguished based on differences in voltage dependence, kinetics, and pharmacological sensitivity. T-type current was half-maximal at -31 mV, showed steady-state voltage-dependent inactivation that was half-maximal at -57 mV, inactivated with a voltage-dependent time constant that reached a minimum of 20 ms at potentials positive to -20 mV, and deactivated with a single time constant of approximately 2 ms at -80 mV. Reactivation of inactivated channels occurred with a time constant of 1.26 s at -90 mV. T current was selectively blocked by Ni2+ at concentrations between 5 and 50 microM. La3+ and Y3+ blocked the T current at 10- to 20-fold lower concentrations. Dihydropyridine-sensitive L-type current was half-maximal at a test potential of -3 mV and was approximately doubled in size when Ba2+ replaced Ca2+ as the charge carrier. Unlike L-type Ca2+ current in many cells, this current in C-cells displayed little Ca(2+)-dependent inactivation. N-type current was composed of inactivating and sustained components that were inhibited by omega-conotoxin. The inactivating component was half-maximal at +9 mV and could be fitted by two exponentials with time constants of 22 and 142 ms. A slow inactivation of N current with a time constant of 24.9 s was observed upon switching the holding potential from -80 to -40 mV. These results demonstrate that, similar to other neural crest derived cells, thyroid C-cells express multiple Ca2+ channels, including one previously observed only in neurons.
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Shimizu, H., K. Yamada, and S. Oiki. "An ion binding site competing with C-type inactivation." Seibutsu Butsuri 41, supplement (2001): S214. http://dx.doi.org/10.2142/biophys.41.s214_1.
Full textDissertations / Theses on the topic "C-type inactivation"
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Nikouee, Azadeh [Verfasser]. "Structural changes of the outer and inner vestibule of hKv1.3 channels during C type inactivation / Azadeh Nikouee." Ulm : Universität Ulm. Medizinische Fakultät, 2012. http://d-nb.info/1024534308/34.
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Pettini, Francesco. "Molecular Dynamics simulation of the hERG channel assisting Precision Medicine in Channelopathies." Doctoral thesis, Università di Siena, 2023. https://hdl.handle.net/11365/1227475.
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In the last two decades, a revolution in biology has shifted the traditional reductive approach to a bottom-up study of virtual models. This discipline, known as System Biology, integrates information coming from individual components, in order to predict the functioning of biological systems, with the idea that complex systems are made up of many independent components that can interact within well-structured networks changing over time, and that the functional properties of biological systems emerge as a consequence of interactions among their components. This paradigm shift is enabled by rapid advancements in technologies providing high-throughput instruments able to analyse in detail biological processes at the single molecule and single cell scale. The vast amount of data produced by these experimental techniques asks for adequate methods of analyses. The present dissertation focues on structural based methods for simulating the functioning of biological molecules, and in particular on the role of Molecular Dynamics simulations. The advantage of Molecular Dynamics simulations is that it is based on physical description of the systems, and consequently it might offer an atomistic description of the process under investigation.
The first chapter of this thesis will provide an introduction on the role of Molecular Genetics and Biology in Medicine, also considering new challenges for the prediction of protein interactions and for development of Precision Medicine. In the Second Chapters, Molecular Dynamics simulations will be discussed, with an emphasis on the methods for data analysis adopted in the research projects presented in the second part of the thesis. The third Chapter will be present the main research project produced during my PhD: the study of inactivation and drug binding in the hERG potassium channel. Side project and parallel collaborations are briefly discussed in the fourth Chapter, followed by concluding remarks.
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Pang, Chunyan. "REGULATION OF L-TYPE VOLTAGE-DEPENDNET CALCIUM CHANNELS BY THE REM GTPASE." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/656.
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The Rem, Rem2, Rad, and Gem/Kir GTPases, comprise a novel subfamily of the small Ras-related GTP-binding proteins known as the RGK GTPases, and have been shown to function as potent negative regulators of high voltage-activated (HVA) Ca2+ channels upon overexpression. HVA Ca2+ channels modulate Ca2+ influx in response to membrane depolarization to regulate a wide variety of cellular functions and they minimally consist of a pore-forming α1 subunit, an intracellular β subunit, and a transmembrane complex α2/δ subunit. While the mechanisms underlying RGK-mediated Ca2+ channel regulation remain poorly defined, it appears that both membrane localization and the binding of accessory Ca2+ channel β subunits (CaVβ) are required for suppression of Ca2+ channel currents. We identified a direct interaction between Rem and the L-type Cavα1 C-terminus (CCT), but not the CCT from CaV3.2 T-type channels. Deletion mapping studies suggest that the conserved CB-IQ domain is required for Rem:CCT association, a region known to contribute to both Ca2+-dependent channel inactivation and facilitation through interactions of Ca2+-bound calmodulin (CaM) with the proximal CCT. Furthermore, both Rem2 and Rad GTPases display similar patterns of CCT binding, suggesting that CCT represents a common binding partner for all RGK proteins. While previous studies have found that association of the Rem C-terminus with the plasma membrane is required for channel inhibition, it is not required for CaVβ- subunit binding. However, Rem:CCT association is well correlated with the plasma membrane localization of Rem and more importantly, Rem-mediated channel inhibition upon overexpression. Moreover, co-expression of the proximal CB-IQ containing region of CCT (residues 1507-1669) in HIT-T15 cells partially relieves Rem blockade of ionic current. Interestingly, Ca2+/CaM disrupts Rem:CCT association in vitro. Moreover, CaM overexpression partially relieves Rem-mediated L-type Ca2+ channel inhibition and Rem overexpression alters the kinetics of calcium-dependent inactivation. Together, these data suggest that the association of Rem with the CCT represents a crucial molecular determinant for Rem-mediated L-type Ca2+ channel regulation and provides new insights into this novel channel regulatory process. These studies also suggest that instead of acting as complete Ca2+ channel blockers, RGK proteins may function as endogenous regulators for the channel inactivation machinery.
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Lin, Chia-Huei, and 林佳慧. "Dependence of 6β-acetoxy-7α-hydroxyroyleanone block of Kv1.2 channel on C-type inactivation." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/86944920781987811088.
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碩士中國醫藥大學
神經科學與認知科學研究所
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Voltage-gated K+ (Kv) channels repolarize excitable cells by providing a pathway for K+ efflux. Kv1.2 channels are present in many neurons and exhibit slow inactivation during continuous depolarization. Here we reported that 6β-acetoxy-7α-hydroxyroyleanone (AHR) could affect slow inactivation of Kv1.2 channels heterologously expressed in HEK293 cells. Extracellular AHR (50 μM) dramatically speeded up the slow decay of Kv currents and left-shifted the steady-state inactivation curve. Intracellular dialysis of AHR did not accelerate the slow current decay, suggesting that this drug acted extracellularly. AHR did not affect at all the kinetics and voltage-dependence of Kv1.2 channel activation. Blockade of currents by AHR required an open state of channels. AHR inhibited Kv1.2 currents with an IC50 of 17.7 μM. In addition, % block of Kv currents by AHR was independent of the intracellular K+ concentration. Therefore, AHR did not appear to directly block the outer channel pore; rather, results suggest that it drastically accelerated Kv channel slow inactivation. The effects of AHR on a slow inactivation defective Kv1.2 mutant (V370G) were much attenuated. In addition, AHR did not affect ATP-sensitive potassium (KATP) channels, which do not display slow inactivation. These results suggest that AHR effects are dependent on Kv channel slow inactivation. Thus, our results show that AHR selectively accelerate Kv slow inactivation without affecting the activation gate, and could be used as a pharmacological probe for Kv slow inactivation gate.
Book chapters on the topic "C-type inactivation"
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"C-Type Inactivation." In Encyclopedia of Biophysics, 404. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-16712-6_100205.
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Wybranska, Iwona. "Genetic Markers of Endothelial Dysfunction." In Endothelial Dysfunction - A Novel Paradigm [Working Title]. IntechOpen, 2023. http://dx.doi.org/10.5772/intechopen.109272.
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The rate of endothelial dysfunction is influenced by genetic variation and thus inherited in families. Genetic disorders, such as familial hypercholesterolemia and homocystinuria, are at risk for premature atherosclerosis, and exhibit early endothelial dysfunction. The known spectrum of mutations in LDL receptor, APOB and PCSK9 gene represent the monogenic dominant hypercholesterolemia. An autosomal recessive form of hypercholesterolaemia in the caused by homozygous mutations in the LDL-R adaptor protein. The polygenic hypercholesterolaemia for patients with a clinical diagnosis of FH is based on the cumulative effect of LDL-C-raising alleles with a cumulative effect, in a complex interaction with the environment that leads to an increase in LDL-C, producing an FH-like phenotype and presenting this type of hypercholesterolaemia as a typical complex disease. The various causes of homocysteinaemia like genetic causes include mutations and enzyme deficiencies such as the most frequently mentioned 5, 10-methylenetetrahydrofolate reductase (MTHFR), but also methionine synthase (MS) and cystathionine β-synthase (CβS) but also by deficiencies of folate, vitamin B12 and, to a lesser extent, deficiencies of vitamin B6, which affects methionine metabolism, and leads also to endothelial disfunction in different mechanismms. Mutations in genes coding enzymes in homocysteine metabolism and also in nitric oxide (NO) synthesis, the main vasodilatator is also presented in this chapter. The crucial importance of microRNAs in endothelial physiology following EC-specific inactivation of the enzyme Dicer which is involved in altered expression of key regulators of endothelial function, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor receptor 2 (VEGF), interleukin-8, Tie-1 and Tie-2. The new discoveries based on genome-wide screening (GWAS) complement the knowledge of the topic.
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Aparecida Matoso, Daniele, Maelin da Silva, Hallana Cristina Menezes da Silva, Eliana Feldberg, and Roberto Ferreira Artoni. "Heterochromatin Dynamics in Response to Environmental Stress in Amazonian Fish." In Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.94929.
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Transcriptionally inactive portions of genomic DNA, condensed with histones and architectural proteins, are known as heterochromatic regions, often positive C band. The advent of epigenetics and new methodological approaches, showed that these regions are extremely dynamic and responsive to different types of environmental stress. The relationship of the constitutive heterochromatin with the transposable elements inactivation, especially from the Rex family, seems to be a frequent condition in fish. In this manuscript we review the existing knowledge of the nature and function of these genomic regions, based on species-based studies, with a focus on species of fish from the Amazon region.
Conference papers on the topic "C-type inactivation"
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Pittman, Debra D., Louise C. Wasley, Beth L. Murray, Jack H. Wang, and Randal J. Kaufman. "ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644044.
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Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.
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Fong, K. L. L., K. E. Boyle, C. S. Crysler, M. S. Landi, H. E. Griffin, and R. K. Lynn. "EXTRAHEPATIC METABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA) IN DOGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644398.
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Hepatic uptake has been proposed as the major mechanism of tPA clearance from systemic circulation. However, our recent studies demonstrated that tPA was rapidly Inactivated through complexation with protease Inhibitors in dog plasma In vitro, and that tPA-inhibitor complexes were present in plasma of dogs receiving tPA. Therefore, the present work was undertaken to differentiate hepatic from extrahepatlc clearance of tPA. Pharmacokinetics of tPA were determined in anesthetized beagle dogs with either Intact hepatic circulation or with Interrupted hepatic blood flow achieved by hepatic artery ligation and portal caval shunt.Recombinant two-chain tPA was administered as an Intravenous bolus dose (80 μg/kg) and plasma active tPA concentrations were measured using a modified and validated S-2251 chromogenlc assay. Following tPA administration to Intact dogs, plasma active tPA concentration declined blexponentlally with time with 84% of the active tPA eliminated during the α-phase. The t1/2's of the a and β-phase were 1.76 ± 0.74 and 6.23 ± 1.56 min, respectively. The systemic clearance was 25.98 ± 1.13 ml/min/kg and the volume of distribution at steady state (VDss) was 73.9 ± 15.1 ml/kg. Upon the elimination of hepatic blood flow, the systemic clearance was reduced by 54% while VDss was unaffected. The contribution of plasma Inactivation of tPA to the systemic clearance was estimated from in vitro Inactivation studies In 37°C plasma. Based on the pseudo first order Inactivation rate constants of 0.184 min-1 and 0.095 min-1 at Initial tPA concentrations of 25 and 250 IU/ml respectively, clearance rates from 5.02 to 9.2 ml/min/kg were calculated. These data suggest that (1) in Intact dogs, 46% of the tPA clearance occurs extrahepatlcally and (2) Inactivation of tPA in plasma accounts for a major portion of the extrahepatlc clearance.
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Elbashir, Israa, Aisha Aisha Nasser J. M. Al-Saei, Paul Thornalley, and Naila Rabbani. "Evaluation of antiviral activity of Manuka honey against SARS-CoV-2." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2021. http://dx.doi.org/10.29117/quarfe.2021.0113.
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Background and aims: In 2020 a global pandemic was declared caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). The pandemic is still ongoing and continues to cause considerable mortality and morbidity world-wide and new variants of the virus are emerging. Rapid development and rollout of vaccines for SARS-CoV-2 is in progress to counter the pandemic but has been tempered by the emergence of new SARS-CoV-2 variants, many of which exhibit reduced vaccine effectiveness. To date there is no approved antiviral treatment for coronavirus disease 2019 (COVID-19). Several studies have shown that Manuka honey has virucidal/antiviral effect. Methylglyoxal (MG), a bioactive component in Manuka honey, has antiviral activity in vitro. MG may modify arginine residues in the functional domains of viral spike and nucleocapsid proteins, resulting in loss of charge, protein misfolding and inactivation. The aim of this study was to characterize the antiviral activity of Manuka honey against SARS-CoV-2 in vitro Materials and methods: Wild-type SARS-CoV-2 with titers of multiplicities of infection (MOI) 0.1 and 0.05 were incubated with 2-fold serial dilutions of 250+ Manuka honey (equivalent to 250 to 31 µM) in infection medium (Dulbecco's Modified Eagle Medium + 2% fetal bovine serum + 100 units/ml penicillin + 100 µg/ml streptomycin) for 3 h. Manuka honey treated and untreated control SARS-CoV-2 was incubated with confluent cultures of Vero cells in vitro for 1 h, cultures washed with phosphate-buffered saline and incubated in fresh infection medium at 37°C for 4 - 5 days until 70% of virus control cells displayed cytopathic effect. We also studied the effect of scavenging MG in Manuka Honey with aminoguanidine (AG; 500 µM) on virucidal activity. The antiviral activity of MG was judged by median tissue culture infectious dose (TCID50) assays. Data analysis was by logistic regression. TCID50 (mean ± SD) was deduced by interpolation. Results: Diluted Manuka honey inhibited SARS-CoV-2 replication in Vero cells. SARS-CoV-2 was incubated in diluted Manuka honey in medium at 37°C for 3 h before adding to Vero cells. Manuka honey dilutions down to 125 µM MG equivalents completely inhibited cytopathic effect of SARS-CoV-2 whereas 31.25 µM and 62.5 µM MG equivalents had limited effect. Logistic regression and interpolation of the cytopathic effect indicated that the TCID50 = 72 ± 2 µM MG equivalents for MOI of 0.1. Prior scavenging of MG by addition of AG resulted in virus replication levels equivalent to those seen in the virus control without AG. Conclusion: Manuka honey has antiviral activity against SARS-CoV-2 when incubated with the virus in cell-free media at no greater than ca. 40-fold dilutions of 250+ grade. Anti-viral activity was inhibited by AG, consistent with the anti-viral effect being mediated by MG. Manuka honey dilutions in MG equivalents had similar antiviral effect compared to authentic MG, also consistent with MG content of Manuka honey mediating the antiviral effect. Whilst Manuka honey may inactivate SARS-CoV-2 in cell-free culture medium, its antiviral activity in vivo for other than topical application may be limited because of the rapid metabolism of MG by the glyoxalase system and limited bioavailability of oral MG.
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Nicham, F., and J. L. Martinoli. "AMIDOLYTIC DETERMINATION OF ANTI-ACTIVATED PROTEIN C ACTIVITY IN PLASMA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644316.
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Anti-activated protein C (anti-APC) potency of plasma was studied using purified bovine activated protein C (Bovine APC) and the chromogenic peptide substrate CBS 65.25. The choice of bovine instead of human APC was justified by a better sensitivity (Km = 0.14 and 0.42 mM respectively). Inhibition was shown to be dramatically enhanced by the presence of Heparin and calcium. No significant difference occurred for pH values up to 8.2 for both inhibition and hydrolysis reactions.In the final test, O.l ml of 1:5 diluted plasma (Tris buffer saline, pH 8.4, containing 5 U/ml of Heparin) were incubated at 37°C with 0.2 ml of Bovine APC (0.125 U/ml). After 10 minutes of inhibition, 0.2 ml of CBS 65.25 (1.5 mM/1) were added to the mixture and the change in absorbance was recorded at 405 nm for 2 minutes. In these conditions linearity of the dose-response curve was ensured from O up to 130 % of activity (normal plasma pool being assigned to 100 %) ; day to day precision was 1.9 %. When a normal plasma was overloaded with different purified inhibitors such as antithrombin III, cl-esterase inactivator, alpha 2 macroglobulin, the measured anti-APC activities were not affected at all. It could be concluded that this test measures protein C inhibitor described by Suzuki.Levels in 23 normal individuals averaged 97.7 %, giving a normal range of 77 - 118 %. Levels were below normal in 6 of 10 patients after surgery (54.1 +/- 4.8 %), in 18 of 19 patients with liver disease (49.5 +/- 9.6 %) and in 4 of 18 coumarin treated patients (54.9 +/- 6.5 %). In 9 of 10 patients previou sly characterized as type I protein C deficient, a statistically significant increase in anti-APC activity was observed (mean 110.7 +/- 7.7 %).The use of a chromogenic peptide substrate has led to a sensitive and fast assay for anti-APC activity in plasma. That could be of interest in clinical investigations and knowledge of regulatory mechanisms in thrombotic disorders.
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Leung, L., K. Saigo, and D. Grant. "HEPARIN BINDING TO HUMAN MONOCYTES: MODULATION BY HISTIDINE-RICH GLYCOPROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643847.
Full textAbstract:
Heparin and its related glycosaminoglycans interact with a variety of cell types and, irrespective of their anticoagulant activities, have a complex and biologically important influence in blood vessel wall biology. In this study, the binding of heparin to the human monocytoid cell line U937 was characterized. 35s-Heparin bound to U937 cells in a specific, concentration-dependent, and saturable manner, while binding to human erythrocytes was minimal. The binding was rapid, reaching a steady state at 30 min at 4°C and was reversible. Excess unlabeled heparin, but not chondroitin sulfate or dermatan sulfate, inhibited heparin binding, demonstrating the specificity for heparin. Scatchard plot analysis revealed a single class of heparin binding sites, with an apparent Kd of 0.32 uM and approx. 2.0 × 106 sites/cell. When binding was performed at 37°C, significant amount of cell-bound heparin was not removed by cell surface trypsinization, indicating endocytosis of heparin by U937 cells. Taken together the binding characteristics suggested the presence of specific heparin receptors on U937 cell surface. Histidine-rich glycoprotein (HRGP), a potent heparin-binding protein found in human plasma and platelets, decreased the affinity of heparin for U937 cells without a significant change in maximal binding. This negative modulation of heparin binding was specific for HRGP since antithrombin III was much less effective. While EDTA abolished the HRGP-heparin interaction, it had no effect on the HRGP modulation of heparin cell binding, suggesting that HRGP interacted directly with the U937 cell and altered its affinity for heparin. Specific binding of labeled HRGP to U937 cells was demonstrated. Cell-surface bound heparin markedly accelerated the inactivation of thrombin by antithrombin III. Its anticoagulant potency was equivalent to heparin in the fluid phase. Human monocytes and macrophages provide an optimal cell surface for the assembly of prothrombinase and the generation of thrombin. The specific binding of anticoagulantly active heparin to monocytes and macrophages may modulate the procoagulant properties of these cells at sites of inflammation and thrombosis. Furthermore, since heparin fractions with high or low affinity for antithrombin III bound equally well to U937 cells, heparin may have a direct effect on monocyte cell biology. The current study also raises the possibility that the significant modulation of heparin cell binding by HRGP may represent a major function for this protein in vivo.
Reports on the topic "C-type inactivation"
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Ohad, Itzhak, and Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, December 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
Full textAbstract:
The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.
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Bryant, C. A., S. A. Wilks, and C. W. Keevil. Survival of SARS-CoV-2 on the surfaces of food and food packaging materials. Food Standards Agency, November 2022. http://dx.doi.org/10.46756/sci.fsa.kww583.
Full textAbstract:
COVID-19, caused by the SARS-CoV-2 virus, was first reported in China in December 2019. The virus has spread rapidly around the world and is currently responsible for 500 million reported cases and over 6.4 million deaths. A risk assessment published by the Foods Standards Agency (FSA) in 2020 (Opens in a new window) concluded that it was very unlikely that you could catch coronavirus via food. This assessment included the worst-case assumption that, if food became contaminated during production, no significant inactivation of virus would occur before consumption. However, the rate of inactivation of virus on products sold at various temperatures was identified as a key uncertainty, because if inactivation does occur more rapidly in some situations, then a lower risk may be more appropriate. This project was commissioned to measure the rate of inactivation of virus on the surface of various types of food and food packaging, reducing that uncertainty. The results will be used to consider whether the assumption currently made in the risk assessment remains appropriate for food kept at a range of temperatures, or whether a lower risk is more appropriate for some. We conducted a laboratory-based study, artificially contaminating infectious SARS-CoV-2 virus onto the surfaces of foods and food packaging. We measured how the amount of infectious virus present on those surfaces declined over time, at a range of temperatures and relative humidity levels, reflecting typical storage conditions. We tested broccoli, peppers, apple, raspberry, cheddar cheese, sliced ham, olives, brine from the olives, white and brown bread crusts, croissants and pain au chocolat. The foods tested were selected as they are commonly sold loose on supermarket shelves or uncovered at deli counters or market stalls, they may be difficult to wash, and they are often consumed without any further processing i.e. cooking. The food packaging materials tested were polyethylene terephthalate (PET1) trays and bottles; aluminium cans and composite drinks cartons. These were selected as they are the most commonly used food packaging materials or consumption of the product may involve direct mouth contact with the packaging. Results showed that virus survival varied depending on the foods and food packaging examined. In several cases, infectious virus was detected for several hours and in some cases for several days, under some conditions tested. For a highly infectious agent such as SARS-CoV-2, which is thought to be transmissible by touching contaminated surfaces and then the face, this confirmation is significant. For most foods tested there was a significant drop in levels of virus contamination over the first 24 hours. However, for cheddar cheese and sliced ham, stored in refrigerated conditions and a range of relative humidity, the virus levels remained high up to a week later, when the testing period was stopped. Both cheddar cheese and sliced ham have high moisture, protein and saturated fat content, possibly offering protection to the virus. When apples and olives were tested, the virus was inactivated to the limit of detection very quickly, within an hour, when the first time point was measured. We suggest that chemicals, such as flavonoids, present in the skin of apples and olives inactivate the virus. The rate of viral decrease was rapid, within a few hours, for croissants and pain au chocolat. These pastries are both coated with a liquid egg wash, which may have an inhibitory effect on the virus. Food packaging materials tested had variable virus survival. For all food packaging, there was a significant drop in levels of virus contamination over the first 24 hours, in all relative humidity conditions and at both 6°C and 21°C; these included PET1 bottles and trays, aluminium cans and composite drinks cartons.
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VanderGheynst, Jean, Michael Raviv, Jim Stapleton, and Dror Minz. Effect of Combined Solarization and in Solum Compost Decomposition on Soil Health. United States Department of Agriculture, October 2013. http://dx.doi.org/10.32747/2013.7594388.bard.
Full textAbstract:
In soil solarization, moist soil is covered with a transparent plastic film, resulting in passive solar heating which inactivates soil-borne pathogen/weed propagules. Although solarization is an effective alternative to soil fumigation and chemical pesticide application, it is not widely used due to its long duration, which coincides with the growing season of some crops, thereby causing a loss of income. The basis of this project was that solarization of amended soil would be utilized more widely if growers could adopt the practice without losing production. In this research we examined three factors expected to contribute to greater utilization of solarization: 1) investigation of techniques that increase soil temperature, thereby reducing the time required for solarization; 2) development and validation of predictive soil heating models to enable informed decisions regarding soil and solarization management that accommodate the crop production cycle, and 3) elucidation of the contributions of microbial activity and microbial community structure to soil heating during solarization. Laboratory studies and a field trial were performed to determine heat generation in soil amended with compost during solarization. Respiration was measured in amended soil samples prior to and following solarization as a function of soil depth. Additionally, phytotoxicity was estimated through measurement of germination and early growth of lettuce seedlings in greenhouse assays, and samples were subjected to 16S ribosomal RNA gene sequencing to characterize microbial communities. Amendment of soil with 10% (g/g) compost containing 16.9 mg CO2/g dry weight organic carbon resulted in soil temperatures that were 2oC to 4oC higher than soil alone. Approximately 85% of total organic carbon within the amended soil was exhausted during 22 days of solarization. There was no significant difference in residual respiration with soil depth down to 17.4 cm. Although freshly amended soil proved highly inhibitory to lettuce seed germination and seedling growth, phytotoxicity was not detected in solarized amended soil after 22 days of field solarization. The sequencing data obtained from field samples revealed similar microbial species richness and evenness in both solarized amended and non-amended soil. However, amendment led to enrichment of a community different from that of non-amended soil after solarization. Moreover, community structure varied by soil depth in solarized soil. Coupled with temperature data from soil during solarization, community data highlighted how thermal gradients in soil influence community structure and indicated microorganisms that may contribute to increased soil heating during solarization. Reliable predictive tools are necessary to characterize the solarization process and to minimize the opportunity cost incurred by farmers due to growing season abbreviation, however, current models do not accurately predict temperatures for soils with internal heat generation associated with the microbial breakdown of the soil amendment. To address the need for a more robust model, a first-order source term was developed to model the internal heat source during amended soil solarization. This source term was then incorporated into an existing “soil only” model and validated against data collected from amended soil field trials. The expanded model outperformed both the existing stable-soil model and a constant source term model, predicting daily peak temperatures to within 0.1°C during the critical first week of solarization. Overall the results suggest that amendment of soil with compost prior to solarization may be of value in agricultural soil disinfestations operations, however additional work is needed to determine the effects of soil type and organic matter source on efficacy. Furthermore, models can be developed to predict soil temperature during solarization, however, additional work is needed to couple heat transfer models with pathogen and weed inactivation models to better estimate solarization duration necessary for disinfestation.