Academic literature on the topic 'Dose of pro-mutation'
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Journal articles on the topic "Dose of pro-mutation"
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Horowitz, Netanel A., Elizabeth A. Blevins, Whitney M. Miller, Ashely R. Perry, Kathryn E. Talmage, Eric S. Mullins, Brett P. Monia, Jay L. Degen, and Joseph S. Palumbo. "Thrombin-Thrombomodulin Interactions Are An Important Determinant of Metastatic Potential." Blood 116, no. 21 (November 19, 2010): 822. http://dx.doi.org/10.1182/blood.v116.21.822.822.
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Abstract Abstract 822 A substantial body of evidence indicates that tumor cell-associated (i.e., tissue factor) and circulating hemostatic system components (i.e., thrombin, fibrinogen, platelets) play a cooperative role in supporting metastasis. However, the role of endothelial regulators of hemostasis in metastasis remains largely unexplored. Thrombomodulin (TM) is the endothelial thrombin receptor central to thrombin-mediated activation of protein C. To test the hypothesis that thrombin-TM interactions are an important determinant of metastasis, we used mice carrying the Glu387Pro mutation in thrombomodulin (TMPro) known to decrease thrombin affinity ∼100 fold and APC generation ∼1000 fold. TMPro/Pro and control mice were intravenously injected with either a low dose (3 × 104 cells/mouse) or high dose (3 × 105 cells/mouse) of Lewis lung carcinoma cells (LLC) in separate experiments. At the low cell dose, the majority of wild-type mice developed no discernable pulmonary metastases, while TMPro/Pro mice each developed ∼100 grossly apparent pulmonary metastases. At the high cell dose the outcome was possibly even more striking, with few metastatic foci apparent in wild-type mice and fully confluent surface metastases too numerable to count in TMPro/Pro mice. Histological analyses confirmed that lung tissue from TMPro/Pro mice had been largely replaced with tumor. The dramatic augmentation in metastasis observed in TMPro/Pro mice did not appear to be due to genotype dependent differences in tumor growth potential as LLC cells transplanted into the dorsal subcutis of TMPro/Pro and control mice grew at similar rates and were histologically indistinguishable. Rather, tumor cell fate analyses using 125I-radiolabeled LLC cells revealed that the imposition of the TMPro mutation dramatically improved the early survival of tumor cells in the lung. Twenty minutes after tumor cell injection >80% of the tumor cells were localized within the lungs regardless of animal genotypes, indicating that the TMPro mutation did not have a major impact on initial tumor cell adhesion/stabilization within the pulmonary vasculature. In contrast, 6 hours post-injection <20% of the initial inoculum remained in the lungs of control mice, while 75% remained in the lungs of TMPro/Pro mice. To determine whether the prometastatic phenotype conferred by the TMPro mutation is directly dependent on tumor cell-associated procoagulant function, TMPro/Pro and control mice were challenged with previously described fibrosarcoma cells genetically incapable of tissue factor (TF) expression, or fibrosarcoma cells in which TF expression had been genetically restored. The number of metastatic foci formed by TF-expressing cells was dramatically higher in TMPro/Pro mice relative to wild-type animals, whereas TF-deficient tumor cells were essentially incapable of forming metastases in mice of either genotype. Thus, the prometastatic effect of the TMPro mutation is contingent upon TF expression by the tumor cell. Depletion of circulating prothrombin levels to <5% of normal by an anti-sense oligonucleotide approach also profoundly limited the formation of metastatic foci in both TMPro/Pro and control mice, consistent with the conclusion that thrombin-TM interactions are a key determinant of metastasis. Taken together, these studies demonstrate for the first time that endothelial thrombin-thrombomodulin interactions strongly control metastatic potential and suggest that intervention at the level of thrombomodulin could represent a novel therapeutic strategy for preventing or treating metastatic disease. Disclosures: No relevant conflicts of interest to declare.
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Deng, Wei, Mengting Yang, Zhiwen Duan, Cheng Peng, Zhiming Xia, and Shuzhong Yuan. "Molecular basis of resistance to bensulfuron-methyl and cross-resistance patterns to ALS-inhibiting herbicides in Ludwigia prostrata." Weed Technology 35, no. 4 (June 21, 2021): 656–61. http://dx.doi.org/10.1017/wet.2021.47.
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AbstractLudwigia prostrata is a problematic weed in rice fields in China, where acetolactate synthase (ALS)-inhibiting herbicides (e.g., bensulfuron-methyl) are widely used for the management of broadleaf weeds. Recently, an L. prostrata biotype (JS-R) that failed to be controlled with ALS-inhibiting herbicides was found in Jiangsu Province, China. This study aims to determine the level and molecular mechanism of resistance to bensulfuron-methyl in this JS-R biotype and to evaluate its spectrum of cross-resistance to other ALS-inhibiting herbicides. The dose–response assays indicated that the JS-R L. prostrata biotype had evolved 21.2-fold resistance to bensulfuron-methyl compared with the susceptible biotype (JS-S). ALS gene sequencing revealed that a nucleotide mutation (CCA to TCA) at codon 197, resulting in a Pro-197-Ser mutation, was detected in the resistant plants. Moreover, while the JS-R biotype contained the Pro-197-Ser resistance mutation and showed cross-resistance to pyrazosulfuron-ethyl (12.0-fold), it was sensitive to penoxsulam, bispyribac-sodium, and imazethapyr, which may serve as alternative herbicides to control the resistant L. prostrata biotype. This is the first confirmation of an L. prostrata biotype resistant to bensulfuron-methyl due to a Pro-197-Ser resistance mutation in the ALS gene.
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Weijer, Sebastiaan, Catharina W. Wieland, Sandrine Florquin, and Tom van der Poll. "A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis." Blood 106, no. 8 (October 15, 2005): 2761–68. http://dx.doi.org/10.1182/blood-2004-12-4623.
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AbstractThrombomodulin (TM) plays an essential role in the generation of activated protein C (APC), a mediator with both anticoagulant and anti-inflammatory properties, and is preferentially expressed in lungs. To investigate the role of TM in the coagulant and inflammatory response in the lung during tuberculosis, mice with a mutation in the TM gene (Thbd), which results in a minimal capacity for APC generation (TMpro/pro mice), were intranasally infected with live virulent Mycobacterium tuberculosis. Whereas pulmonary tuberculosis was not associated with activation of coagulation in either wild-type or TMpro/pro mice, 5 weeks after infection TMpro/pro mice displayed an uncontrolled inflammatory response in their lungs, as reflected by higher lung weights, a diminished ability to form well-shaped granulomas, elevated levels of proinflammatory cytokines, and concurrently reduced concentrations of anti-inflammatory cytokines. During a 36-week follow-up after infection with a lower dose of M tuberculosis, 35% of TMpro/pro mice died from week 28 onward versus none of the wild-type mice, and the surviving TMpro/pro mice displayed increased lung inflammation accompanied by higher mycobacterial loads in liver and spleen. These data suggest that a TM mutation that impairs APC generation results in uncontrolled lung inflammation during tuberculosis.
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Alwarnaidu Vijayarajan, Vijaya Bhaskar, Patrick D. Forristal, Sarah K. Cook, David Schilder, Jimmy Staples, Michael Hennessy, and Susanne Barth. "First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland." Agriculture 11, no. 12 (December 14, 2021): 1272. http://dx.doi.org/10.3390/agriculture11121272.
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Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.
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Takano, Hudson K., Rafael R. Mendes, Leonardo B. Scoz, Ramiro F. Lopez Ovejero, Jamil Constantin, Todd A. Gaines, Philip Westra, Franck E. Dayan, and Rubem S. Oliveira. "Proline-106 EPSPS Mutation Imparting Glyphosate Resistance in Goosegrass (Eleusine indica) Emerges in South America." Weed Science 67, no. 1 (December 18, 2018): 48–56. http://dx.doi.org/10.1017/wsc.2018.71.
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AbstractGlyphosate-resistant (GR) goosegrass [Eleusine indica(L.) Gaertn.] was recently identified in Brazil, but its resistance mechanism was unknown. This study elucidated the resistance mechanism in this species and developed a molecular marker for rapid detection of this target-site resistance trait. The resistance factor for the resistant biotype was 4.4-fold compared with the glyphosate-susceptible (GS) in greenhouse dose–response experiments. This was accompanied by a similar (4-fold) difference in the levels of in vitro andin plantashikimate accumulation in these biotypes. However, there was no difference in uptake, translocation, or metabolism of glyphosate between the GS and GR biotypes. Moreover, both biotypes showed similar values for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) copy number and transcription. Sequencing of a 330-bp fragment of theEPSPSgene identified a single-nucleotide polymorphism that led to a Pro-106-Ser amino acid substitution in the enzyme from the GR biotype. This mutation imparted a 3.8-fold increase in the amount of glyphosate required to inhibit 50% of EPSPS activity, confirming the role of this amino acid substitution in resistance to glyphosate. A quantitative PCR–based genotyping assay was developed for the rapid detection of resistant plants containing this Pro-106-Ser mutation.
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Pan, Lang, Haitao Gao, Han Wu, and Liyao Dong. "Molecular basis of multiple resistance to herbicides inhibiting acetyl-CoA carboxylase and acetolactate synthase in American sloughgrass (Beckmannia syzigachne) from China." Crop and Pasture Science 67, no. 11 (2016): 1208. http://dx.doi.org/10.1071/cp16109.
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American sloughgrass (Beckmannia syzigachne Steud.) is a problematic grass that is widely distributed in wheat and oilseed rape fields in China. The herbicides fenoxaprop-P-ethyl and mesosulfuron-methyl failed to control B. syzigachne JCWJ-R populations collected from a wheat field in Jiangsu Province. Dose-response experiments showed that JCWJ-R was resistant to the acetyl-CoA carboxylase (ACCase) inhibitors fenoxaprop-P-ethyl (33.8-fold), haloxyfop-R-methyl (12.7-fold), clethodim (7.8-fold) and pinoxaden (11.6-fold), and to the acetolactate synthase (ALS) inhibitors mesosulfuron-methyl (15.9-fold), pyroxsulam (17.6-fold), flucarbazone-Na (10.7-fold) and imazethapyr (7-fold). Resistance to ALS inhibitors was due to a Pro-197-Ser mutation in the ALS gene and resistance to ACCase inhibitors was due to an Ile-1781-Leu mutation in the ACCase gene. A derived cleaved amplified polymorphic sequence method was developed to detect the ALS mutation in B. syzigachne. This was combined with a previously established method to detect Ile-1781-Leu, and the mutation frequency and homozygous mutation rates in the JCWJ-R population were determined. The evolution of multiple resistance to ACCase and ALS inhibitors in this B. syzigachne population indicated that alternative methods should be developed to control resistant weeds.
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Lethagen, Stefan, Christina Isaksson, Charlotta Schaedel, and Lars Holmberg. "Von Willebrand’s Disease caused by Compound Heterozygosity for a Substitution Mutation (T1156M) in the D3 Domain of the Von Willebrand Factor and a Stop Mutation (Q2470X)." Thrombosis and Haemostasis 88, no. 09 (2002): 421–26. http://dx.doi.org/10.1055/s-0037-1613232.
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SummaryHereditary defects of the von Willebrand factor (VWF) gene cause von Willebrand’s disease (VWD) which shows great variability dependent on the nature and location of the mutation. We here describe the characteristics of a substitution of methionine for threonine 1156 in the D3 domain of the VWF, i.e. the domain involved in the intracellular multimerization of pro-VWF dimers. A VWD patient with severe symptoms was a compound heterozygote for the T1156M mutation and a null allele (Q2470X) on the other chromosome. This led to marked reduction of plasma VWF concentration to about 0.05 U/ml and an abnormality of VWF multimers as in type 2A VWD. Expression in vitro of the mutation demonstrated that 1156M-VWF is secreted from COS-7 cells in a much reduced amount and lacking large multimers. When coexpressed with normal VWF 1156M-VWF decreased the secretion of normal VWF in a dose-dependent manner, the secreted VWF showing all the multimers. Two relatives of the propositus were single heterozygotes for the T1156M mutation and were either asymptomatic or had the manifestations of mild type 1 VWD. The expression data and studies of platelet VWF indicate that the T1156M mutation results in intracellular retention of VWF rather than impaired synthesis. Three other members of the family were heterozygotes for the Q2470X mutation and demonstrated the variable expressivity of a null allele.
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Manshouri, Taghi, Zeev Estrov, Alfonso Quintas-Cardama, Jorge Cortes, Francis Giles, David Harris, Waldemar Priebe, Hagop Kantarjian, and Srdan Verstovsek. "WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation." Blood 108, no. 11 (November 16, 2006): 4885. http://dx.doi.org/10.1182/blood.v108.11.4885.4885.
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Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.
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Salgotra, Romesh, and Bhagirath Singh Chauhan. "The First Report of Target-Site Resistance to Glyphosate in Sweet Summer Grass (Moorochloa eruciformis)." Plants 10, no. 9 (September 11, 2021): 1885. http://dx.doi.org/10.3390/plants10091885.
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Sweet summer grass is a problematic weed in the central Queensland region of Australia. This study found glyphosate resistance in two biotypes (R1 and R2) of sweet summer grass. The level of resistance in these biotypes was greater than 8-fold. The glyphosate dose required to reduce dry matter by 50% (GR50) for the resistant populations varied from 1993 to 2100 g ha−1. A novel glyphosate resistance double point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was identified for the first time in sweet summer grass. Multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon, were observed. Both resistant biotypes exhibited a nucleotide change of CAA to ACA in codon 106, which predicts an amino acid change of proline to a threonine (Pro-106-Thr). In addition, the R1 biotype also possessed a mutation at codon 100, where a nucleotide substitution of T for G occurred (GCT to TCT), resulting in a substitution of serine for alanine (Ala-100-Ser). Understanding the molecular mechanism of glyphosate resistance will help to design effective management strategies to control invasive weeds.
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Gerakari, Maria, Nikolina Cheimona, Eleni Tani, Ilias Travlos, Demosthenis Chachalis, Donato Loddo, Solvejg Kopp Mathiassen, et al. "Biochemical and Rapid Molecular Analyses to Identify Glyphosate Resistance in Lolium spp." Agronomy 12, no. 1 (December 25, 2021): 40. http://dx.doi.org/10.3390/agronomy12010040.
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Lolium spp. are troublesome weeds mainly found in winter cereal crops worldwide, including Europe. In recent years resistant mechanisms have been evolved to several important herbicides. In this study we investigated the mechanisms responsible for conferring glyphosate resistance in some Lolium spp. populations. A holistic approach was used, based on dose-response experiments, determination of shikimic acid concentration in plant leaf tissue, as well as molecular analyses. More specifically, in three Lolium spp. populations the existence of a mutation in the Pro-106 codon of the 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) gene was investigated as well as the relative transcript levels of four ABC-transporter genes were monitored at three time points after glyphosate application. The results demonstrated that glyphosate resistance is a multifactor phenomenon. Relative transcript levels of the ABC-transporter genes were abundant at very early time points after glyphosate treatments. Dose-response experiments and shikimate analyses were in accordance with the findings of the quantitative PCR (qPCR) analyses. We suggest that relative expression ratio of ABC-transporter genes can be a useful tool to rapidly identify Lolium spp. populations resistant to glyphosate.
Dissertations / Theses on the topic "Dose of pro-mutation"
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Санін, Юрій Костянтинович. "Методи та засоби впливу УФ випромінюванням для знезараження варроатозу бджіл." Thesis, Національний технічний університет "Харківський політехнічний інститут", 2020. http://repository.kpi.kharkov.ua/handle/KhPI-Press/48429.
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Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 05.11.17 – біологічні та медичні прилади і системи. ‒ Харківський національний технічний університет сільського господарства імені Петра Василенка Міністерства освіти і науки України. – Харків, 2020. Літературний аналіз показує, що сучасна технологія боротьби з варроатозом бджіл є не завжди ефективною та на додаток не екологичною. У зв'язку з цим виникає необхідність в розробці нових, більш доступних і менш витратних технологій боротьби. У дисертаційній роботі на основі теоретичних і експериментальних досліджень вирішене науково-прикладне завдання, яке направлене на підвищення ефективності біопотенциалу бджолосім’ї шляхом обгрунтування процесу знищення кліща Варроа деструктор та розробки льоткової приставки забезпеченої світлодіодними модулями УФ випромінювання при живленні від сонячних фотоелементів, в якій за рахунок дії УФ опромінювання забезпечується санація бджіл від варроатозу без порушення їх природного ритму життєдіяльності. Експериментальні дослідження показали, що для звільнення медоносної бджоли від кліща Варроа деструктор слід опромінювати її в тунелі льоткової приставки інстальованої поза розплідним гніздом з системою світлодіодних модулів УФ випромінювання з параметрами: частота 0,99‧10¹⁵ Гц (300 нм); потужність потоку УФ випромінювання (0,54 ± 0,05) Вт; експозиція (6 ± 1) с; температура навколишнього середовища (25 ± 1)°С. Виробничими випробуваннями розробленої льоткової приставки при боротьбі з варроатозом для бджолосім’ї встановлено, що ефективність застосування льоткової приставки становить 75,8 %, зростання медопродукції на 3 % і отримання додаткового річного економічного ефекту, за рахунок оздоровлення бджіл від варроатозу, становить більше 10 тис. гривень.Thesis for the degree of candidate of technical sciences on specialty 05.11.17 ‒ biological and medical devices and systems. ‒ Kharkiv National Technical University of Agriculture named after Petr Vasilenko, Ministry of Education and Science of Ukraine. ‒ Kharkiv, 2020. In the dissertation the scientific and applied task is solved, which is aimed at increasing the efficiency of the biopotential of bees through the substantiation of the process of destruction of the Varrova mite and the development of a pilot console equipped with LED modules of UV radiation when powered from solar photocells, which provides for the rehabilitation of bees from arrotoza without disturbing their natural rhythm of life. To calculate the technological parameters of the irradiation process of the Varrova mite, mathematical dependences of the electrical and structural characteristics of the air console were first obtained. Complex substantiation of the parameters of the aviation console, which provide the harmful effect of UV radiation on the physiological functions of the Varrova tick, is fulfilled. The production tests of the developed airliner in the fight against warochotomy for bee colony found that the effectiveness of the use of the air sacs is 75.8%, the increase in the medoproduction by 3%, and the additional annual economic effect, due to the improvement of the bees from the varroastosis, is more than 10 thousand UAH.
Reports on the topic "Dose of pro-mutation"
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Whitham, Steven A., Amit Gal-On, and Tzahi Arazi. Functional analysis of virus and host components that mediate potyvirus-induced diseases. United States Department of Agriculture, March 2008. http://dx.doi.org/10.32747/2008.7591732.bard.
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The mechanisms underlying the development of symptoms in response to virus infection remain to be discovered in plants. Insight into symptoms induced by potyviruses comes from evidence implicating the potyviral HC-Pro protein in symptom development. In particular, recent studies link the development of symptoms in infected plants to HC-Pro's ability to interfere with small RNA metabolism and function in plant hosts. Moreover, mutation of the highly conserved FRNK amino acid motif to FINK in the HC-Pro of Zucchini yellow mosaic virus (ZYMV) converts a severe strain into an asymptomatic strain, but does not affect virus accumulation in cucurbit hosts. The ability of this FINK mutation to uncouple symptoms from virus accumulation creates a unique opportunity to study symptom etiology, which is usually confounded by simultaneous attenuation of both symptoms and virus accumulation. Our goal was to determine how mutations in the conserved FRNK motif affect host responses to potyvirus infection in cucurbits and Arabidopsis thaliana. Our first objective was to define those amino acids in the FRNK motif that are required for symptoms by mutating the FRNK motif in ZYMV and Turnip mosaic virus (TuMV). Symptom expression and accumulation of resulting mutant viruses in cucurbits and Arabidopsis was determined. Our second objective was to identify plant genes associated with virus disease symptoms by profiling gene expression in cucurbits and Arabidopsis in response to mutant and wild type ZYMV and TuMV, respectively. Genes from the two host species that are differentially expressed led us to focus on a subset of genes that are expected to be involved in symptom expression. Our third objective was to determine the functions of small RNA species in response to mutant and wild type HC-Pro protein expression by monitoring the accumulation of small RNAs and their targets in Arabidopsis and cucurbit plants infected with wild type and mutant TuMV and ZYMV, respectively. We have found that the maintenance of the charge of the amino acids in the FRNK motif of HC-Pro is required for symptom expression. Reduced charge (FRNA, FRNL) lessen virus symptoms, and maintain the suppression of RNA silencing. The FRNK motif is involved in binding of small RNA species including microRNAs (miRNA) and short interfering RNAs (siRNA). This binding activity mediated by the FRNK motif has a role in protecting the viral genome from degradation by the host RNA silencing system. However, it also provides a mechanism by which the FRNK motif participates in inducing the symptoms of viral infection. Small RNA species, such as miRNA and siRNA, can regulate the functions of plant genes that affect plant growth and development. Thus, this binding activity suggests a mechanism by which ZYMVHC-Pro can interfere with plant development resulting in disease symptoms. Because the host genes regulated by small RNAs are known, we have identified candidate host genes that are expected to play a role in symptoms when their regulation is disrupted during viral infections. As a result of this work, we have a better understanding of the FRNK amino acid motif of HC-Pro and its contribution to the functions of HC-Pro, and we have identified plant genes that potentially contribute to symptoms of virus infected plants when their expression becomes misregulated during potyviral infections. The results set the stage to establish the roles of specific host genes in viral pathogenicity. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.
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Grumet, Rebecca, and Benjamin Raccah. Identification of Potyviral Domains Controlling Systemic Infection, Host Range and Aphid Transmission. United States Department of Agriculture, July 2000. http://dx.doi.org/10.32747/2000.7695842.bard.
Full textAbstract:
Potyviruses form one of the largest and most economically important groups of plant viruses. Individual potyviruses and their isolates vary in symptom expression, host range, and ability to overcome host resistance genes. Understanding factors influencing these biological characteristics is of agricultural importance for epidemiology and deployment of resistance strategies. Cucurbit crops are subject to severe losses by several potyviruses including the highly aggressive and variable zucchini yellow mosaic virus (ZYMV). In this project we sought to investigate protein domains in ZYMV that influence systemic infection and host range. Particular emphasis was on coat protein (CP), because of known functions in both cell to cell and long distance movement, and helper component-protease (HC-Pro), which has been implicated to play a role in symptom development and long distance movement. These two genes are also essential for aphid mediated transmission, and domains that influence disease development may also influence transmissibility. The objectives of the approved BARD project were to test roles of specific domains in the CP and HC-Pro by making sequence alterations or switches between different isolates and viruses, and testing for infectivity, host range, and aphid transmissibility. These objectives were largely achieved as described below. Finally, we also initiated new research to identify host factors interacting with potyviral proteins and demonstrated interaction between the ZYMV RNA dependent RNA polymerase and host poly-(A)-binding protein (Wang et al., in press). The focus of the CP studies (MSU) was to investigate the role of the highly variable amino terminus (NT) in host range determination and systemic infection. Hybrid ZYMV infectious clones were produced by substituting the CP-NT of ZYMV with either the CP-NT from watermelon mosaic virus (overlapping, but broader host range) or tobacco etch virus (TEV) (non- overlapping host range) (Grumet et al., 2000; Ullah ct al., in prep). Although both hybrid viruses initially established systemic infection, indicating that even the non-cucurbit adapted TEV CP-NT could facilitate long distance transport in cucurbits, after approximately 4-6, the plants inoculated with the TEV-CPNT hybrid exhibited a distinct recovery of reduced symptoms, virus titer, and virus specific protection against secondary infection. These results suggest that the plant recognizes the presence of the TEV CP-NT, which has not been adapted to infection of cucurbits, and initiates defense responses. The CP-NT also appears to play a role in naturally occurring resistance conferred by the zym locus in the cucumber line 'Dina-1'. Patterns of virus accumulation indicated that expression of resistance is developmentally controlled and is due to a block in virus movement. Switches between the core and NT domains of ZYMV-NAA (does not cause veinal chlorosis on 'Dina-1'), and ZYMV-Ct (causes veinal chlorosis), indicated that the resistance response likely involves interaction with the CP-NT (Ullah and Grumet, submitted). At the Volcani Center the main thrust was to identify domains in the HC-Pro that affect symptom expression or aphid transmissibility. From the data reported in the first and second year report and in the attached publications (Peng et al. 1998; Kadouri et al. 1998; Raccah et al. 2000: it was shown that: 1. The mutation from PTK to PAK resulted in milder symptoms of the virus on squash, 2. Two mutations, PAK and ATK, resulted in total loss of helper activity, 3. It was established for the first time that the PTK domain is involved in binding of the HC-Pro to the potyvirus particle, and 4. Some of these experiments required greater amount of HC-Pro, therefore a simpler and more efficient purification method was developed based on Ni2+ resin.