Intra-pulpal connective tissue formation and the advanced carious lesion: Is chondrogenesis and heterotopic ossification a response to pulpal inflammation?

AIMS: (a) The aim of this study was to investigate both the formation of dense connective tissue within the dental pulp, and its association with pulpal inflammation in teeth with advanced carious lesions; and (b) to investigate in vitro whether inflammation affects the expression of markers related to chondrogenesis/osteogenesis in pulp cells. MATERIALS AND METHODS: Radiology and Histology: Forty-six teeth with advanced carious lesions were radiographically investigated for intra-pulpal radiodense structures. Specimens were processed for histology and stained with haematoxylin/eosin and proteoglycan-specific stains. The intra-pulpal connective tissue was scored as pulp stones or ectopic connective tissue. Cell culture: pulpal cells from human third molars (n = 5) were cultured in chondrogenic medium +/- TLR2/4 agonists. Expression of the genes IL6, TLR2/4, SOX9, COL1A1, COL2A1, TGFB1, RUNX2 and ALPL was assessed by qPCR. Proteoglycan content within cultures was assessed spectrophotometrically. RESULTS: Radiodense structures were discovered in about half of all pulps. They were associated with ectopic connective tissue (χ2  = 8.932, p = .004, OR = 6.80, 95% CI: [1.84, 25.19]) and with pulp stones (χ2  = 12.274, df = 1, p < .001, OR = 22.167, 95% CI: [2.57, 200.00]). The morphology of the ectopic tissue resembled cartilage and was associated with inflammatory infiltration of the pulp (χ2  = 10.148, p = .002, OR = 17.77, 95% CI: [2.05, 154.21]). After continuous stimulation of cultured cells with TLR2/4 agonists, the expression of two inflammatory markers increased: IL6 at Days 7 (p = .020) and 14 (p = .008); TLR2 at Days 7 (p = .023) and 14 (p = .009). Similarly, expression of chondrogenic markers decreased: SOX9 at Day 14 (p = .035) and TGFB1 at Day 7 (p = .004), and the osteogenic marker COL1A1 at Day 7 (p = .007). Proteoglycan content did not differ between unstimulated and stimulated cells. CONCLUSIONS: Ectopic connective tissue resembling cartilage can form in teeth affected by advanced carious lesions. This tissue type is radiographically visible and is associated with inflammatory infiltration of the pulp. Although TLR2/4 agonists led to an inflammatory response in cell culture of pulp cells, the effect on the expression of osteogenic/chondrogenic markers was limited, suggesting that immune cells are needed for connective tissue formation in vivo.

Validation of Muscle Fiber Architecture of the Human Tongue Revealed by Diffusion Magnetic Resonance Imaging With Histology Verification.

PURPOSE: The goal of this study is to validate the muscle architecture derived from both ex vivo and in vivo diffusion-weighted magnetic resonance imaging (dMRI) of the human tongue with histology of an ex vivo tongue. METHOD: dMRI was acquired with a 200-direction high angular resolution diffusion imaging (HARDI) diffusion scheme for both a postmortem head (imaged within 48 hr after death) and a healthy volunteer. After MRI, the postmortem head was fixed and the tongue excised for hematoxylin and eosin (H&E) staining and histology imaging. Structure tensor images were generated from the stained images to better demonstrate muscle fiber orientations. The tongue muscle fiber orientations, estimated from dMRI, were visualized using the tractogram, a novel representation of crossing fiber orientations, and compared against the histology images of the ex vivo tongue. RESULTS: Muscle fibers identified in the tractograms showed good correspondence with those appearing in the histology images. We further demonstrated tongue muscle architecture in in vivo tractograms for the entire tongue. CONCLUSION: The study demonstrates that dMRI can accurately reveal the complex muscle architecture of the human tongue and may potentially benefit planning and evaluation of oral surgery and research on speech and swallowing.

Morphometric and gut microbial evaluation of Tilapia (Oreochromis niloticus) fed on different levels of Moringa oleifera.

In current study, different feeding levels of Moringa oleifera formulated diet was compared to analyze the growth performance, feed conversion ratio, feed conversion efficiency and gut microbiology of Oreochromis niloticus. The study was comprised of four treatment groups including 4%, 8% and 12% Moringa oleifera and one control group which was devoid of Moringa leaves. The experimental trial was conducted at the Zoology laboratory of Pakistan Institute of Applied and Social Sciences, (PIASS) Kasur. The physicochemical parameters of water such as temperature, dissolve oxygen, pH, total dissolved solids and salinity in all aquaria were found non-significantly different from each other. In control condition T1, the average weight gain was 14.89±16.90a grams, while average length gain was 11.52±7.444a cm. However, the total viable count on Eosin methylene blue was 7.4×107, 5.8×107 on Tryptic soy agar and 5.8×107on Nutrient agar. In T2, the average weight gain was 16.22±16.09b grams and average length gain was 12.97±7.79b cm. The total viable count on Eosin methylene blue was 7×107, 5.5×107 on Tryptic soy agar and 5.8×107on Nutrient agar. In T3, the average weight gain was 37.88±27.43c grams, while the average length gain was recorded as 16.48±12.56c cm. However, the total viable count for treatment 3 was 6.4×10 on Eosin methylene blue, 4.8×107 on Tryptic soy agar and 5.2×107on Nutrient agar. In T4, the average weight gain was 44.22±31.67d grams, while the average length gain was 15.25±10.49d cm. The total viable count was 4.3×107on Eosin methylene blue, 3.1×107 on Tryptic soy agar and 3.8×107 on Nutrient agar. The effect of Moringa oleifera on the growth of Oreochromis niloticus was found to be significant and 12% Moringa extract showed maximum length and weight gain and minimum feed conversion ratio with the least microbial count in fish intestine.

Segmentation of HE-stained meningioma pathological images based on pseudo-labels.

Biomedical research is inseparable from the analysis of various histopathological images, and hematoxylin-eosin (HE)-stained images are one of the most basic and widely used types. However, at present, machine learning based approaches of the analysis of this kind of images are highly relied on manual labeling of images for training. Fully automated processing of HE-stained images remains a challenging task due to the high degree of color intensity, size and shape uncertainty of the stained cells. For this problem, we propose a fully automatic pixel-wise semantic segmentation method based on pseudo-labels, which concerns to significantly reduce the manual cell sketching and labeling work before machine learning, and guarantees the accuracy of segmentation. First, we collect reliable training samples in a unsupervised manner based on K-means clustering results; second, we use full mixup strategy to enhance the training images and to obtain the U-Net model for the nuclei segmentation from the background. The experimental results based on the meningioma pathology image dataset show that the proposed method has good performance and the pathological features obtained statistically based on the segmentation results can be used to assist in the clinical grading of meningiomas. Compared with other machine learning strategies, it can provide a reliable reference for clinical research more effectively.

Laboratory Indices in Patients with Positive and Borderline Flow Cytometry Eosin-5-Maleimide-Screening Test Results for Hereditary Spherocytosis.

OBJECTIVE: To evaluate laboratory indices in patients with hereditary spherocytosis, with positive and borderline flow cytometry eosin-5-melamide (EMA)-bound red blood cells screening test. STUDY DESIGN: We compared laboratory indices of 151 samples obtained from 139 different individual patients with negative, borderline, or positive EMA-test results. We also compared the clinical data of the patients in each EMA test results group. RESULTS: Borderline EMA-test results were obtained for 13 patients and were associated with more severe anemia, and lower reticulocyte count and reticulocyte production index compared with samples with positive EMA-test results. A receiving operator characteristic analysis identified mean corpuscular hemoglobin concentration of <32.5 g/dL as a cut-off, between positive/borderline and negative test results with 100% sensitivity. A higher prevalence of clinical markers typical of hereditary spherocytosis was found in patients with borderline or positive compared with negative EMA test samples. CONCLUSIONS: Based on laboratory data, borderline EMA-test results may be an indication of a more severe form of hereditary spherocytosis. Using mean corpuscular hemoglobin concentration as a cut-off may help predict and reduce negative EMA tests without compromising sensitivity. This finding needs to be further validated in other flow cytometry laboratories with a large EMA test sample pool.

Change in rat meibomian gland structure with external pressure on the eyelid.

PURPOSE: This study investigated structural changes in rat meibomian glands following repeated and sustained application of external pressure on the eyelids using a magnet and then subsequent removal of the external pressure. METHODS: Twenty-eight Sprague-Dawley rats were used. The upper eyelid was externally compressed using a pair of magnets. One magnet was placed inside the upper eyelid, another was placed outside the eyelid, and varying periods of pressure were investigated. Untreated eyes were used as controls. Meibography was performed, and the transverse eyelid tissue sections were stained with hematoxylin and eosin and anti-cytokeratin 5 antibody at one hour, two and four weeks after removing the magnets. RESULTS: Meibography showed increased meibomian gland loss (30.0 ± 5.0%), and tissue sections showed decreased area of secretory acini (0.04 ± 0.08 mm2) at one hour after applying external pressure using magnets versus in the control eyes (5.0 ± 5.0% and 0.08 ± 0.08 mm2, respectively). On the other hand, there was no meibomian gland loss or reduction of the area of secretory acini at two and four weeks after removing the magnets in comparison with the control eyes. CONCLUSIONS: Repeated and sustained application of external pressure on the eyelid could induce meibomian gland loss; however, this meibomian gland loss can be restored when the external pressure is removed. Therefore, the repeated application of external pressure on the eyelid is a safe treatment method for obstructive MGD.

Dual Photoredox Catalysis Strategy for Enhanced Photopolymerization-Based Colorimetric Biodetection.

Catalytic redox reactions have been employed to enhance colorimetric biodetection signals in point-of-care diagnostic tests, while their time-sensitive visual readouts may increase the risk of false results. To address this issue, we developed a dual photocatalyst signal amplification strategy that can be controlled by a fixed light dose, achieving time-independent colorimetric biodetection in paper-based tests. In this method, target-associated methylene blue (MB+) photocatalytically amplifies the concentration of eosin Y by oxidizing deactivated eosin Y (EYH3-) under red light, followed by photopolymerization with eosin Y autocatalysis under green light to generate visible hydrogels. Using the insights from mechanistic studies on MB+-sensitized photo-oxidation of EYH3-, we improved the photocatalytic efficiency of MB+ by suppressing its degradation. Lastly, we characterized 100- to 500-fold enhancement in sensitivity obtained from MB+-specific eosin Y amplification, highlighting the advantages of using dual photocatalyst signal amplification.

Use of Immunostaining for the diagnosis of Lymphovascular invasion in superficial Barrett's esophageal adenocarcinoma.

BACKGROUND: The prevalence of Barrett's esophageal adenocarcinoma (BEA) is increasing in Japan. Accurate assessment of lymphovascular invasion (LVI) after endoscopic resection or surgery is essential in evaluating treatment response. This study aimed to assess the usefulness of immunostaining in determining the extent of LVI in superficial BEA. METHODS: We retrospectively included 41 patients who underwent endoscopic resection or surgery between January 2007 and July 2018. In all cases, 3-µm serial sections from paraffin-embedded resected specimens were used for hematoxylin and eosin (H-E) staining and immunostaining for D2-40 and CD31. Two specialized gastrointestinal pathologists (T.Y. and T.T.), blinded to clinical information, independently evaluated the extent of LVI from these specimens. The LVI-positivity rate was evaluated with respect to the depth of invasion, changes in the positivity rate on immunostaining, pathological characteristics of patients with LVI, lymph node metastasis or relapse, and course after treatment. RESULTS: H-E staining alone identified LVI in 7 patients (positivity rate: 17.1%). Depths of invasion were categorized based on extension to the submucosa (SM) or deeper. On immunostaining for D2-40 and CD31, additional positivity was detected in 2 patients with SM1 and 1 SM3, respectively; LVI was detected in 10 patients (positivity rate: 24.4%). LVI-positivity rates with invasion of the superficial muscularis mucosa (SMM)/lamina propria mucosa (LPM)/deep muscularis mucosa (DMM), SM 1, 2, and 3 were 0, 75, 28.6, and 55.6%, respectively. CONCLUSIONS: Combined H-E staining and immunostaining is useful in diagnosing LVI in superficial BEA, particularly in endoscopically resected specimens.

AIM2 gene silencing attenuates diabetic cardiomyopathy in type 2 diabetic rat model.

AIMS: Absent in melanoma 2 (AIM2) is a cytosolic DNA sensor which plays an important role in inflammasome formation and is involved in various cellular functions including pyroptosis, fibrosis, and tissue injury. Our study aimed to investigate whether AIM2 plays a role in diabetic cardiomyopathy (DCM) and to explore its potential molecular mechanism. MAIN METHODS: Sprague-Dawley rats were randomly divided into 4 groups: Control, Diabetes Mellitus (DM), DM + shAIM2, and DM + shNC. The cardiac function of rats was measured. Hematoxylin and eosin staining, Masson's staining, sinus red staining, and immunohistochemistry were performed. H9c2 cardiomyocytes were cultured in DMEM and stimulated with high-glucose treatment (25 mmol/l). The level of reactive oxygen species (ROS) was measured. AIM2-siRNA were used to inhibit the expression of AIM2. TUNEL assay and EthD-III staining were used to measure cell death. The expression levels of AIM2, ASC, caspase-1, IL-1ß, and GSDMD-N were measured by western blotting. KEY FINDINGS: In the streptozotocin-induced diabetic rat model, AIM2 expression was significantly increased in heart tissue compared with the control. Also, diabetic rats exhibited severe left ventricular dysfunction including metabolic disorder, cardiac fibrosis, and cardiomyocyte death. Gene silencing of AIM2 alleviated cardiac dysfunction which resulted from metabolic disorder and ventricular remodelling. In vitro, treatment of H9C2 cardiomyoblasts with HG significantly increased AIM2, while ROS inhibition reduced the level of AIM2. AIM2-siRNA alleviated GSDMD-N-related pyroptosis in H9c2 cardiomyoblasts. SIGNIFICANCE: Our results indicate that AIM2 plays an important role in cell death and fibrosis in HG-induced, ROS-mediated diabetic cardiomyopathy via the GSDMD pathway.

Determination of Phthalic Acid, 2-(3',5'-Dibromo-2',4'-Dihydroxybenzoyl) Benzoic Acid, and Tribromoresorcinol in the Color Additives D&C Red No. 21, D&C Red No. 22 (Eosin Y), and Their Lakes Using UHPLC.

Background: Certain impurities in the color additives drug and cosmetic (D&C) Red No. 21 (R21), D&C Red No. 22 (R22), and their lakes are limited to levels specified in the Code of Federal Regulations (CFR) and are quantified by the U.S. Food and Drug Administration in batches of these color additives submitted for certification. Currently, a lengthy and tedious method based on gravity flow elution column chromatography is used to quantify the following CFR-specified impurities: the intermediate, phthalic acid (PhthAc); the manufacturing by-products, 2-(3',5'-dibromo-2',4'-dihydroxybenzoyl)benzoic acid (Br2BBA); and brominated resorcinol. "Brominated resorcinol" implies the sum of all possible brominated resorcinols, but the current work focused on 2,4,6-tribromoresorcinol (Br3R) as the most probable side-reaction product. Objective: An improved method was needed to quantify PhthAc, Br2BBA, and Br3R in R21, R22, and their lakes. Methods: A rapid ultra-HPLC (UHPLC) method was developed to replace the gravity flow method for quantitative determination of PhthAc, Br2BBA, and Br3R. Results: PhthAc, Br2BBA, and Br3R were quantified by using five-point calibration curves with data point ranges of 0.11-1.55, 0.06-0.77, and 0.04-0.61% by weight, respectively. LODs for the analytes ranged from 0.01 to 0.03%. Recoveries of the analytes ranged from 90.6 to 99.9%. Conclusions: The UHPLC method is accurate and significantly more rapid than the gravity -flow method, requiring approximately 7 min as compared with 6 h to detect PhthAc, Br2BBA, and Br3R in one sample. Highlights: A rapid UHPLC method was developed to determine CFR-specified impurities in the color additives D&C Red Nos. 21 and 22 and their lakes.

Highly selective and sensitive macrocycle-based dinuclear foldamer for fluorometric and colorimetric sensing of citrate in water.

The selective detection of citrate anions is essential for various biological functions in living systems. A quantitative assessment of citrate is required for the diagnosis of various diseases in the human body; however, it is extremely challenging to develop efficient fluorescence and color-detecting molecular probes for sensing citrate in water. Herein, we report a macrocycle-based dinuclear foldamer (1) assembled with eosin Y (EY) that has been studied for anion binding by fluorescence and colorimetric techniques in water at neutral pH. Results from the fluorescence titrations reveal that the 1·EY ensemble strongly binds citrate anions, showing remarkable selectivity over a wide range of inorganic and carboxylate anions. The addition of citrate anions to the 1·EY adduct led to a large fluorescence enhancement, displaying a detectable color change under both visible and UV light in water up to 2 µmol. The biocompatibility of 1·EY as an intracellular carrier in a biological system was evaluated on primary human foreskin fibroblast (HF) cells, showing an excellent cell viability. The strong binding properties of the ensemble allow it to be used as a highly sensitive, detective probe for biologically relevant citrate anions in various applications.

Validated spectroscopic methods for determination of anti-histaminic drug azelastine in pure form: Analytical application for quality control of its pharmaceutical preparations.

Two simple, sensitive, rapid, validated and cost effective spectroscopic methods were established for quantification of antihistaminic drug azelastine (AZL) in bulk powder as well as in pharmaceutical dosage forms. In the first method (A) the absorbance difference between acidic and basic solutions was measured at 228nm, whereas in the second investigated method (B) the binary complex formed between AZL and Eosin Y in acetate buffer solution (pH3) was measured at 550nm. Different criteria that have critical influence on the intensity of absorption were deeply studied and optimized so as to achieve the highest absorption. The proposed methods obeyed Beer's low in the concentration range of (2.0-20.0µg·mL-1) and (0.5-15.0µg·mL-1) with % recovery±S.D. of (99.84±0.87), (100.02±0.78) for methods (A) and (B), respectively. Furthermore, the proposed methods were easily applied for quality control of pharmaceutical preparations without any conflict with its co-formulated additives, and the analytical results were compatible with those obtained by the comparison one with no significant difference as insured by student's t-test and the variance ratio F-test. Validation of the proposed methods was performed according the ICH guidelines in terms of linearity, limit of quantification, limit of detection, accuracy, precision and specificity, where the analytical results were persuasive.

Is it time for one-step nucleic acid amplification (OSNA) in colorectal cancer? A systematic review and meta-analysis.

BACKGROUND: Lymph node metastasis (LNM) is prognostic in colorectal cancer (CRC). However, evaluation by routine haematoxylin and eosin histology (HE) limits nodal examination and is subjective. Missed LNMs from tissue allocation bias (TAB) might under-stage disease, leading to under-treatment. One-step nucleic acid amplification (OSNA) for CK19 messenger ribonucleic acid (mRNA), a marker of LNM, analyses the whole node. The aim of the present systematic review and meta-analysis was to assess recent studies on OSNA versus HE and its implications for CRC staging and treatment. METHODS: Databases including OVID, Medline and Google Scholar were searched for OSNA, LNM and CRC. Study results were pooled using a random-effects model. Summary receiver operator curves (SROC) assessed OSNA's performance in detecting LNM when compared to routine HE histology. RESULTS: Five case-control studies analysing 4080 nodes from 622 patients were included. The summary estimates of pooled results for OSNA were sensitivity 0.90 [95% confidence interval (CI) 0.86-0.93], specificity 0.94 (95% CI 0.93-0.95) and diagnostic odds ratio 179.5 (CI 58.35-552.2, p < 0.0001). The SROC curve indicated a maximum joint sensitivity and specificity of 0.88 and area under the curve of 0.94, p < 0.0001. On average, 5.4% HE-negative nodes were upstaged by OSNA. CONCLUSIONS: OSNA is as good as routine HE. It may avoid TAB and offer a more objective and standardised assay of LNM. However, for upstaging, its usefulness as an adjunct to HE or superiority to HE requires further assessment of the benefits, if any, of adjuvant therapy in patients upstaged by OSNA.

Delayed Measurement of Eosin-5'-Maleimide Binding May Affect the Test Results of Highly Hemolyzed Samples In Vivo and In Vitro-A Case Study.

Diagnosis of hereditary spherocytosis (HS) is based on clinical evaluation and eosin-5'-maleimide (EMA) test. A decrease in EMA fluorescence compared with healthy individuals is typical for HS and serves as a basis for HS diagnosis. Sensitivity and specificity of the test is high and false-positive results rarely occur. Studies have shown that anticoagulated blood sample when stored at 4°C for 7 days do not affect the test results. This case study is about an autoimmune hemolytic anemia patient who showed a primary positive result for EMA test (decrease in EMA fluorescence-47% compared with 100% for samples of healthy individual), when the test was performed in the sample stored for 48 hours after venipuncture and before staining. An irrelevant decrease (92.5% compared with 100% for samples of healthy individual) was found when freshly collected sample was analyzed. On the basis of the results obtained, it is recommended that EMA staining should be performed on the same day of blood collection for patients with significant hemolysis.

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats.

Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners.

Evaluating eosin-5-maleimide binding as a diagnostic test for hereditary spherocytosis in newborn infants.

OBJECTIVE: Neonates with undiagnosed hereditary spherocytosis (HS) are at risk for developing hazardous hyperbilirubinemia and anemia. Making an early diagnosis of HS in a neonate can prompt anticipatory guidance to prevent these adverse outcomes. A recent comparison study showed that a relatively new diagnostic test for HS, eosin-5-maleimide (EMA)-flow cytometry, performs better than other available tests in confirming HS. However, reports have not specifically examined the performance of this test among neonates. STUDY DESIGN: We compared EMA-flow cytometry from blood samples of healthy control neonates vs samples from neonates suspected of having HS on the basis of severe Coombs-negative jaundice and spherocytes on blood film. The diagnosis of HS was later either confirmed or excluded based on clinical findings and next generation sequencing (NGS) after which we correlated the EMA-flow results with the diagnosis. RESULT: EMA-flow was performed on the blood of 31 neonates; 20 healthy term newborns and 11 who were suspected of having HS. Eight of the 11 were later confirmed positive for HS and one was confirmed positive for hereditary elliptocytosis (HE). All nine had persistently abnormal erythroid morphology, reticulocytosis and anemia, and eight of the nine had relevant mutations discovered using NGS. The other was confirmed positive for HS on the basis that a parent had HS, and the neonate's spherocytosis, reticulocytosis and anemia persisted. The 20 healthy controls and the 2 in whom HS was initially suspected but later excluded all had EMA-flow results in the range reported in healthy children and adults. In contrast, all nine in whom HS or HE was confirmed had abnormal EMA-flow results consistent with previous reports in older children and adults with HS. CONCLUSION: Although our sample size is small, our findings are consistent with the literature in older children and adults suggesting that EMA-flow cytometric testing performs well in supporting the diagnosis of HS/HE during the early neonatal period.

Study on the fluorescence resonance energy transfer between CdS quantum dots and Eosin Y.

Water-soluble CdS quantum dots (QDs) were prepared using mercaptoacetic acid (TGA) as the stabilizer in an aqueous system. A fluorescence resonance energy transfer (FRET) system was constructed between water-soluble CdS QDs (donor) and Eosin Y (acceptor). Several factors that impacted the fluorescence spectra of the FRET system, such as pH (3.05-10.10), concentration of Eosin Y (2-80 mg/L) and concentration of CdS QDs (2-80 mg/L), were investigated and refined. Donor-to-acceptor ratios, the energy transfer efficiency (E) and the distance (r) between CdS QDs and Eosin Y were obtained. The results showed that a FRET system could be established between water-soluble CdS QDs and Eosin Y at pH 5.0; donor-to-acceptor ratios demonstrated a 1: 8 proportion of complexes; the energy transfer efficiency (E) and the distance (r) between the QDs and Eosin Y were 20.07% and 4.36 nm,respectively.

Resolution of fluorophore mixtures in biological media using fluorescence spectroscopy and Monte Carlo simulation.

In excitation-emission fluorescence spectroscopy, the simultaneous quantitative prediction and qualitative resolution of mixtures of fluorophores using chemometrics is a major challenge because of the scattering and reabsorption effects (turbidity) presented mainly in biomaterials. The measured fluorescence spectra are distorted by multiple scattering and reabsorption events in the surrounding medium, thereby diminishing the performance of the commonly used three-way resolution methods such as parallel factor (PARAFAC) analysis or multivariate curve resolution-alternating least squares (MCR-ALS). In this work we show that spectral loadings and concentration profiles from model mixtures provided using PARAFAC and MCR-ALS are severely distorted by reabsorption and scattering phenomena, although both models fit rather well the experimental data in terms of percentage of the explained variance. The method to correct the fluorescence excitation-emission matrix (EEM) consisted in measuring the optical properties (absorption parameter µa , scattering parameter µs, and anisotropy factor g) of samples and calculating the corresponding transfer function by means of the Monte Carlo simulation method. By applying this transfer function to the measured EEM, it was possible to compensate for reabsorption and scattering effects and to restore the ideal EEM, i.e., the EEM that is due only to fluorophores, without distortions from the absorbers and scatterers that are present. The PARAFAC and MCR-ALS decomposition of the resulting ideal EEMs provided spectral loadings and concentration profiles that matched the true profiles.

Aspect ratio dependent fluorescence quenching of eosin Y by gold nanorods.

Gold nanorods of different aspect ratios had been synthesized using seed mediated growth method. The formed gold nanorods had been characterized by the absorption and transmission electron microscopy (TEM) measurements. The obtained gold nanorods were used to study the quenched effect on fluorescence of Eosin Y. Experimental results revealed that Eosin Y molecules adsorbed on the metallic surfaces, suffering strong quenching of their fluorescence and the quenching efficiency was different for different aspect ratio. Using dielectric coated gold nanorods model, the probable mechanism of aspect ratio dependent quenching efficiency was obtained by numerical calculation based on fluorescence resonance energy transfer and quasi-static theory. The calculation results showed that the non-monotonic changing of fluorescence quenching was attributed to competing effects of aspect ratio and the dielectric constant of coated shell on surface plasmon resonance.

Analysis of tumor mitotic rate in thin metastatic melanomas compared with thin melanomas without metastasis using both the hematoxylin and eosin and anti-phosphohistone 3 IHC stain.

Studies have suggested that elevated tumor mitotic rate (MR) is linked to overall survival in thin melanoma. Recently, promising data regarding anti-phosphohistone 3 (pHH3) immunohistochemistry and its ability to aid in calculation of MR have emerged. The authors retrospectively analyzed original biopsies from 13 thin melanomas with positive sentinel node (SN) status and 16 thin melanomas with negative SN status. Both anti-pHH3 immunohistochemistry and the hematoxylin and eosin (H&E) stain were used to evaluate MR by 2 dermatopathologists blinded to SN status using the "hot spot" method. Intraclass coefficient values were attained to measure interobserver concordance and reliability of the pHH3 stain. By generating a receiver operating characteristic curve and analyzing the overall area under the curve, pHH3 was found to have good interobserver reliability. The relationship between MR and SN involvement was also evaluated, but this correlation was not statistically significant.