Diaminobenzidine Staining Research Articles

A rice transcription factor, OsMADS57, positively regulates high salinity tolerance in transgenic Arabidopsis thaliana and Oryza sativa plants.

MADS-box transcription factors (TFs) play indispensable roles in various aspects of plant growth, development as well as in response to environmental stresses. Several MADS-box genes have been reported to be involved in the salt tolerance in different plant species. However, the role of the transcription factor OsMADS57 under salinity stress is still unknown. Here, the results of this study showed that OsMADS57 was mainly expressed in roots and leaves of rice plants (Oryza sativa). Gene expression pattern analysis revealed that OsMADS57 was induced by NaCl. Overexpression of OsMADS57 in both Arabidopsis thaliana (A. thaliana) and rice could improve their salt tolerance, which was demonstrated by higher germination rates, longer root length and better growth status of overexpression plants than wild type (WT) under salinity conditions. In contrast, RNA interference (RNAi) lines of rice showed more sensitivity towards salinity. Moreover, less reactive oxygen species (ROS) accumulated in OsMADS57 overexpressing lines when exposed to salt stress, as measured by 3, 3'-diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) staining. Further experiments exhibited that overexpression of OsMADS57 in rice significantly increased the tolerance ability of plants to oxidative damage under salt stress, mainly by increasing the activities of antioxidative enzymes such as superoxide dismutase (SOD) and peroxidase (POD), reducing malonaldehyde (MDA) content and improving the expression of stress-related genes. Taken together, these results demonstrated that OsMADS57 plays a positive role in enhancing salt tolerance by activating the antioxidant system.

Receptor-like kinase OsASLRK regulates methylglyoxal response and content in rice

Receptor-like kinases (RLKs) are essential for plant abiotic stress responses. Methylglyoxal (MG) is a cellular metabolite that is often considered to be a stress signal molecule. However, limited information is available about the relationship between RLKs and MG. Here, we addressed the function of a receptor-like kinase, OsASLRK, in the MG response and content in rice. A typical MG-responsive element (AAAAAAAA) exists in the promoter region of the OsASLRK gene. RTqPCR analysis indicated that the transcript level of OsASLRK was significantly increased by exogenous MG in a time- and dosage-dependent fashion. GUS staining also confirmed that the expression of OsASLRK in rice root was enhanced by exogenous MG treatment. Genetic analysis suggested that the Osaslrk mutant displays increased sensitivity to MG and it showed higher endogenous MG content under exogenous MG treatments, while OsASLRK-overexpressing rice plants showed the opposite phenotypes. Diaminobenzidine (DAB) staining, scavenging enzyme activities and GSH content assays indicate that OsASLRK regulates MG sensitivity and content via the elevation of antioxidative enzyme activities and alleviation of membrane damage. Therefore, our results provide new evidence illustrating the roles that receptor-like kinase OsASLRK plays in MG regulation in rice.

ONTOGENETIC ASPECTS OF MELATONIN RECEPTORS 1A DENSITY IN THE LATERAL PREOPTIC NUCLEUS OF THE HYPOTHALAMUS UNDER VARIOUS LIGHT CONDITIONS

The aim: To find out density of melatonin receptors 1A in the neurons of the lateral preoptic nucleus of the hypothalamus in mature and old rats under various light conditions. Materials and methods: The study was carried out on 72 albino mature and old rats with light conditions appropriate for the experiment. To find out circadian differences of melatonin receptors 1A the material for the study was taken at 2 p.m. and 2 a.m. Visualization of primary antibodies against melatonin receptors 1A (Abcam) was conducted by means of the polymeric system Dako and diaminobenzidine staining under the microscope Delta Optical Evolution 100. The intensity of staining was assessed on the digital copies of images according to computer microdensitometry method. Results: Immunohistochemical examinations conducted enable to suggest that melatonin receptors 1A in the neurons of the lateral preoptic nucleus of the hypothalamus respond to different light conditions. In particular, intensity of immunohistochemical staining to melatonin receptors 1A under conditions of light deprivation increases both in mature and old rats, but it decreases under conditions of light stimulation. The parameter is higher at 2 a.m. as compared with 2 p.m. Intensity of immunohistochemical staining to melatonin receptors 1A in the neurons of the lateral preoptic nucleus of the hypothalamus is always lower in old rats than in mature ones. Conclusions: Density of melatonin receptors 1A in the neurons of the lateral preoptic nucleus of the hypothalamus is subordinated to the circadian rhythm: it increases at night and decreases in the daytime. At the same time, light stimulation results in disorders of the rhythm and development of desynchronization.

AutoIHC-Analyzer: computer-assisted microscopy for automated membrane extraction/scoring in HER2 molecular markers.

Human epidermal growth factor receptor 2 (HER2) is one of the widely used Immunohistochemical (IHC) markers for prognostic evaluation amongst the patient of breast cancer. Accurate quantification of cell membrane is essential for HER2 scoring in therapeutic decision making. In modern laboratory practice, expert pathologist visually assesses the HER2-stained tissue sample under the bright field microscope for cell membrane assessment. This manual assessment is time consuming, tedious and quite often results in interobserver variability. Further, the burden of increasing number of patients is a challenge for the pathologists. To address these challenges, there is an urgent need with a rapid HER2 cell membrane extraction method. The proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. A series of image processing approaches have been used to automatically extract the stained cells and membrane region, followed by automatic assessment of complete and broken membrane. Finally, a set of features are used to automatically classify the tissue under observation for the quantitative scoring as 0/1+, 2+ and 3+. In a set of surgically extracted cases of HER2-stained tissues, obtained from collaborative hospital for the testing and validation of the proposed approach AutoIHC-Analyzer and publicly available open source ImmunoMembrane software are compared for 90 set of randomly acquired images with the scores by expert pathologist where significant correlation is observed [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001)] respectively. The output shows promising quantification in automated scoring. LAY DESCRIPTION: In cancer prognosis amongst the patient of breast cancer, human epidermal growth factor receptor 2 (HER2) is used as Immunohistochemical (IHC) biomarker. The correct assessment of HER2 leads to the therapeutic decision making. In regular practice, the stained tissue sample is observed under a bright microscope and the expert pathologists score the sample as negative (0/1+), equivocal (2+) and positive (3+) case. The scoring is based on the standard guidelines relating the complete and broken cell membrane as well as intensity of staining in the membrane boundary. Such evaluation is time consuming, tedious and quite often results in interobserver variability. To assist in rapid HER2 cell membrane assessment, the proposed study aims at developing an automated IHC scoring system, termed as AutoIHC-Analyzer, for automated cell membrane extraction followed by HER2 molecular expression assessment from stained tissue images. The input image is preprocessed using modified white patch and CMYK and RGB colour space were used in extracting the haematoxylin (negatively stained cells) and diaminobenzidine (DAB) stain observed in the tumour cell membrane. Segmentation and postprocessing are applied to create the masks for each of the stain channels. The membrane mask is then quantified as complete or broken using skeletonisation and morphological operations. Six set of features were assessed for the classification from a set of 180 training images. These features are: complete to broken membrane ratio, amount of stain using area of Blue and Saturation channels to the image size, DAB to haematoxylin ratio from segmented masks and average R, G and B from five largest blobs in segmented DAB-masked image. These features are then used in training the SVM classifier with Gaussian kernel using 5-fold cross-validation. The accuracy in the training sample is found to be 88.3%. The model is then used for 90 set of unknown test sample images and the final labelling of stained cells and HER2 scores (as 0/1+, 2+ and 3+) are compared with the ground truth, that is expert pathologists' score from the collaborative hospital. The test sample images were also fed to ImmunoMembrane software for a comparative assessment. The results from the proposed AutoIHC-Analyzer and ImmunoMembrane software were compared with the expert pathologists' score where significant agreement using Pearson's correlation coefficient [(r = 0.9448; p < 0.001) and (r = 0.8521; p < 0.001) respectively] is observed. The results from AutoIHC-Analyzer show promising quantitative assessment of HER2 scoring.

Abstract 3192: Quanticell, a novel histopathology technology for visualization and quantitation of membrane PSMA demonstrated in cell lines and clinical tissue

Abstract Prostate specific membrane antigen (PSMA), being both a biomarker and a therapeutic target, is important in prostate cancer (PC) diagnosis and treatment. Imaging and pathology are two of the most widely used approaches for PSMA detection in the diagnosis of PC. However, these techniques lack sensitivity for accurate assessment of low levels of PSMA expression and lack the capabilities to properly characterize heterogeneous PSMA expression. When using anti-PSMA imaging ligands, radiotherapies or immunotherapies, these drawbacks may ultimately prevent proper diagnosis and response prediction and could deny anti-PSMA treatment to qualified patients that have low PSMA expression. Better, more sensitive detection methods are needed for improved PSMA-based diagnosis and prediction of response. Quanticell is a novel histopathology technology utilizing fluorescent nanoparticles that are compatible with standard immunostaining practices. Combined with a dedicated software, Quanticell provides increased sensitivity and quantification and a wide dynamic range for improved detection and characterization of a broad range of target expression. A pilot study was conducted to demonstrate this technology on PSMA-expressing cell lines and clinical samples, and to evaluate its utility for discovery research and clinical studies. An anti-PSMA Quanticell protocol was developed and optimized on several PSMA-expressing cell lines, focusing on the lowest expressing sample. A dual-staining protocol for PSMA and a membrane marker was further developed to allow for detection and analysis of membrane-specific PSMA. Staining of negative and positive controls demonstrate high specificity to PSMA with high signal-to-noise that is not affected with increased primary concentration. When compared to DAB (diaminobenzidine) staining, we observed increased sensitivity in a low PSMA-expressing cell line, VCAP, indicated by increased detection of percent PSMA-positive cells. Additionally, using Quanticell, we demonstrate increased dynamic range in high expressing PSMA clinical samples compared to DAB staining, which is subject to saturation. Citation Format: Hemi Dimant, Ankit Gandhi, Micheal Maker, Adam Markman, Jeanette Reinhardt, Stefan Collins, Jacob Hesterman, Joseph Krueger, Kenneth Bloom, Julie Bailis, Gloria Juan. Quanticell, a novel histopathology technology for visualization and quantitation of membrane PSMA demonstrated in cell lines and clinical tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3192.

Physiological and antioxidant responses of cultivated and wild barley under salt stress

Saline soil is a critical environmental problem affecting crop yield worldwide. Tibetan wild barley is distinguished for its vast genetic diversity and high degree of tolerance to abiotic stress, including salinity. The present study compared the response of antioxidant defense system in the XZ16 wild and CM72 cultivated barleys to salt stress. Wild barley was relatively more tolerant than cultivated CM72, salt-tolerant cultivar, with less Na&lt;sup&gt;+&lt;/sup&gt; uptake and more K&lt;sup&gt;+&lt;/sup&gt;, Ca&lt;sup&gt;2+&lt;/sup&gt;, and Mg&lt;sup&gt;2+&lt;/sup&gt; retention in plant tissues. The results of diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining showed that XZ16 had significantly lower H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; and O&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;−&lt;/sup&gt; concentrations than a salt-sensitive cultivar Gairdner, suggesting that the salt-tolerant genotype suffer from less oxidative damage. Moreover, XZ16 and Gairdner had the highest and lowest anti-oxidative enzyme activities and proline content in plant tissues. In addition, the microscopic examination revealed that DNA damage in cv. Gairdner was closely correlated to oxidative stress, representing that more reactive oxygen species accumulation in plants tissues leads to subsequent DNA damage. The present results show that higher salt tolerance of wild barley XZ16 is attributed to less Na&lt;sup&gt;+&lt;/sup&gt; accumulation and stronger anti-oxidative capacity.

Abstract A40: Induction of oxidative/nitrosative stress, proinflammatory cytokines and dysregulation of Wnt/beta-catenin signaling by benzo(a)pyrene in colons of BALB/c mice

Abstract Epidemiologic surveys and experimental data have revealed that environmental and dietary factors contribute to the etiology of colon cancer, which ranks third in cancer-related mortalities in the United States and fourth worldwide. In particular, human diet containing environmental carcinogens such as benzo(a)pyrene (B(a)P), a polycyclic aromatic hydrocarbon, has been the focus of investigators recently in relation to generated interest in looking at how it exerts its colorectal cancer. The present study investigated the effects of B(a)P on biomarkers of oxidative stress, inflammation and Wnt-signaling in colon of BALB/c mice following exposure to varying doses of 62.5, 125, and 250 mg/kg B(a)P for 7 days by oral gavage. Exposure to B(a)P induced marked oxidative stress accompanied by decrease in the colonic antioxidant enzyme activities and glutathione level with concomitant increase in myeloperoxidase activity, nitric oxide, and lipid peroxidation levels. Colon histopathology results showed B(a)P-related lesions characterized by atrophy, mucosal ulceration, and gland erosion in the mice. Immunohistochemistry analysis showed that B(a)P treatment increased the protein expression of nuclear factor kappa B, proinflammatory cytokines, namely tumor necrosis factor alpha and interleukin-1 β, as well as cyclooxygenase-2 and inducible nitric oxide synthase in the mice colon. Altered canonical Wnt signaling was confirmed by strong diaminobenzidine staining for p38 mitogen-activated protein kinase, b-catenin expression, and absence of adenomatous polyposis coli following B(a)P administration. Overall, our results show that exposure of mice to B(a)P induces colonic toxicity by mechanism involving oxidative and nitrosative stress, inflammation, and dsyregulation of Wnt/b-catenin signaling, and emphasizes the role of environmental polycyclic aromatic hydrocarbons like B(a)P in the pathology of colonic toxicity and tumorigenesis. Citation Format: Ebenezer O. Farombi, Babajide O. Ajayi, Isaac A. Adedara, Solomon E. Owumi. Induction of oxidative/nitrosative stress, proinflammatory cytokines and dysregulation of Wnt/beta-catenin signaling by benzo(a)pyrene in colons of BALB/c mice [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A40.

An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat.

Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3′diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.

Impaired Expression of Chloroplast HSP90C Chaperone Activates Plant Defense Responses with a Possible Link to a Disease-Symptom-Like Phenotype.

RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast- and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death.

A comparative study of phytotoxic effects of metal oxide (CuO, ZnO and NiO) nanoparticles on in-vitro grown Abelmoschus esculentus

The extensive use of nanoparticles (NPs) in various industrial and household products pose a threat of their release into the environment. Plants being sessile are prone to be directly affected by these nanomaterials in polluted soils, which may represent possible risks to food safety and human health. In the present study, we compared the phytotoxic effects of metal oxide NPs namely nickel oxide (NiO), copper oxide (CuO) and zinc oxide (ZnO) NPs at four varying concentrations (100, 250, 500 and 1000 mg/L) on in-vitro grown seedlings of Abelmoschus esculentus (okra). All the NPs tested demonstrated a suppressed plant growth in a concentration-dependent manner which was evidenced from the repression of shoot and root length. A decrease in chlorophyll content was recorded in the NP-treated plants. Anthocyanin contents, total phenolic and total flavonoid contents were significantly altered in the NP-treated plants. Moreover, reactive oxygen species (ROS) generation was monitored in the roots of NP-treated plants using 4-nitro blue tetrazolium chloride (NBT) and 3, 3′-diaminobenzidine (DAB) staining, demonstrated an increase in ROS production in a dose-dependent manner in plants exposed to all three NPs. Enhanced ROS and malondialdehyde (MDA) production was recorded in A. esculentus seedlings when exposed to NPs. Among the three metal oxide NPs compared in the study, NiO NPs were found to be more toxic than CuO and ZnO ones.

Combining Physiological and Metabolomic Analysis to Unravel the Regulations of Coronatine Alleviating Water Stress in Tobacco (Nicotiana tabacum L.).

Drought is a major abiotic stress that restricts plants growth, development, and yield. Coronatine (COR), a mimic of JA-Ile, functions in plant tolerance to multiple stresses. In our study, we examined the effects of COR in tobacco under polyethylene glycol (PEG) stress. COR treatment improved plant growth under stress as measured by fresh weight (FW) and dry weight (DW). The enzyme activity assay indicated that, under osmotic stress conditions, the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by COR treatment. Histochemical analyses via nitrotetrazolium blue chloride (NBT) and 3,3′-diaminobenzidine (DAB) staining showed that COR reduced reactive oxygen species (ROS) accumulation during osmotic stress. Metabolite profiles revealed that COR triggered significant metabolic changes in tobacco leaves under osmotic stress, and many essential metabolites, such as sugar and sugar derivatives, organic acids, and nitrogen-containing compounds, which might play active roles in osmotic-stressed tobacco plants, were markedly accumulated in the COR-treated tobacco. The work presented here provides a comprehensive understanding of the COR-mediated physiological, biochemical, and metabolic adjustments that minimize the adverse impact of osmotic stress on tobacco.

Vitis vinifera bZIP14 functions as a transcriptional activator and enhances drought stress resistance via suppression of reactive oxygen species

Background: Drought stress affects grapevine growth and development and reduces berry yield and quality. Identifying genes that are involved in the plant response to drought stress will enable the development of new grape strains that are tolerant to drought. Objective: We cloned the VvibZIP14 gene from Vitis vinifera and analyzed its role in drought resistance. Methods: Gene expression was analyzed by quantitative real-time PCR. Subcellular localization was assessed with a transient expression assay. The transactivation activity of the protein was evaluated in yeast. The physiologic role of VvibZIP14 was analyzed by overexpressing VvibZIP14 in Arabidopsis following drought stress. Hydrogen peroxide accumulation in Arabidopsis was visualized by diaminobenzidine staining. Results: Drought stress caused the accumulation of VvibZIP14, which was localized in the nucleus and had transcriptional activity. Transgenic plants showed improved resistance to drought stress and reduced electrolyte leakage compared to plants overexpressing empty vector, whereas chlorophyll content, photosystem II maximal photochemical efficiency, and net photosynthetic rate were higher. Catalase, peroxidase, and superoxide dismutase activities were also increased in VvibZIP14-overexpressing plants subjected to drought stress. Conclusions: VvibZIP14 functions as a transcription factor that confers resistance to drought stress in grape by enhancing the antioxidant response.

Characterization and Genetic Mapping of White-Spotted Leaf (wspl) Mutant in Rice

Spotted leaf mutants which produce necrotic lesions spontaneously are important sources to study programmed cell death in plant defense responses. A novel white-spotted leaf (wspl) mutant was induced from Ilpum, Korean japonica rice cultivar by the treatment of ethyl methane sulfonate (EMS). The phenotype of wspl mutant differed from that of other spotted leaf mutants in that not only brown spots but also white lesion mimic spots were observed on the tip of the leaves from the vegetative stage. Strong nitro blue tetrazolium (NBT) and 3, 3′-diaminobenzidine (DAB) staining were observed on the older leaf of wspl mutant in microscopic reactive oxygen species (ROS) assay, and the chlorophyll content of wspl mutant maintained longer than wild-type in the old leaves. Genetic analysis revealed that the wspl mutant trait was controlled by a single recessive gene and the locus of wspl gene was mapped on the long arm of chromosome 5 between the flanking markers S05100 and S05112 (4.1 Mb). Through the combination of the genetic mapping and SNP analysis, two candidate genes for white-spotted leaf were identified in the genic region. A novel phenotype of white-spotted leaf mutant has not yet been reported, thus further study of the wspl mutant will contribute to understanding of the molecular mechanisms involved in lesion mimic phenotype in rice.

Microscopic, Biochemical, and Molecular Comparisons of Moderately Resistant and Susceptible Populus Genotypes Inoculated with Sphaerulina musiva.

Sphaerulina musiva, the causal agent of Septoria leaf spot and stem canker, is responsible for mortality and yield loss in Populus plantations. However, little is known about the mode of infection and the mechanisms of resistance in this pathosystem. To characterize these phenomena, microscopic, biochemical, and transcriptome comparisons were performed between leaves of moderately resistant and susceptible genotypes of Populus inoculated with S. musiva conidia. Using scanning electron, cryofracture, and laser-scanning confocal microscopy, the infection and colonization of Populus leaves by S. musiva were examined across five time points (48 h, 96 h, 1 week, 2 weeks, and 3 weeks). The infection process was similar regardless of the host genotype. Differences in host colonization between susceptible and moderately resistant genotypes were apparent by 1 week postinoculation. However, the germination of conidia was greater on the susceptible than on the moderately resistant genotype (P < 0.008). Diaminobenzidine staining, a measure of hydrogen peroxide accumulation, was different (P < 0.001) between the host genotypes by 2 weeks postinoculation. Transcriptome differences between genotypes indicated that the speed and amplitude of the defense response were faster and more extensive in the moderately resistant genotype. Changes in gene expression support the microscopic and biochemical observations.

AMELIORATION WITH TITANIUM DIOXIDE NANOPARTICLE FOR REGULATION OF OXIDATIVE STRESS IN MAIZE (Zea mays L.)

In the present experiment, Zea mays L. was evaluated for its C4 modality through oxidative stress responses. Moreover, under variable concentrations (0, 50, 100µM) of hydrogen peroxide, the impacts were variable and modulated with titanium dioxide nanoparticle (TiO2-NP). Plants scored with elevated concentrations of H2O2 and superoxide (O2.-) maximum by 1.31 &amp; 1.52-fold respectively over 0µM H2O2. TiO2-NP recovered those maximums with 8.69 &amp; 7.89% as compared to control plants. The effects of H2O2 were moderated with malondialdehyde and carbonyl content by peak values 7.45 &amp; 5.91% under TiO2-NP treatment respectively. A depletion in proline content recorded under H2O2 stress but recovered through highest 1.23-fold with TiO2-NP application. TiO2-NP recorded NADP-ME activities up-regulated in concentration gradient of H2O2. Oxidative exposure was also documented by in-vivo detection of H2O2 and O2.- by 3,3′-diaminobenzidine and nitroblue tetrazolium staining. For the anti-oxidative cascades superoxide dismutase activity had induced by 1.42-fold under H2O2 when pre-treated with TiO2-NP. However, glutathione reductase activity was differentially modulated under H2O2 through interaction with TiO2-NP. Activity of guaiacol peroxidase was significantly up-regulated by 1.07-fold when TiO2-NP applied. The inductions of oxidative stress recorded more evident when a distinct polymorphism for GPX protein resolved variations in band intensities, but not in number. Activity of catalase experience a down-regulated trend all through the H2O2 exposure and had significant sensitivity to TiO2-NP. The results depict the nature and reactivity of C4 modules under oxidative stress that would be the selection pressures and that might be modulated with TiO2-NP application.

Comparative transcriptome combined with physiological analyses revealed key factors for differential cadmium tolerance in two contrasting hemp (Cannabis sativa L.) cultivars

Cadmium(Cd) is a widespread and one of the most toxic heavy metals, threatening human health. As a pioneer fiber crop, hemp (Cannabis sativa L.) has a great potential in Cd-polluted soil repair, but molecular mechanisams of hemp supporting the process of soil decontamination are still unknown. In this study, the key factors for differential Cd tolerance in two contrasting hemp cultivars (Cd-tolerant: Ym, Cd-sensitive: Nx) were investigated. Ym has a stronger transport and accumulation capacity than Nx. Furthermore, Cd detoxification in Ym is stronger than in Nx. Phenotype and 3,3′-diaminobenzidine(DAB) staining observation indicated that Ym was more tolerant to cadmium stress than Nx, as well as malondialdehyde(MDA) contents, superoxide dismutase(SOD) and peroxidase(POD) activities showed Ym has a stronger detoxification ability than Nx. Compared the transcriptome analysis between Ym and Nx, many genes involved in heavy metal transport and oxidation-reduction progress were identified between the two varieties. A schematic representation of main processes about differential Cd transport and detoxification was described, based on these results, which suggested that the higher Cd tolerance in Ym due to the key genes encoding some specific Cd transporter, defense system, special proteins that have a high expression in Ym.

H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3′-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Abstract 1964: C-C chemokine receptor type 1 is a prognostic indicator in ovarian cancer

Abstract Targeting CC-chemokine receptor type 1 (CCR1) in pre-clinical cancer models has shown reduction in tumor burden. Similar agents have also been safely used in clinical trials for autoimmune conditions. Despite promising results in cancer models and adequate safety profiles, the role of targeting CCR1 in ovarian cancer is poorly understood, warranting further evaluation. We have identified a macrophage-driven chemokine receptor axis, CCL23/CCR1 that may play a critical role in ovarian cancer metastasis. We hypothesize that CCR1 expressed by ovarian cancer cells and the differential expression of this chemokine receptor may have a prognostic role in survival and response to chemotherapy. We performed immunofluorescence and immunohistochemistry to profile the expression of CCR1 in a cell array containing 53 ovarian cancer cell lines and in three tissue microarrays containing greater than 1000 cores from 192 patients diagnosed with serous, endometrioid, or clear cell ovarian carcinoma. We measured the percent of CCR1 3,3′-Diaminobenzidine (DAB) staining using HALO image analysis software’s area quantification algorithm (Indica Labs, Corrales, NM) and correlated with survival. We analyzed three tissue microarrays containing varied histologic subtypes of ovarian cancer and found that CCR1 was highly expressed, defined as greater than 30% DAB staining, in 53% of serous (N=93), 68% of endometrioid (N=66), and 45% of clear cell carcinoma (N=22) averaged tissue cores. In comparing different ovarian cancer histologic subtypes, averaged tissue cores from patients with endometrioid carcinoma have significantly higher expression of CCR1, compared to serous carcinoma and clear cell carcinoma, 10.5% (p≤0.02) and 12.5% (p≤0.002), respectively. In all histotypes, we observed a significant lower median progression-free survival (p≤0.05) in patients with high CCR1 expression. Interestingly, in evaluating the level of CCR1 expression and response to platinum chemotherapy, we found that patients with platinum resistance (N=36) (defined as recurrence after 12 months of completing chemotherapy) had significantly higher expression of CCR1 compared to patients that were platinum sensitive (N=92) (p≤0.04). In addition, we analyzed a cell array and found differential expression of CCR1 across phenotypically different lines. For example, the cell lines OVCA420, OVSAHO, and OVCAR3 showed higher expression of CCR1 compared to TOV-21G, MOVCAR18, and OVCAR5. Given the potential prognostic significance of CCR1 in ovarian cancer, our future studies are focused on determining the functional role of CCR1 inhibition using both in vitro migration and in vivo colonization assays. Outcomes from pre-clinical studies will inform us on the clinical translatability for targeting CCR1 in patients with epithelial ovarian cancer. Citation Format: Anita M. Chanana, Venkatesh Krishnan, Supreeti Tallapragada, Oliver Dorigo. C-C chemokine receptor type 1 is a prognostic indicator in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1964.

Development of a PD-L1 multiplex immunofluorescence assay with advanced visual analysis for understanding the tumor microenvironment.

e14283 Background: Current PD-L1 Immunohistochemistry (IHC) assays utilizing conventional brightfield, chromogenic 3,3′-Diaminobenzidine (DAB) staining modalities are the norm in both translational and clinical research. With the rise of immuno-oncology, multiplexed immunofluorescence (mIF) has the potential to revolutionize pathology research as it enables the identification of complex cell phenotypes and their potential interactions in the tumor microenvironment (TME). Ultivue has developed a mIF assay for profiling PD-L1, CD8, CD68, pan-Cytokeratin, and Sox10 enabling whole-slide imaging and data analysis in a single workday. Methods: With an expanding number of biomarkers being interrogated, the complexity of the data analysis and visualization tasks increase exponentially. For N markers, a total of 2N phenotypes are possible. To address this problem and reveal the biologically relevant information embedded in the data, we have developed software tools to reduce the complexity, visualize, and quantify spatial distributions of cells across the full spectrum of possible phenotypes. In this study we demonstrate the utility, robustness, and ability to derive meaningful biological insights by validating the accuracy, precision, sensitivity, and specificity of the PD-L1 multiplex immunofluorescence assay. Results: Here we present results that identify all distinct binary phenotypes within cohorts of lung cancer and melanoma samples using qualitative pathology review and an image processing technique called Phenotypic Surface Density Mapping (PSDM). Important features of this quantitative technique are robustness, use of real physical units (e.g. cells/µm2 or intensity/µm2), and data generation in an unbiased fashion to reveal information about every possible phenotype. Conclusions: Applying the pathology review and PSDM to the cohorts of samples confirmed a high level of concordance demonstrating sensitivity and specificity with high degrees of confidence above 85%. Analysis of intra-run, inter-run, and inter-technician runs revealed that cell counts were within a CV less than 15% across all markers. Data presented here indicates the potential for use in a high throughput testing laboratory to aid in collection and interpretation of meaningful biological insights.

A novel stress-responsive BnaNAL1 transcriptional activator in oilseed rape positively modulates reactive oxygen species production and cell death

Programmed cell death (PCD) is an active, genetically controlled process associated with a number of developmental processes including response/adaptation to abiotic and biotic stresses and leaf senescence. Reactive oxygen species (ROS) are important modulators of PCD and leaf senescence. However, the transcriptional control of ROS and PCD in oilseed rape (Brassica napus L.) is largely unknown. In this study, we identified a NAC transcription factor gene BnaNAL1 from oilseed rape, which encodes a transcriptional activator and plays an important role in modulating ROS and cell death. BnaNAL1 was localized exclusively to the nucleus. Expression profiling of the gene showed increased transcript levels in early senescent and mature leaves. Expression of BnaNAL1 resulted in hypersensitive response (HR)-like cell death symptoms. Further analysis of various physiological indices such as diaminobenzidine (DAB) staining, malondialdehyde contents and electrolyte leakage supported the role of BnaNAL1. Furthermore, we also observed an increase in DNA fragmentation and ROS levels. A quantitative reverse transcription PCR (qRT-PCR) analysis indicated genes involved in ROS production (RbohB), cell death (VPE1a, ZEN1), defense (PR2) and leaf senescence (HIN1, ZAT12) as putative downstream targets. We further verified that BnaNAL1 activated promoters of ZEN1, HIN1, ZAT12 and VPEs using a dual luciferase reporter assay. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR assays, we further confirmed that BnaNAL1 could bind to the promoters of ZEN1, ZAT12, HIN1 and four VPEs for direct transcriptional modulation of these genes. Collectively our results demonstrate that BnaNAL1 positively modulates cell death and leaf senescence.