Short communication: The rate of release of Cry1Ab protein from Bt maize leaves into water

Transgenic Bt maize plants are genetically modified to contain genes of Bacillus thuringiensis that encode for δ-endotoxins (Cry1Ab protein) that have insecticidal properties. These endotoxins target certain lepidopteran pests of maize. There are several entry routes by which Cry proteins enter the aquatic ecosystem in which aquatic organisms are exposed to these proteins. The main route is through plant debris such as leaves, stalks and postharvest detritus that are transported by means of run-off, rain and wind. While several studies have been conducted on the fate of Cry1Ab protein in terrestrial ecosystems, little is known of the release rates of Cry1Ab proteins from maize plant tissues that end up in aquatic ecosystems. In this study, leaves of Bt-maize and its isoline were submerged in containers filled with deionised or borehole water for a period of 16 days, and kept at 3 different temperatures (10±1, 21±1 and 30±1°C). Samples were collected at 1, 2, 4, 8, 16, 24, 48, 96, 192 and 384 h post submersion and analysed for Cry protein content using enzyme-linked immunosorbent assay (ELISA). The release of Cry protein from submerged maize leaves was influenced by temperature, and duration of immersion. An increase in Cry protein levels in the water was observed from the first hour onwards in both water types until the end of the experiment. The highest concentration of Cry protein was found at 30°C. This study showed that temperature and time period influence the release rate of Cry proteins from dried leaf matter into the aquatic environment.

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Development of a Model System Approach for Generating Fragments of the Cry1Ab Protein

Journal of AOAC International ◽

10.5740/jaoacint.15-0229 ◽

2016 ◽

Vol 99 (3) ◽

pp. 792-805

Author(s):

Vurtice C Albright ◽

Richard L Hellmich ◽

Joel R Coats

Keyword(s):

System Approach ◽

Environmental Fate ◽

Model System ◽

Model Systems ◽

Cry Protein ◽

Cry1ab Protein ◽

Cry Proteins ◽

Insect Management ◽

Protein Fragments ◽

Different Types

Abstract The use of transgenic crops expressing one or more crystal (Cry) proteins for insect management has grown dramatically since their introduction nearly 2 decades ago. However, many questions surrounding the environmental fate of these proteins still persist. One area of particular interest is the possible detection of Cry protein fragments by the antibodies used in ELISA kits. A model system approach is used to generate environmentally relevant fragments by simulating conditions and digestive enzymes that are known to exist in environments in which Cry proteins may be present. Seven different types of model systems were screened for their ability to generate fragments of the Cry1Ab protein; five of these model systems reliably generated Cry1Ab fragments. These fragments were analyzed in a subsequent study to determine whether the fragments were still detectable by ELISA and whether they retained any bioactivity.

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Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2017.0440 ◽

2017 ◽

Vol 284 (1859) ◽

pp. 20170440 ◽

Cited By ~ 14

Author(s):

Zdeňka Svobodová ◽

Yinghua Shu ◽

Oxana Skoková Habuštová ◽

Jörg Romeis ◽

Michael Meissle

Keyword(s):

Harmonia Axyridis ◽

Rhopalosiphum Padi ◽

Spider Mites ◽

Genetically Engineered ◽

Acute Effects ◽

Target Species ◽

Cry Protein ◽

Cry Proteins ◽

Arthropod Predators ◽

Maize Leaves

Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites ( Tetranychus urticae ), aphids ( Rhopalosiphum padi ), predatory spiders ( Phylloneta impressa ), ladybeetles ( Harmonia axyridis ) and lacewings ( Chrysoperla carnea ) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.

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The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656315 ◽

1992 ◽

Vol 68 (01) ◽

pp. 040-047 ◽

Cited By ~ 3

Author(s):

C Scott Jamison ◽

Bryan F Burkey ◽

Sandra J Friezner Degen

Keyword(s):

Total Protein ◽

Hepg2 Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Response Analysis ◽

Secreted Protein ◽

Mrna Levels ◽

Vitamin K1 ◽

Maximal Increase ◽

Protein Levels ◽

Warfarin Treatment

SummaryCultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 µg vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 µg/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 µg/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 µg/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240–350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on γ-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

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Road Salt Application in Highland Creek Watershed, Toronto, Ontario - Chloride Mass Balance

Water Quality Research Journal ◽

10.2166/wqrj.2010.044 ◽

2010 ◽

Vol 45 (4) ◽

pp. 451-461 ◽

Cited By ~ 21

Author(s):

Nandana Perera ◽

Bahram Gharabaghi ◽

Peter Noehammer ◽

Bruce Kilgour

Keyword(s):

Mass Balance ◽

Management Practices ◽

Chloride Concentration ◽

Aquatic Organisms ◽

Terrestrial Ecosystems ◽

Road Salt ◽

Winter Period ◽

Chloride Mass Balance ◽

Creek Watershed ◽

Chloride Concentrations

Abstract Occurrence of increasing chloride concentrations in urban streams of cold climates, mainly due to road salt application, has raised concerns on its adverse effects on aquatic and terrestrial ecosystems. Therefore, there is a need for a better understanding of processes associated with road salt application and subsequent discharge into the environment in order to develop management practices to minimize detrimental effects of chlorides. The chloride mass analysis for the Highland Creek watershed based on four years of hourly monitoring data indicates that approximately 60% of the chlorides applied on the watershed enter streams prior to subsequent salting period, 85% of which occurs during the period between November and March. Contribution of private de-icing operations on chloride mass input within Highland Creek watershed was estimated to be approximately 38%, indicating its significance in overall chloride mass balance. Salt application rates, as well as chloride output in the streams, vary spatially based on land use, influencing chloride concentrations in surface waters. The estimated groundwater chloride concentration of 275 mg/L indicates that some aquatic organisms in Highland Creek would potentially be at risk even outside the winter period under dry weather flow conditions.

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Eff ects of hyperbaric oxygen on NLRP3 infl ammasome activation in the brain aft er carbon monoxide poisoning

Undersea and Hyperbaric Medicine ◽

10.22462/10.12.2020.10 ◽

2020 ◽

pp. 607-619

Author(s):

Ya’nan Qi ◽

◽

Zhibao Guo ◽

Huijun Hu ◽

Xiang’en Meng ◽

...

Keyword(s):

Carbon Monoxide ◽

Nadph Oxidase ◽

Hyperbaric Oxygen ◽

Nlrp3 Inflammasome ◽

Carbon Monoxide Poisoning ◽

Enzyme Linked Immunosorbent Assay ◽

Inflammasome Activation ◽

Protein Levels ◽

Nlrp3 Inflammasome Activation ◽

Myelin Injury

Neuroinflammation plays an important role in brain damage after acute carbon monoxide poisoning (ACOP). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 inflammasome triggers the activation of inflammatory caspases and maturation of interleukin (IL)-1β and -18, and has been linked to various human autoinflammatory and autoimmune diseases. In this study we investigated the effects of hyperbaric oxygen (HBO2) on NLRP3 inflammasome activation after ACOP. Mice were randomly divided into four groups: sham group (exposure to normobaric air – i.e., 21% O2 at 1 atmosphere absolute); HBO2-only group; CO + normobaric air group; and CO + HBO2 group. Cognitive function was evaluated with the Morris water maze; myelin injury was assessed by Fluoro-Myelin GreenTM fluorescent myelin staining and myelin basic protein (MBP) immunostaining; and mRNA and protein levels of NLRP3 inflammasome complex proteins were measured by quantitative real-time PCR and Western blot, respectively. Additionally, serum and brain levels of IL-1β and -18 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined by enzyme-linked immunosorbent assay. It was found that HBO2 improved learning and memory, and alleviated myelin injury in mice subjected to acute CO exposure. Furthermore, HBO2 decreased NLRP3, absent in melanoma 2 (AIM2), caspase-1, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain mRNA and protein levels, and reduced brain and serum concentrations of IL-1β and -18 and NADPH oxidase. These results indicate that HBO2 suppresses the inflammatory response after ACOP by blocking NLRP3 inflammasome activation, thereby alleviating cognitive deficits.

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miR-128-3p inhibits apoptosis and inflammation in LPS-induced sepsis by targeting TGFBR2

Open Medicine ◽

10.1515/med-2021-0222 ◽

2021 ◽

Vol 16 (1) ◽

pp. 274-283

Author(s):

Peng Yang ◽

Jianhua Han ◽

Shigeng Li ◽

Shaoning Luo ◽

Xusheng Tu ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Quantitative Polymerase Chain Reaction ◽

Luciferase Reporter ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Aberrant Expression ◽

Protein Levels ◽

Apoptosis And Inflammation ◽

Hk2 Cells

Abstract Background Sepsis is a systemic inflammatory response that can lead to the dysfunction of many organs. The aberrant expression of miRNAs is associated with the pathogenesis of sepsis. However, the biological functions of miR-128-3p in sepsis remain largely unknown, and its mechanism should be further investigated. This study aimed to determine the regulatory network of miR-128-3p and TGFBR2 in lipopolysaccharide (LPS)-induced sepsis. Methods The expression levels of miR-128-3p and transforming growth factor beta receptors II (TGFBR2) were detected by quantitative polymerase chain reaction (qPCR). The protein levels of TGFBR2, Bcl-2, Bax, cleaved caspase 3, Smad2, and Smad3 were measured by western blot. Cell apoptosis was analyzed by flow cytometry. Cytokine production was detected by enzyme-linked immunosorbent assay (ELISA). The binding sites of miR-128-3p and TGFBR2 were predicted by Targetscan online software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Results The level of miR-128-3p was decreased, and TGFBR2 expression was increased in serum samples of sepsis patients and LPS-induced HK2 cells. Overexpression of miR-128-3p or knockdown of TGFBR2 ameliorated LPS-induced inflammation and apoptosis. Moreover, TGFBR2 was a direct target of miR-128-3p, and its overexpression reversed the inhibitory effects of miR-128-3p overexpression on inflammation and apoptosis in LPS-induced HK2 cells. Besides, overexpression of miR-128-3p downregulated TGFBR2 to suppress the activation of the Smad signaling pathway. Conclusion miR-128-3p could inhibit apoptosis and inflammation by targeting TGFBR2 in LPS-induced HK2 cells, which might provide therapeutic strategy for the treatment of sepsis.

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Abstract TP274: Orosomucoid-1 Protein Increases Following Ischemic Stroke in the Brain and Periphery

Stroke ◽

10.1161/str.47.suppl_1.tp274 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Hetal Mistry ◽

Madeline Levy ◽

Meaghan Roy-O'Reilly ◽

Louise McCullough

Keyword(s):

Sex Differences ◽

Ischemic Stroke ◽

Rna Sequencing ◽

Multiple Testing ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Stroke Patients ◽

Post Stroke ◽

Protein Levels ◽

The Brain

Background and Purpose: Orosomucoid-1 (ORM-1) is an abundant protein with important roles in inflammation and immunosuppression. We utilized RNA sequencing to measure mRNA levels in human ischemic stroke patients, with confirmation by serum ORM-1 protein measurements. A mouse model of ischemic stroke was then used to examine post-stroke changes in ORM-1 within the brain itself. Hypothesis: We tested the hypothesis that ORM-1 levels increase following ischemic stroke, with sex differences in protein dynamics over time. Methods: RNA sequencing was performed on whole blood from ischemic stroke patients (n=23) and controls (n=12), with Benjamini-Hochberg correction for multiple testing. Enzyme-linked immunosorbent assay was performed on serum from ischemic stroke patients (n=28) and controls (n=8), with analysis by T-test. For brain analysis, mice (n=14) were subjected to a 90-minute middle cerebral artery occlusion (MCAO) surgery and sacrificed 6 or 24 hours after stroke. Control mice underwent parallel “sham” surgery without occlusion. Western blotting was used to detect ORM-1 protein levels in whole brain, with analysis by two-way ANOVA. Results: RNA sequencing showed a 2.8-fold increase in human ORM-1 at 24 hours post-stroke (q=.0029), an increase also seen in serum ORM-1 protein levels (p=.011). Western blot analysis of mouse brain revealed that glycosylated (p=0.0003) and naive (p=0.0333) forms of ORM-1 were higher in female mice compared to males 6 hours post-stroke. Interestingly, ORM-1 levels were higher in the brains of stroke mice at 6 hours (p=.0483), while at 24 hours ORM-1 levels in stroke mice were lower than their sham counterparts (p=.0212). In both human and mouse data, no sex differences were seen in ORM-1 levels in the brain or periphery at 24 hours post-stroke. Conclusion: In conclusion, ORM-1 is a sexually dimorphic protein involved in the early (<24 hour) response to ischemic stroke. This research serves as an initial step in determining the mechanism of ORM-1 in the ischemic stroke response and its potential as a future therapeutic target for both sexes.

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Pharmacological Basis for Use of a Novel Compound in Hyperuricemia: Anti-Hyperuricemic and Anti-Inflammatory Effects

Frontiers in Pharmacology ◽

10.3389/fphar.2021.772504 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lei Zhao ◽

Yihang Li ◽

Dahong Yao ◽

Ran Sun ◽

Shifang Liu ◽

...

Keyword(s):

Uric Acid ◽

Western Blot ◽

Serum Uric Acid ◽

Glucose Transporter ◽

Serum Uric Acid Level ◽

Enzyme Linked Immunosorbent Assay ◽

The Novel ◽

Mice Model ◽

Protein Levels ◽

Anti Inflammatory

Background: The prevalence of hyperuricemia is considered high worldwide. Hyperuricemia occurs due to decreased excretion of uric acid, increased synthesis of uric acid, or a combination of both mechanisms. There is growing evidence that hyperuricemia is associated with a decline of renal function.Purpose: This study is aimed at investigating the effects of the novel compound on lowering the serum uric acid level and alleviating renal inflammation induced by high uric acid in hyperuricemic mice.Methods: Hyperuricemic mice model was induced by potassium oxonate and used to evaluate the effects of the novel compound named FxUD. Enzyme-linked immunosorbent assay was used to detect the related biochemical markers. Hematoxylin-eosin (HE) staining was applied to observe pathological changes. The mRNA expression levels were tested by qRT-PCR. The protein levels were determined by Western blot. In parallel, human proximal renal tubular epithelial cells (HK-2) derived from normal kidney was used to further validate the anti-inflammatory effects in vitro.Results: FxUD administration significantly decreased serum uric acid levels, restored the kidney function parameters, and improved the renal pathological injury. Meanwhile, treatment with FxUD effectively inhibited serum and liver xanthine oxidase (XOD) levels. Reversed expression alterations of renal inflammatory cytokines, urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were observed in hyperuricemic mice. Western blot results illustrated FxUD down-regulated protein levels of inflammasome components. Further studies showed that FxUD inhibited the activation of NF-κB signaling pathway in the kidney of hyperuricemic mice. In parallel, the anti-inflammatory effect of FxUD was also confirmed in HK-2.Conclusion: Our study reveals that FxUD exhibits the anti-hyperuricemic and anti-inflammatory effects through regulating hepatic XOD and renal urate reabsorption transporters, and suppressing NF-κB/NLRP3 pathway in hyperuricemia. The results provide the evidence that FxUD may be potential for the treatment of hyperuricemia with kidney inflammation.

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LncRNA NRON alleviates atrial fibrosis through suppression of M1 macrophages activated by atrial myocytes

Bioscience Reports ◽

10.1042/bsr20192215 ◽

2019 ◽

Vol 39 (11) ◽

Cited By ~ 5

Author(s):

Fei Sun ◽

Zhixiang Guo ◽

Chengxin Zhang ◽

Hong Che ◽

Wenhui Gong ◽

...

Keyword(s):

Macrophage Polarization ◽

Underlying Mechanism ◽

Enzyme Linked Immunosorbent Assay ◽

Ang Ii ◽

Atrial Myocytes ◽

Atrial Fibrosis ◽

M1 Macrophages ◽

Protein Levels ◽

Raw264.7 Macrophages ◽

Non Coding Rna

Abstract The aim of the present study was to explore the role of long non-coding RNA (lncRNA) non-coding repressor of NFAT (NRON) in the atrial fibrosis and to explore whether its underlying mechanism was associated with macrophage polarization. Enzyme-linked immunosorbent assay (ELISA) analysis of pro-inflammatory cytokines revealed that NRON overexpression suppressed, whereas NRON silencing facilitated the angiotensin II (Ang II)-induced inflammatory response in primary cultured atrial myocytes. The chromatin immunoprecipitation (ChIP) results showed that nuclear factor of activated T cell 3 (NFATc3) was recruited to the promoter region of interleukin (IL) 12 (IL-12) in atrial myocytes. Further data showed that NRON overexpression suppressed, whereas NRON silencing further promoted the Ang II-induced NFATc3 nuclear transport and IL-12 expression in atrial myocytes. Moreover, RAW264.7 macrophages were incubated with the conditioned medium from the Ang II-treated atrial myocytes transfected with NRON and IL-12 overexpression vectors. IL-12 overexpression abrogated the NRON overexpression-mediated inhibition of RAW264.7 macrophage polarization to the M1-like phenotype. Additionally, mouse atrial fibroblasts were incubated with the culture medium from RAW264.7 macrophages treated as described above. IL-12 overexpression rescued the NRON overexpression-inhibited protein levels of fibrosis markers Collagen I/III in mouse atrial fibroblasts. Collectively, our data indicate that lncRNA NRON alleviates atrial fibrosis through suppression of M1 macrophages activated by atrial myocytes.

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The effect of exposure of SO2 in high concentrations on CD19+ cells in reactive airway dysfunction syndrome in rat

European Journal of Inflammation ◽

10.1177/2058739218791905 ◽

2018 ◽

Vol 16 ◽

pp. 205873921879190

Author(s):

Zhiyuan Zhang ◽

Zhuang Ma ◽

Wenwu Sun ◽

Debin Ma ◽

Jianping Cao

Keyword(s):

Flow Cytometry ◽

Bronchoalveolar Lavage Fluid ◽

Lavage Fluid ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Protein Levels ◽

Reactive Airway ◽

Reactive Airway Dysfunction Syndrome ◽

High Concentrations ◽

Cd19 Expression

Reactive airway dysfunction syndrome (RADS) has a clinical manifestation similar to asthma, but some features are different between both the diseases. To probe the effect of CD19+ cells in RADS pathogenesis by inhalation of sulfur dioxide (SO2), rats were exposed to SO2 at 600 ppm for 2 h per day for 7 days and the CD19 expression in lung tissue was detected both at mRNA and protein levels by RT-PCR and western blot. The percentages of CD19+ and CD19+ CD23+ cells were measured by flow cytometry. IgG, IgA, and IgE in serum and bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). Histological analysis was performed. The results showed that expression of CD19 in SO2 exposure group was lower than that in the control both at mRNA and protein levels ( P < 0.05). Flow cytometry analysis showed that the percentages of CD19+ and CD19+ CD23+ were significantly lower in the SO2 exposed group than that in the control ( P < 0.05). There was no difference between the control and SO2 exposed groups in both serum and BALF levels of IgG, IgA, and IgE. Pathological changes, such as chronic bronchitis, local alveolar hemorrhage, and lymphocytes infiltration were observed in SO2 exposed. RADS is a non-immunogenicity, chronic airway inflammatory disease caused by irritation of harmful factor and manifests as airway hyperresposiveness.

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