A flow cytometric cell-cycle assay using methyl green.

Methyl green (MG), a conventional, low-cost histological stain, was used to design a flow cytometric cell-cycle/DNA-ploidy assay. On fluorometry, MG absorbed maximally at 633-nm, showed negligible fluorescence in free-state, but emitted brightly when bound to DNA. Optimal dye and cell concentrations for staining and effects of time and photobleaching on stained cells were determined for a lyse-permeabilize-stain protocol. Linearity of DNA-binding, coefficients-of-variation of G0/G1-peaks and minimal carryover were confirmed. Assay results correlated highly with a propidium iodide-based kit in 29 acute lymphoblastic leukemia specimens. The MG-based DNA-ploidy assay represented an accurate and inexpensive alternative to conventional PI-based assays.

High-throughput quantitative histology in systemic sclerosis skin disease using computer vision.

BACKGROUND: Skin fibrosis is the clinical hallmark of systemic sclerosis (SSc), where collagen deposition and remodeling of the dermis occur over time. The most widely used outcome measure in SSc clinical trials is the modified Rodnan skin score (mRSS), which is a semi-quantitative assessment of skin stiffness at seventeen body sites. However, the mRSS is confounded by obesity, edema, and high inter-rater variability. In order to develop a new histopathological outcome measure for SSc, we applied a computer vision technology called a deep neural network (DNN) to stained sections of SSc skin. We tested the hypotheses that DNN analysis could reliably assess mRSS and discriminate SSc from normal skin. METHODS: We analyzed biopsies from two independent (primary and secondary) cohorts. One investigator performed mRSS assessments and forearm biopsies, and trichrome-stained biopsy sections were photomicrographed. We used the AlexNet DNN to generate a numerical signature of 4096 quantitative image features (QIFs) for 100 randomly selected dermal image patches/biopsy. In the primary cohort, we used principal components analysis (PCA) to summarize the QIFs into a Biopsy Score for comparison with mRSS. In the secondary cohort, using QIF signatures as the input, we fit a logistic regression model to discriminate between SSc vs. control biopsy, and a linear regression model to estimate mRSS, yielding Diagnostic Scores and Fibrosis Scores, respectively. We determined the correlation between Fibrosis Scores and the published Scleroderma Skin Severity Score (4S) and between Fibrosis Scores and longitudinal changes in mRSS on a per patient basis. RESULTS: In the primary cohort (n = 6, 26 SSc biopsies), Biopsy Scores significantly correlated with mRSS (R = 0.55, p = 0.01). In the secondary cohort (n = 60 SSc and 16 controls, 164 biopsies; divided into 70% training and 30% test sets), the Diagnostic Score was significantly associated with SSc-status (misclassification rate = 1.9% [training], 6.6% [test]), and the Fibrosis Score significantly correlated with mRSS (R = 0.70 [training], 0.55 [test]). The DNN-derived Fibrosis Score significantly correlated with 4S (R = 0.69, p = 3 × 10- 17). CONCLUSIONS: DNN analysis of SSc biopsies is an unbiased, quantitative, and reproducible outcome that is associated with validated SSc outcomes.

The Diagnostic Role of Methyl Green-Pyronin Y Staining in Oral Leukoplakia and Oral Squamous Cell Carcinoma: An Exfoliative Cytology-Based Cytomorphometric Analysis.

BACKGROUND: Oral exfoliative cytology is a noninvasive and nonpainful technique for early diagnosis of oral potentially malignant disorders and oral cancer, and the use of cytomorphometry ameliorates its diagnostic reliability. The objective of the present study was to analyze methyl green-pyronin Y (MGP)-stained oral exfoliated cells (OECs) of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) by cytomorphometry. MATERIALS AND METHOD: An observational study was conducted on 150 individuals equally divided into three groups: normal mucosa, OL, and OSCC. Smears were prepared from OECs and stained with MGP. Cytomorphometry was done for 100 cells per subject, and various cell and nuclear parameters were measured and calculated. RESULTS: The Kruskal-Wallis test with post hoc correlation showed significant differences in nucleus and cell diameter (ND, CD), nucleus and cell area (NA, CA), nucleus and cell perimeter (NP, CP), and nucleus to cytoplasmic (N:C) ratio for diameter, perimeter, and area. Spearman's ρ correlation of various N:C ratio methods showed good correlation between N:C perimeter and diameter ratio, N:C diameter and ellipse ratio, and N:C area and ellipse ratio. Additional morphological factors showed significant relations for both cell and nuclear regularity factor, shape factor, and nuclear contour index. DISCUSSION: MGP-based cytomorphometry showed a significant decrease in CD, CA, and CP and increase in ND, NA, NP, and N:C ratio from normal mucosa to OL and OSCC. MGP proved its worth as an effective stain for OECs, despite its strict standardization.

A histochemical comparison of methyl green-pyronin, and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma, oral leukoplakia, oral submucous fibrosis and normal oral mucosa.

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.

Solvatochromism, DNA binding, antitumor activity and molecular modeling study of mixed-ligand copper(II) complexes containing the bulky ligand: bis[N-(p-tolyl)imino]acenaphthene.

Four mixed-ligand copper(II) complexes of the nitrogen ligand bis[N-(p-tolyl)imino]acenaphthene 1 (p-Tol-BIAN). These complexes, namely [Cu(p-Tol-BIAN)2](ClO4)2 2, [Cu(p-Tol-BIAN)(acac)](ClO4) 3, [Cu(p-Tol-BIAN)Cl2] 4 and [Cu(p-Tol-BIAN)(AcOH)(2)](ClO4)2 5, were prepared and characterized. Solvatochromism of the novel copper complexes in various solvents has been studied. Molecular mechanics (MM+) and molecular dynamic simulations have been performed to learn more about the solvatochromic behaviour and the DNA binding affinity of these complexes.

Reproductive management of silver foxes (Vulpes vulpes) in captivity.

Specific protocols need to be developed that take into consideration the requirements of silver foxes. This study was designed to investigate the reproduction of 26 pairs of silver foxes (Vulpes vulpes). Reproduction parameters (breeding season, age at puberty, fecundity and fertility) of the foxes were examined under experimental conditions, and new techniques to optimize fox breeding were assessed. Staining of the vaginal smears with the double Harris-Schorr stain allowed for the precise detection of estrus and proved to be more reliable than simply measuring vaginal resistance. Ultrasonography was demonstrated to be useful for pregnancy diagnosis and prediction of parturition. Results demonstrate that the methods reported in this paper are easily applicable to similar studies carried out on small groups of animals within the framework of fox animal experimentation.

Nemaline rods in canine myopathies: 4 case reports and literature review.

The diagnosis of nemaline rod myopathy (NM) is based on the presence of numerous pathognomonic rods within a fresh frozen muscle biopsy specimen. Three forms of congenital NM have been described in humans, and rods have been found to occur in various other conditions. A similar myopathy was described in 1986 in a family of cats. In this report, we describe a case of congenital NM in a 10-month-old Border Collie, an adult-onset NM in an 11-year-old Schipperke, and 2 acquired myopathies with nemaline rods in adult dogs associated with hypothyroidism and Cushing's syndrome. Common clinical features included exercise intolerance, abnormal electromyography, and the presence of nemaline rods in fresh, frozen, and glutaraldehyde-fixed biopsies from proximal appendicular limb muscles. Staining of cryostat sections of muscle biopsy specimens by the modified Gomori trichrome technique disclosed numerous rod bodies that were localized to type 1 fibers by the histochemical adenosine triphosphatase reaction. Accumulation of rods also was demonstrated by electron microscopy in 2 of the cases with localized enlargement and streaming of Z lines. Documentation of NM in a young Border Collie and the adult-onset form in the Schipperke alerts clinicians to the existence of this disorder in these breeds.

Experimental infection of inbred mouse strains with Spironucleus muris.

Four inbred mouse strains: BALB/c ByJ, 129/J, C3H/HeJ, and DBA/lJ, differing in major histocompatibility type, were orally inoculated with 2 x 10(5) infectious cysts of Spironucleus muris. Fecal samples were collected for fecal cyst output prior to infection, and on days 2, 6, 7, 8, 9, 11, 13, 15, and 17 after infection. Following necropsy, formalin-fixed intestinal sections were examined for the presence of trophozoites. On post-inoculation days 6 and 8, mice of the 129/J strain shed significantly (p<0.05) fewer cysts than other strains. This pilot study suggests that major histocompatibility haplotype may influence susceptibility of inbred mouse strains to S. muris.

Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism.

Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.