A new Eosin Y-based 'turnon' fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity.

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

Quantitative assessment of H&E staining for pathology: development and clinical evaluation of a novel system.

BACKGROUND: Staining tissue samples to visualise cellular detail and tissue structure is at the core of pathology diagnosis, but variations in staining can result in significantly different appearances of the tissue sample. While the human visual system is adept at compensating for stain variation, with the growth of digital imaging in pathology, the impact of this variation can be more profound. Despite the ubiquity of haematoxylin and eosin staining in clinical practice worldwide, objective quantification is not yet available. We propose a method for quantitative haematoxylin and eosin stain assessment to facilitate quality assurance of histopathology staining, enabling truly quantitative quality control and improved standardisation. METHODS: The stain quantification method comprises conventional microscope slides with a stain-responsive biopolymer film affixed to one side, called stain assessment slides. The stain assessment slides were characterised with haematoxylin and eosin, and implemented in one clinical laboratory to quantify variation levels. RESULTS: Stain assessment slide stain uptake increased linearly with duration of haematoxylin and eosin staining (r = 0.99), and demonstrated linearly comparable staining to samples of human liver tissue (r values 0.98-0.99). Laboratory implementation of this technique quantified intra- and inter-instrument variation of staining instruments at one point in time and across a five-day period. CONCLUSION: The proposed method has been shown to reliably quantify stain uptake, providing an effective laboratory quality control method for stain variation. This is especially important for whole slide imaging and the future development of artificial intelligence in digital pathology.

Optimized Degradation of Eosin Dye Through UV-ZnO NPs Catalyzed Reaction.

The present study is set out to determine the photocatalytic degradation potential of ZnO nanoparticles for effective degradation of Eosin dye. The heterogeneous photocatalytic experiments were carried out by irradiating aqueous dye solutions with ultraviolet light. The influence of effective parameters like flow rate, pH, catalyst dose, and dye concentration was examined. The best degradation efficiency (66.82%) of ZnO Nanoparticles against Eosin dye was achieved within 90 min of reaction time. The Box-Behnken design under the Response Surface Methodology (RSM) was chosen as a statistical tool to obtain the correlation of influential parameters. The optimum values were recorded as follows: 0.59 g, 15.75 ppm and 136.12 ml/min for amount of catalyst, dye concentration and flow rate, respectively. The maximum percent degradation achieved at these conditions was 71.44%.

(Rose Bengal)/(Eosin Yellow)-Gold-Polypyrrole Hybrids: A Design for Dual Photo-Active Nano-System with Ultra-High Loading Capacity.

PURPOSE: Enhancement of the photodynamic/photothermal efficiency of two water-soluble dyes, rose bengal (RB) and eosin yellow (EY), via conjugation to a polymeric nano-system gold-polypyrrole nanoparticle (AuPpy NPs). METHODOLOGY: A multi-step synthesis method and an in situ one-pot synthesis method were used. Loading percentage, particle size, zeta potential, morphology, UV-Vis-NIR spectrophotometry and in vitro photothermal activity were measured. Then, both hybrid nanocomposites were examined for their cytotoxicity and photocytotoxicity on HepG2 cell line as a model for cancer cells. RESULTS: Dyes loaded in the traditional multi-step method did not exceed 9% w/w, while in the one-pot synthesis method they reached ~67% w/w and ~75% w/w for EY-AuPpy NPs and RB-AuPpy NPs, respectively. UV-Vis-NIR spectrophotometry showed that both nano-systems exhibited intense absorption in the NIR region. The mean size of the nanoparticles was ~31.5 nm (RB-AuPpy NPs) and ~33.6 nm (EY-AuPpy NPs) with zeta potential values of -26.5 mV and -33 mV, respectively. TEM imaging revealed the morphology of both hybrids, showing ultra-nano spherical-shaped gold cores in the case of RB-AuPpy NPs, and different shapes of larger gold cores in the case of EY-AuPpy NPs, both embedded in the polymer film. Conjugation to AuPpy was found to significantly reduce the dark cytotoxicity of both RB and EY, preserving the photocytotoxicity of EY and enhancing the photocytotoxicity of RB. CONCLUSION: Gold-polypyrrole nanoparticles represent an effective delivery system to improve the photodynamic and photothermal properties of RB and EY. The in situ one-pot synthesis method provided a means to greatly increase the loading capacity of AuPpy NPs. While both hybrid nanocomposites exhibited greatly diminished dark cytotoxicity, RB-AuPpy NPs showed significantly enhanced photocytotoxicity compared to the free dyes. This pattern enables the safe use of both dyes in high concentrations with sustained action, reducing dose frequency and side effects.

Lignin-Induced CaCO3 Vaterite Structure for Biocatalytic Artificial Photosynthesis.

The vaterite phase of CaCO3 exhibits unique characteristics, such as high porosity, surface area, dispersivity, and low specific gravity, but it is the most unstable polymorph. Here, we report lignin-induced stable vaterite as a support matrix for integrated artificial photosynthesis through the encapsulation of key active components such as the photosensitizer (eosin y, EY) and redox enzyme (l-glutamate dehydrogenase, GDH). The lignin-vaterite/EY/GDH photobiocatalytic platform enabled the regeneration of the reduced nicotinamide cofactor under visible light and facilitated the rapid conversion of α-ketoglutarate into l-glutamate (initial conversion rate, 0.41 mM h-1; turnover frequency, 1060 h-1; and turnover number, 39,750). The lignin-induced vaterite structure allowed for long-term protection and recycling of the active components while facilitating the photosynthesis reaction due to the redox-active lignin. Succession of stability tests demonstrated a significant improvement of GDH's robustness in the lignin-vaterite structure against harsh environments. This work provides a simple approach for solar-to-chemical conversion using a sustainable, integrated light-harvesting system.

Detection of malignant melanoma in H&E-stained images using deep learning techniques.

Histopathological images are widely used to diagnose diseases including skin cancer. As digital histopathological images are typically of very large size, in the order of several billion pixels, automated identification of all abnormal cell nuclei and their distribution within multiple tissue sections would assist rapid comprehensive diagnostic assessment. In this paper, we propose a deep learning-based technique to segment the melanoma regions in Hematoxylin and Eosin (H&E) stained histopathological images. In this technique, the nuclei in the image are first segmented using a Convolutional Neural Network (CNN). The segmented nuclei are then used to generate melanoma region masks. Experimental results with a small melanoma dataset show that the proposed method can potentially segment the nuclei with more than 94 % accuracy and segment the melanoma regions with a Dice coefficient of around 85 %. The proposed technique also has a small execution time making it suitable for clinical diagnosis with a fast turnaround time.

Influence of deep-freezing and MGG staining on DNA and RNA quality in different types of lung adenocarcinoma cytological smears.

BACKGROUND: Preserving the optimal quality of DNA and RNA is mandatory for molecular testing in lung adenocarcinoma cytological smears (LACSs). METHODS: DNA and RNA were isolated from 90 frozen unstained and 46 May Grünwald Giemsa (MGG) stained LACSs prepared from bronchial washing (BW), bronchial brushing (BB), and pleural effusion (PE) samples during 3 years. Concentrations of nucleic acids in all LACSs were assessed by spectrophotometric analysis. Fragmentation of DNA and RNA was determined by PCR amplification of selected genes. Amplicons of 100, 200, 300, 400, and 600 bp were used for DNA and 108 bp-long HPRT1 transcript fragment for RNA fragmentation analysis. RESULTS: Among 90 frozen LACSs, significantly lower DNA concentrations of BB and RNA concentrations of BW samples frozen for 6-10 months were observed in comparison with samples frozen for longer periods (p < .05). Among 46 paired LACSs, 44 (95.7%) frozen and 15 (32.6%) MGG-stained samples showed 600 bp-long DNA amplicons. Statistically significant difference (p < .05) in the fragmentation of DNA between frozen and MGG-stained LACSs was observed (p < .05), with DNA being less fragmented in frozen LACSs. In addition, 33 (71.7%) frozen and 36 (78.2%) MGG-stained LASCs showed HPRT1 gene amplicon of 108 bp. RNA was less fragmented in 3-year old MGG-stained samples than in LACSs frozen for 3 years. CONCLUSION: DNA and RNA extracted from frozen and MGG-stained LACSs showed different results depending on the time of storage and/or type of samples, but in general all samples had adequate quantity and quality for downstream molecular testing.

Modulating photochemical reactions in Langmuir monolayers of Escherichia coli lipid extract with the binding mechanisms of eosin decyl ester and toluidine blue-O photosensitizers.

Photodynamic damage to the cell envelope can inactivate microorganisms and may be applied to combat super-resistance phenomenon, empowered by the indiscriminate use of antibiotics. Efficiency in microbial inactivation is dependent on the incorporation of photosensitizers (PS) into the bacterial membranes to trigger oxidation reactions under illumination. Herein, Langmuir monolayers of Escherichia coli lipid extract were built to determine the binding mechanisms and oxidation outcomes induced by eosin decyl ester (EosDEC) and toluidine blue-O (TBO) PSs. Surface-pressure isotherms of the E. coli monolayers were expanded upon EosDEC and TBO, suggesting incorporation of both PSs. Fourier-transform infrared spectroscopy (FTIR) of Langmuir-Schaefer (LS) films reveled that the EosDEC and TBO binding mechanisms are dominated by electrostatic interactions with the anionic polar groups, with limited penetration into the chains. Light-irradiation reduced the relative area of E. coli monolayer on TBO, indicating an increased loss of material to the subphase owing to the chain cleavage, generated by contact-dependent reactions with excited states of TBO. In contrast, the increased relative area of E. coli monolayers containing EosDEC suggests lipid hydroperoxidation, which is PS contact-independent. Even considering a small chain penetration, the saturated EosDEC may have partitioned towards saturated reach domains, avoiding direct contact with membrane unsaturations.

Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

Investigating eosin Y as a photocatalyst for the radical-dependent activity-based probing of deubiquitinating enzymes.

Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.

A resonance Rayleigh scattering and fluorescence quenching dual-channel sensor for sensitive detection of chitosan based on Eosin Y.

The sensitive chitosan (CTS) detection methods based on the resonance Rayleigh scattering (RRS) quenching method and fluorescence quenching of Eosin Y were put forward. In the HAC-NaAC buffer solution, Eosin Y interacted with Triton X-100 to generate the binary complex which served as the RRS spectral probe. When CTS was interacted with the binary complex, the RRS intensity decreased with the increase of CTS. At the same time, the fluorescence intensity of Eosin Y decreased in the presence of Triton X-100, and the fluorescence intensity of "Eosin Y+Triton X-100" system further decreased when CTS was added. So it was further proved that there was a forming complex in "Eosin Y+Triton X100+CTS" system. The interaction was characterized by zeta potential, RRS, fluorescence spectrum, and UV-Vis spectroscopy. Under optimal conditions, there was a good linear relationship between the RRS decreased intensity (ΔI) and the concentration of CTS in the range of 0.05-1.30 µg/mL, with a regression equation of ΔI = 1325c + 73.66 and correlation coefficient (R2) of 0.9907. The detection limit was 0.0777 µg/mL. Likewise, the linear range of the fluorescence quenching was 0.03-1.30 µg/mL; the regression equation was ΔF = 1926c + 294.0 with R2 = 0.9800 under fluorescence quenching. The detection limit was 0.0601 µg/mL. Therefore, the dual-channel sensor for the determination of CTS was applied to the health products, and the results were satisfactory. The t test result showed that there was no statistical difference between the two methods.

Metal-free visible-light-mediated organophotoredox catalysis: synthesis of 3-functionalized indole via C-C, C-N bond formation.

A visible-light-mediated, mild and one-pot three-component reaction in the presence of organophotoredox catalyst Eosin Y using EtOH:H2O as reaction medium for the synthesis of 3-functionalized indole derivatives was developed. Visible light used in the protocol is green, inexpensive, readily available energy source. The sustainable reagents make the protocol compatible with green chemistry demands.

The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1-E2 conformational changes assessed by eosin fluorescence measurements.

H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1-E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K â†’ E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that-during dephosphorylation-the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.

Malignancy is in the eye of the beholder: Pathologic diagnosis of challenging follicular neoplasms in the era of noninvasive follicular thyroid neoplasms with papillary-like nuclear features and immunohistochemical and molecular adjuncts.

BACKGROUND: Classification of thyroid follicular neoplasms can be challenging for pathologists. Introduction of noninvasive follicular thyroid neoplasms with papillary-like nuclear features, the utilization of immunohistochemistry, and molecular analysis are all thought to be valuable diagnostic adjuncts. Our aim was to determine whether interobserver variability for follicular neoplasms has improved since the application of these adjuncts. METHODS: One representative section from a cohort of follicular neoplasms previously proven difficult for pathologists were examined independently by 7 pathologists and assigned to 1 of 3 diagnostic categories (benign, neoplasms with papillary-like nuclear features, or malignant). This process was carried out separately 3 times: (1) after viewing hematoxylin and eosin stain slides, (2) hematoxylin and eosin stain in conjunction with immunohistochemistry, and (3) hematoxylin and eosin stain/immunohistochemistry in conjunction with molecular analysis. The interobserver variability and overall agreement were then calculated using the free-marginal kappa coefficient. RESULTS: Agreement on hematoxylin and eosin stain was 57%, with a kappa coefficient of 0.36 (minimal agreement). The agreement improved slightly with the application of immunohistochemistry (kappa coefficient = 0.49 [weak agreement] and a percentage agreement 67%). The level of agreement decreased slightly after the addition of molecular analysis (kappa coefficient = 0.43 [weak agreement] and percentage agreement 62%). CONCLUSION: Despite attempts to standardize the diagnostic criteria for neoplasms with papillary-like nuclear features and the utilization immunohistochemistry and molecular analysis, attaining pathologic consensus for difficult follicular neoplasms of the thyroid remains a challenge.

Digital Multiplexed Gene Expression Analysis of mRNA and miRNA from Routinely Processed and Stained Cytological Smears: A Proof-of-Principle Study.

OBJECTIVE: Although transcriptomic assessments of small samples using high-throughput techniques are usually performed on fresh or frozen tissues, there is a growing demand for those performed on stained cellular specimens already used for diagnostic purposes. STUDY DESIGN: The possibility of detecting mRNAs and microRNAs (miRNAs) from routinely processed cytological samples using nCounter® technology was explored. Fresh samples from pleural and peritoneal effusions were analyzed using 2 parallel methods: samples were smeared and routinely stained using the May-Grünwald-Giemsa or Diff-Quik® method and mounted using conventional methods, and they were also studied following a snap freezing method, in which samples were maintained at -80°C until use. mRNAs and miRNAs were assessed and compared after total RNA extraction from both routinely processed samples and their matched frozen controls. RESULTS: A good concordance was found between the gene expression measured in routinely processed samples and their matched frozen controls for the majority of mRNAs and miRNAs tested. However, the standard deviation of low-expressed miRNA was high. CONCLUSIONS: Although nCounter® technology is a robust method to measure and characterize both mRNAs and miRNAs from routinely processed cytological samples, caution is recommended for the interpretation of low-expressed miRNA.

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Antibiotic Treatment in an Animal Model of Inflammatory Lung Disease.

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

Adsorption Characteristics of Activated Carbon for the Reclamation of Eosin Y and Indigo Carmine Colored Effluents and New Isotherm Model.

The adsorption response of eosin Y and indigo carmine acid dyes on activated carbon as a function of system temperature for a fixed concentration was investigated at various temperatures via adsorption isotherms and their thermodynamic quantities such as enthalpy, entropy, and Gibbs free energy changes. The adsorption data were exploited to develop a new adsorption isotherm. The new isotherm was developed with the spirit of solid-liquid phase equilibrium and regular solution theory. The proposed model has four adjustable constants and correlates adsorption isotherm in terms of the system temperature and melting temperature of the dye. The effect of pH on the removal of acid dyes was reported. The pH variation was observed to affect the adsorption efficiency. The removal of eosin Y and indigo carmine decreased from 99.4% to 82.6% and 92.38% to 79.48%, respectively, when the pH of the solution varied from 2 to 12. The thermodynamic analysis of the process reveals that the process of the removal of acid dyes is exothermic and spontaneous. Moreover, the kinetics parameters of the batch process are reported.

A deep learning diagnostic platform for diffuse large B-cell lymphoma with high accuracy across multiple hospitals.

Diagnostic histopathology is a gold standard for diagnosing hematopoietic malignancies. Pathologic diagnosis requires labor-intensive reading of a large number of tissue slides with high diagnostic accuracy equal or close to 100 percent to guide treatment options, but this requirement is difficult to meet. Although artificial intelligence (AI) helps to reduce the labor of reading pathologic slides, diagnostic accuracy has not reached a clinically usable level. Establishment of an AI model often demands big datasets and an ability to handle large variations in sample preparation and image collection. Here, we establish a highly accurate deep learning platform, consisting of multiple convolutional neural networks, to classify pathologic images by using smaller datasets. We analyze human diffuse large B-cell lymphoma (DLBCL) and non-DLBCL pathologic images from three hospitals separately using AI models, and obtain a diagnostic rate of close to 100 percent (100% for hospital A, 99.71% for hospital B and 100% for hospital C). The technical variability introduced by slide preparation and image collection reduces AI model performance in cross-hospital tests, but the 100% diagnostic accuracy is maintained after its elimination. It is now clinically practical to utilize deep learning models for diagnosis of DLBCL and ultimately other human hematopoietic malignancies.

A pH-sensitive eosin-block copolymer delivers proteins intracellularly.

There is great interest in developing strategies to deliver proteins into the cytoplasm of cells. We report here a PEG-poly-eosin block copolymer (PEG-pEosin) that can encapsulate proteins and release them in active form under mildly acidic conditions. A PEG-pEosin formulation composed of Cre and the endosomolytic protein LLO efficiently performed gene editing in cells and in the brains of mice after an intracranial injection.