Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3'-diaminobenzidine-stained esophageal biopsies.

Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) share many histopathological features; therefore, markers for differentiation are of diagnostic interest and may add to the understanding of the underlying mechanisms. The nitrergic system is upregulated in GERD and probably also in EoE. Esophageal biopsies of patients with EoE (n = 20), GERD (n = 20), and healthy volunteers (HVs) (n = 15) were exposed to antibodies against inducible nitric oxide synthase (iNOS), nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3. The stained object glasses were randomized, digitized, and blindly analyzed regarding the expression of DAB (3,3'-diaminobenzidine) by a protocol developed in QuPath software. A statistically significant overexpression of iNOS was observed in patients with any of the two inflammatory diseases compared with that in HVs. Eotaxin-3 could differentiate HVs versus inflammatory states. Gastroesophageal reflux patients displayed the highest levels of nitrotyrosine. Neither iNOS nor nitrotyrosine alone were able to differentiate between the two diseases. For that purpose, eosinophil peroxidase was a better candidate, as the mean levels increased stepwise from HVs via GERD to EoE. iNOS and nitrotyrosine are significantly overexpressed in patients with EoE and GERD compared with healthy controls, but only eosinophil peroxidase could differentiate the two types of esophagitis. The implications of the finding of the highest levels of nitrotyrosine among gastroesophageal reflux patients are discussed.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.

Optimizing the Composition of the Substrate Enhances the Performance of Peroxidase-like Nanozymes in Colorimetric Assays: A Case Study of Prussian Blue and 3,3'-Diaminobenzidine.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

Exponential Amplification Using Photoredox Autocatalysis.

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.

Fluorescent nanoparticle-mediated semiquantitative MYC protein expression analysis in morphologically diffuse large B-cell lymphoma.

The current World Health Organization (WHO) classification defines a new disease entity of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, making fluorescence in situ hybridization (FISH) screening for these genes mandatory. In addition, the prognostic significance of MYC expression was reported, with a cut-off value of 40%. However, interobserver discrepancies arise due to the heterogeneous intensity of MYC expression by immunohistochemistry. Moreover, a cut-off value of positivity for MYC protein in diffuse large B-cell lymphoma (DLBCL) varies among studies at present. Here, we applied a high-sensitivity semiquantitative immunohistochemical technique using fluorescent nanoparticles called phosphor-integrated dots (PID) to evaluate the MYC expression in 50 de novo DLBCL cases, and compared it with the conventional diaminobenzidine (DAB)-developing system. The high MYC expression detected by the PID-mediated system predicted poor overall survival in DLBCL patients. However, we found no prognostic value of MYC protein expression for any cut-off value by the DAB-developing system, even if the intensity was considered. These results indicate that the precise evaluation of MYC protein expression can clarify the prognostic values in DLBCL, irrespective of MYC rearrangement.

Peroxidase-mimicking nanozyme with surface-dispersed Pt atoms for the colorimetric lateral flow immunoassay of C-reactive protein.

Platinum-containing nanozymes with peroxidase-mimicking activity (PMA) have found a broad application in bioanalytical methods and are potentially able to compete with enzymes as the labels. However, traditionally used methods for the synthesis of nanozymes result in only a small fraction of surface-exposed Pt atoms, which participate in catalysis. To overcome this limitation, we propose a new approach for the synthesis of nanozymes with the efficient dispersion of Pt atoms on particles' surfaces. The synthesis of nanozymes includes three steps: the synthesis of gold nanoparticles (Au NPs), the overgrowth of a silver layer over Au NPs (Au@Ag NPs, 6 types of NPs with different thicknesses of Ag shell), and the galvanic replacement of silver with PtCl62- leading to the formation of trimetallic Au@Ag-Pt NPs with uniformly deposited catalytic sites and high Pt-utilization efficiency. Au@Ag-Pt NPs (23 types of NPs with different concentrations of Pt) with various sizes, morphology, optical properties, and PMA were synthesized and comparatively tested. Using energy-dispersive spectroscopy mapping, we confirm the formation of core@shell Au@Ag NPs and dispersion of surface-exposed Pt. The selected Au@Ag-Pt NPs were conjugated with monoclonal antibodies and used as the colorimetric and catalytic labels in lateral flow immunoassay of the inflammation biomarker: C-reactive protein (CRP). The colorimetric signal enhancement was achieved by the oxidation of 3,3'-diaminobenzidine by H2O2 catalyzed by Au@Ag-Pt NPs directly on the test strip. The use of Au@Ag-Pt NPs as the catalytic label produces a 65-fold lower limit of CRP detection in serum (15 pg mL-1) compared with Au NPs and ensures the lowest limit of detection for equipment-free lateral flow immunoassays. The assay shows a high correlation with data of enzyme-linked immunosorbent assay (R2 = 0.986) and high recovery (83.7-116.2%) in serum and plasma. The assay retains all the benefits of lateral flow immunoassay as a point-of-care method.

Inhibitory Effect of Thai Purple Rice Husk Extract on Chemically Induced Carcinogenesis in Rats.

This study investigated the cancer chemopreventive effects of an acidic methanol extract of purple rice husk on chemically induced carcinogenesis in rats. This purple rice husk extract (PRHE) had high polyphenol contents. Vanillic acid was a major phenolic compound in PRHE. Three major anthocyanins found in PRHE were malvidin-3-glucoside, peonidin-3-glucoside and cyanidin-3-glucoside. PRHE was not toxic and clastogenic in rats. The LD50 of PRHE was greater than 2000 mg kg-1 body weight (BW). The oral administration of 300 or 1000 mg kg-1 BW of PRHE for 28 days significantly decreased the number of micronucleated hepatocytes in diethylnitrosamine-initiated rats. The inhibitory mechanisms were associated with the reduction of cytochrome P450 2E1 expression and induction of some detoxifying enzymes in the liver. In addition, treatment with 500 mg kg-1 BW of PRHE for eight weeks did not induce preneoplastic lesions in the liver and colon. It significantly inhibited hepatic glutathione-S-transferase positive foci formation induced by diethylnitrosamine and 1,2-dimethylhydrazine by suppression of hepatocyte proliferation and induction of apoptosis. In conclusion, PRHE did not present toxicity, clastogenicity or carcinogenicity in rats. It exhibited cancer chemopreventive properties against chemically induced early stages rat hepatocarcinogenesis. Anthocyanins and vanillic acid might be candidate anticarcinogenic compounds in purple rice husk.

A field-applicable colorimetric assay for notorious explosive triacetone triperoxide through nanozyme-catalyzed irreversible oxidation of 3, 3'-diaminobenzidine.

A field-applicable colorimetric assay for fast detection of notorious explosive triacetone triperoxide (TATP) has been developed through the selective irreversible oxidation of 3, 3'-diaminobenzidine by hydrogen peroxide (HP) liberated during the acidic hydrolysis/degradation of TATP in the presence of MnO2 nanozymes. The generated HP was detected by probing the absorbance of the product (indamine polymer) of the 3, 3'-diaminobenzidine (DAB) oxidation reaction at 460.0 nm. The UV-Vis measurements provided a linear range from 1.57 to 10.50 mg L-1 TATP with a detection limit of 0.34 mg L-1. The oxidation of DAB cannot proceed by molecular oxygen, thus it is selectively oxidized by H2O2; this prevents false-positive results from laundry detergents (containing O2-releasing substances). Moreover, a naked-eye field test was developed, and a fast spot test analyzing time of 5 s was achieved. The selectivity of the assay was checked by analyzing some synthetic samples prepared with a laundry detergent as camouflage. The results of the developed assay revealed quantitative recoveries for TATP whereas the standard nanozyme-based method showed significant false-positive results. Graphical abstract.

Copper- and Cobalt-Codoped CeO2 Nanospheres with Abundant Oxygen Vacancies as Highly Efficient Electrocatalysts for Dual-Mode Electrochemical Sensing of MicroRNA.

Oxide materials with redox properties have aroused growing interest in many applications. Introducing dopants into crystal lattices provides an effective way to optimize the catalytic activities of the oxides as well as their redox properties. Herein, CeO2 nanospheres codoped with Cu and Co (CuCo-CeO2 NSs) were first synthesized and exploited as efficient electrocatalysts for dual-mode electrochemical sensing of microRNA (miRNA). With the doping of Cu and Co into the CeO2 lattice, large amounts of extra oxygen vacancies were generated, remarkably enhancing the redox and electrocatalytic properties of the CeO2 material. The abundant oxygen vacancies of the CuCo-CeO2 NSs were further identified by X-ray photoelectron spectroscopy (XPS), H2 temperature-programmed reduction (H2-TPR), and electron-energy-loss spectroscopy (EELS). Moreover, Mg2+-induced DNAzyme-assisted target recycling was introduced for ultrasensitive determination. The dual-mode sensing with generality was conducted as follows: First, the CuCo-CeO2 NSs acted as a direct redox mediator to generate a differential-pulse-voltammetry (DPV) signal, which was then greatly amplified by the efficient electrocatalysis of CuCo-CeO2 NSs toward H2O2 decomposition. Second, under the electrocatalysis of CuCo-CeO2 NSs, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive insoluble precipitates (IPs), leading to great amplification of the electrochemical-impedimetric-spectroscopy (EIS) signal. The dual-mode electrochemical sensor showed a wide linear range (0.1 fM to 10 nM) with a low detection limit (33 aM), paving a new way for constructing ultrasensitive electrochemical sensors.

Substrate specificity of IgGs with peroxidase and oxidoreductase activities from sera of patients with systemic lupus erythematosus and multiple sclerosis.

The analysis of IgGs to protect humans from oxidative stress through oxidation of harmful compounds was carried out. We have compared here for the first time peroxidase (in the presence of H2 O2 ) and oxidoreductase (in the absence of H2 O2 ) activities of IgGs from sera of healthy humans and patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, substrate specificity of SLE and MS IgG preparations in the oxidation of different compounds was analyzed: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3'-diaminobenzidine (DAB), homovanillic acid (HVA), o-phenylenediamine (OPD), α-naphthol, 3-amino-9-ethylcarbazole (AEC), p-hydroquinone (pHQ), and adrenaline. IgGs of healthy humans and SLE and MS patients oxidized DAB, ABTS, and OPD due to their peroxidase and oxidoreductase activities, while other compounds were substrates of IgGs only in the presence of H2 O2 : adrenaline was not oxidized by both activities of IgGs. The average SLE IgGs peroxidase activity increased statistically significant in comparison with abzymes from healthy humans in the order (-fold): OPD (1.2) <  DAB (1.7) < α-naphtol (2.2) ≤ AEC (2.4) < ABTS (4.5) < 5-ASA (10.6), while with oxidoreductase activity: OPD (1.8) ≤ DAB (2.1-fold) < ABTS (5.0). Only HVA was oxidized by IgGs with peroxidase activity of healthy donors faster than by SLE (1.3-fold) and MS abzymes (2.4-fold). In the oxidation of several substrates, only three IgGs of MS patients were used. The data speak of a tendency to increase the peroxidase and oxidoreductase activities of MS IgGs in comparison with healthy donors, but to a lesser extent: OPD (1.1 to 1.2-fold) ≤ ABTS (1.2 to 1.8-fold). It was shown that development of SLE and MS leads to increase in peroxidase and oxidoreductase activities of IgGs toward most of classical substrates. Thus, abzymes can serve as an additional factor of reactive oxygen species detoxification protecting of patients with SLE and MS from some harmful compounds somewhat better than healthy peoples.

Mitotic Golgi disassembly is required for bipolar spindle formation and mitotic progression.

During mitosis, the mammalian Golgi vesiculates and, upon partitioning, reassembles in each daughter cell; however, it is not clear whether the disassembly process per se is important for partitioning or is merely an outcome of mitotic entry. Here, we show that Golgi vesiculation is required for progression to metaphase. To prevent Golgi disassembly, we expressed HRP linked to a Golgi-resident protein and acutely triggered the polymerization of 3,3'-diaminobenzidine (DAB) in the Golgi lumen. The DAB polymer does not affect interphase cell viability, but inhibits Golgi fragmentation by nocodazole and brefeldin A and also halts cells in early mitosis. The arrest is Golgi specific and does not occur when DAB is polymerized in the endosomes. Cells with a DAB polymer in the Golgi enter mitosis normally but arrest with an intact Golgi clustered at a monopolar spindle and an active spindle assembly checkpoint (SAC). Mitotic progression is restored upon centrosome depletion by the Polo-like kinase 4 inhibitor, centrinone, indicating that the link between the Golgi and the centrosomes must be dissolved to reach metaphase. These results demonstrate that Golgi disassembly is required for mitotic progression because failure to vesiculate the Golgi activates the canonical SAC. This requirement suggests that cells actively monitor Golgi integrity in mitosis.

Irrigation-Induced Changes in Chemical Composition and Quality of Seeds of Yellow Lupine (Lupinus luteus L.).

The quality and amount of yellow lupine yield depend on water availability. Water scarcity negatively affects germination, flowering, and pod formation, and thus introduction of an artificial irrigation system is needed. The aim of this study was to evaluate the influence of irrigation on the quality of yellow lupine seeds. Raining was applied with a semi-solid device with sprinklers during periods of greatest water demand. It was shown that watered plants produced seeds of lesser quality, having smaller size and weight. To find out why seeds of irrigated plants were of poor quality, interdisciplinary research at the cellular level was carried out. DNA cytophotometry evidenced the presence of nuclei with lower polyploidy in the apical zone of mature seeds. This may lead to formation of smaller cells and reduce depositing of storage materials. The electrophoretic and Fourier transform infrared spectroscopic analyses revealed differences in protein and cuticular wax profiles, while scanning electron microscopy and energy dispersive spectroscopy revealed, among various chemical elements, decreased calcium content in one of seed zones (near plumule). Seeds from irrigated plants showed slightly higher germination dynamics but growth rate of seedlings was slightly lower. The studies showed that irrigation of lupine affected seed features and their chemical composition, an ability to germination and seedlings growth.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Salicylic Acid Induces Resistance in Rubber Tree against Phytophthora palmivora.

Induced resistance by elicitors is considered to be an eco-friendly strategy to stimulate plant defense against pathogen attack. In this study, we elucidated the effect of salicylic acid (SA) on induced resistance in rubber tree against Phytophthora palmivora and evaluated the possible defense mechanisms that were involved. For SA pretreatment, rubber tree exhibited a significant reduction in disease severity by 41%. Consistent with the occurrence of induced resistance, the pronounced increase in H2O2 level, catalase (CAT) and peroxidase (POD) activities were observed. For defense reactions, exogenous SA promoted the increases of H2O2, CAT, POD and phenylalanine ammonia lyase (PAL) activities, including lignin, endogenous SA and scopoletin (Scp) contents. However, SA had different effects on the activity of each CAT isoform in the particular rubber tree organs. Besides, three partial cDNAs encoding CAT (HbCAT1, HbCAT2 and HbCAT3) and a partial cDNA encoding PAL (HbPAL) were isolated from rubber tree. Moreover, the expressions of HbCAT1, HbPAL and HbPR1 were induced by SA. Our findings suggested that, upon SA priming, the elevated H2O2, CAT, POD and PAL activities, lignin, endogenous SA and Scp contents, including the up-regulated HbCAT1, HbPAL and HbPR1 expressions could potentiate the resistance in rubber tree against P. palmivora.

Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.

Establishing the subcellular localization of photodynamically-induced ROS using 3,3'-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration.

The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Feeding on resistant rice leads to enhanced expression of defender against apoptotic cell death (OoDAD1) in the Asian rice gall midge.

BACKGROUND: The Asian rice gall midge (Orseolia oryzae) is a destructive insect pest of rice. Gall midge infestation in rice triggers either compatible or incompatible interactions leading to survival or mortality of the feeding maggots, respectively. In incompatible interactions, generation of plant allelochemicals/defense molecules and/or inability of the maggots to continue feeding on the host initiate(s) apoptosis within the maggots. Unraveling these molecular events, triggered within the maggots as a response to feeding on resistant hosts, will enable us to obtain a better understanding of host resistance. The present study points towards the likely involvement of a defender against apoptotic cell death gene (DAD1) in the insect in response to the host defense. RESULTS: The cDNA coding for the DAD1 orthologue in the rice gall midge (OoDAD1) consisted of 339 nucleotides with one intron of 85 bp and two exons of 208 and 131 nucleotides. The deduced amino acid sequence of OoDAD1 showed a high degree of homology (94.6%) with DAD1 orthologue from the Hessian fly (Mayetiola destructor)--a major dipteran pest of wheat. Southern hybridization analysis indicated that OoDAD1 was present as a single copy in the genomes of the Asian rice gall midge biotypes (GMB) 1, 4 and 4 M. In the interactions involving GMB4 with Jaya (susceptible rice host) the expression level of OoDAD1 in feeding maggots gradually increased to 3-fold at 96 hai (hours after infestation) and peaked to 3.5-fold at 96 hai when compared to that at 24 hai. In contrast, expression in maggots feeding on RP2068 (resistant host) showed a steep increase of more than 8-fold at 24 hai and this level was sustained at 48, 72 and 96 hai when compared with the level in maggots feeding on Jaya at 24 hai. Recombinant OoDAD1, expressed in E. coli cells, when injected into rice seedlings induced a hypersensitive response (HR) in the resistant rice host, RP2068, but not in the susceptible rice variety, Jaya. CONCLUSIONS: The results indicate that the expression of OoDAD1 is triggered in the feeding maggots probably due to the host resistance response and therefore, is likely an important molecule in the initial stages of the interaction between the midge and its rice host.