Staining Properties Research Articles

Comparison Between p16INK4A Immunohistochemistry and Human Papillomavirus Polymerase Chain Reaction Assay in Oral Papillary Squamous Cell Carcinoma

Oral papillary squamous cell carcinoma (OPSCC) is a histologic variant of SCC with a growth pattern suggesting human papillomavirus (HPV) infection. The aim of this study was to investigate the presence of HPV genotypes in OPSCC. All cases with a histologic diagnosis of OPSCC from 1993 through 2008 were retrieved and confirmed. Immunohistochemical evaluation for the surrogate marker p16(INK4A) and HPV polymerase chain reaction were performed in tissue and DNA derived from formalin-fixed paraffin-embedded tissue. Forty-four patients with confirmed OPSCC (mean age, 71.96 yr; female-to-male ratio, 1.75:1) comprised the study population. The most common site of involvement was the gingiva followed by the palate and buccal mucosa. Forty cases exhibited an invasive component, 1 was noninvasive, and in 3 cases invasion could not be confirmed owing to suboptimal thickness of the biopsy. Paraffin tissue blocks were available in 41 cases. Twenty-three cases (56.1%) exhibited positive p16(INK4A) staining, which was primarily weak to moderate with a generally focal pattern. Polymerase chain reaction assays were negative for HPV DNA in all cases. In this study, there was a clinical predilection of OPSCC in older women, with most cases occurring in the masticatory mucosa. Weak to moderate and focal p16(INK4A) staining was appreciated in contrast to reported staining properties in genital and oropharyngeal PSCC. Failure of the polymerase chain reaction assay to exhibit transcriptionally active HPV genotypes suggests that HPV is not associated with OPSCC tumorigenesis.

New fluorescent derivatives of oligopropylamines

New fluorescent-tagged amines have been obtained starting from 4-chloro-7-nitro-2,1,3benzoxadiazole and oligopropylamines containing fragments of N-methylpropylamine close to those found in diatom frustules. Optical properties of new dyes and staining of silica structures in vivo and in vitro are described.

Comparative tissue stainability and localization of active staining principle in fractions of Lawsonia inermis (Henna) leaves

The crude aqueous and ethanolic extracts of the leaves of Lawsonia inermis (Henna) have demonstrated cytoplasmic staining properties in animal tissues. However, to improve the staining efficacy on tissues and to localize the active dyestuff, the extracts were fractionated sequentially in n‐hexane, ethyl acetate, n‐butanol and distilled water using column chromatography. The soluble fractions were subjected to thin layer chromatography to identify the active compounds. Tissue stainability of the fractions was evaluated in formalin‐fixed and paraffin embedded tissues (liver, kidney, cardiac muscle, testis, lungs, brain and intestine) obtained from Wistar rats. Ethyl acetate was the most effective and showed better tissue stainability compared to n‐Butanol, characterized by the deep brown colour intensity on the tissues and remarkable contrast with haematoxylin comparable to haematoxylin and eosin stain. This corroborated the TLC analysis which demonstrated that the ethyl acetate and n‐butanol fractions showed brownish spots which were absent in the hexane and distilled water fractions. Ethyl acetate spot showed deeper colouration than the n‐Butanol spot suggesting that the active staining principle known as lawsone is localized in these fractions. The n‐hexane and distilled water fractions showed weak to no staining activity on tissues. This indicates that the active staining principle of Henna is localized in the ethyl acetate and n‐butanol fractions and these will serve as good solvents for the further purification of the active staining compound.Grant Funding Source: A.B.U Zaria

Secondary metabolite production of Epicoccum sp. isolated from lignite

Abstract We have extracted secondary metabolites from fossil fungus Epicoccum sp. isolated from lignite and characterized changes in production on various growth media. Chromatographic separation yielded more than 7 fractions that were characterized spectroscopically. One secondary metabolite showed antimicrobial activity. Peaks in the NMR spectra of isolated fractions differ from compounds described in literature that were isolated from recent strains of Epicoccum. The remaining water phase after the extraction was shown to have protein staining properties and could be also used for fluorescent viability stain of yeast cells.

The spatial organization and extraction of the wall-forming bodies ofEimeria maxima

Eimeria maxima has been used as a model apicomplexan parasite to study sexual stage development and oocyst wall formation. A complete understanding of the wall's biochemical and biophysical properties is of great interest in research on all apicomplexan parasites. Purified gametocytes, zygotes and oocysts were analysed by three-dimensional confocal microscopy, and wide-field fluorescent microscopy was used to investigate the appearance and spatial organization of the 2 types of wall-forming bodies (WFBs). In addition, a variety of staining procedures and immunoassays were used to assess the biosynthesis, metabolic activity, intactness and molecular composition of the WFBs in situ. WFBs were extracted from gametocytes/zygotes and their composition was assessed by microscopy and SDS-PAGE analysis. It was concluded that isolated gametocytes are intact and metabolically active. Additionally, it was observed that the Type 1 WFBs are aligned at the periphery of the parasite and fuse together producing neutral lipid rich patches that appear to be inserted into the space between 2 parasite-specific membranes. Finally, it was shown that the WFBs extracted from purified gametocytes had the same shape, size and staining properties as those observed in situ, and contained the major glycoprotein antigens known to be present in these organelles.

The Truncated C-terminal RNA Recognition Motif of TDP-43 Protein Plays a Key Role in Forming Proteinaceous Aggregates

TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncated RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycine-rich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two β-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed β-strands, which can oligomerize into high-order inclusions.

Shape Fidelity of Native and Engineered Human Nasal Septal Cartilage

To test engineered and native septal cartilage for resistance to deformation and remodeling under sustained bending loads and to determine the effect of bending loads on the biochemical properties of constructs. Prospective, basic science. Laboratory. Human septal chondrocytes from 6 donors were used to create 12-mm constructs. These were cultured for 10 weeks and subjected to bending for 6 days. Free-swelling controls and native tissue from 6 donors were used for comparison. Shape retention, photo documentation, live-dead staining, and biochemical properties were measured. Live-dead staining showed no difference in cell survival between loaded constructs and free-swelling controls. The immediate shape retention of the constructs was 39.0% versus 24.4% for native tissue (P = .13). After 2 and 24 hours of relaxation, the constructs possessed similar shape retention to native tissue (26.9% and 16.4%; P = .126; 21.7% and 14.4%; P = .153). There was no significant change in construct shape retention from immediately after release to 2 hours of relaxation (39.0% and 26.9%, respectively; P = .238). In addition, the retention did not change significantly between 2 and 24 hours of relaxation (26.9% and 21.7%; P = .48). There was no significant difference in biochemical properties between loaded constructs and controls. The shape retention properties of human septal neocartilage constructs are comparable to human native septal cartilage. In addition, mechanical loading of neocartilage constructs does not adversely affect cell viability or biochemical properties. This study demonstrates that neocartilage constructs possess adequate shape fidelity for use as septal cartilage graft material.

Hydrothermal Synthesis and Characterization of Ni-Al Montmorillonite-Like Phyllosilicates.

This work describes the first hydrothermal synthesis in fluoride medium of Ni-Al montmorillonite-like phyllosilicates, in which the only metallic elements in the octahedral sheet are Ni and Al. X-ray diffraction , chemical analysis, thermogravimetric and differential thermal analysis, scanning electron microscopy and transmission electron microscopy confirm that the synthesized samples are montmorillonite-like phyllosilicates having the expected chemical composition. The specific surface areas of the samples are relatively large (>100 m2 g−1) compared to naturally occurring montmorillonites. 29Si and 27Al nuclear magnetic resonance (NMR) indicate substitutions of Al for Si in the tetrahedral sheet. 19F NMR and Ni K-edge extended X-ray absorption fine structure (EXAFS) local probes highlight a clustering of the metal elements and of the vacancies in the octahedral sheet of the samples. These Ni-Al phyllosilicates exhibit a higher local order than in previously synthesized Zn-Al phyllosilicates. Unlike natural montmorillonites, where the distribution of transition metal cations ensures a charge equilibrium allowing a stability of the framework, synthetic montmorillonites entail clustering and instability of the lattice when the content of divalent element in the octahedral sheet exceeds ca. 20%. Synthesis of Ni-Al montmorillonite-like phyllosilicates, was successfully achieved for the first time. These new synthetic materials may find potential applications as catalysts or as materials with magnetic, optical or staining properties.

Optical imaging with a high‐resolution microendoscope to identify cholesteatoma of the middle ear

High-resolution optical imaging is an imaging modality that allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation, and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Ex vivo imaging of histopathologically confirmed samples of cholesteatoma and uninvolved middle ear epithelium. Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin and eosin staining to allow histopathological correlation. Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, whereas nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin, allowing for discrimination of cholesteatoma from normal middle ear epithelium. This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease. Laryngoscope, 2012.

Isolation of α-Haemolysin-ProducingStaphylococcus aureusfrom the Blood Sample ofGallus domesticus(Chicken) resistant to few Antibiotics

The present study was conducted in blood samples collected from Chicken (Gallus domesticus) during the period of December, 2010, from Anand Poultry, Gujarat, India, for isolation, identification and characterisation of pathogenic bacterial species on basis of their morphology, staining properties, cultural and biochemical properties. Seven different isolates were obtained from the blood. All of them were found to be pathogenic. Antibiotic sensitivity and resistivity of the isolated organisms were also studied. Sodium dodecyl sulfate-polyacrylamide electrophoresis demonstrated the presence of α-haemolysin protein of 33 kDa. Further, 16SrRNA was performed to characterise bacteria, which were further confirmed as streptomycin-, penicillin- and MOX-resistant Staphylococcus aureus.

A new diagnostic algorithm for Burkitt and diffuse large B-cell lymphomas based on the expression of CSE1L and STAT3 and on MYC rearrangement predicts outcome

Aggressive mature B-cell non-Hodgkin's lymphomas (BCL) sharing features of Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) (intermediate BL/DLBCL) but deviating with respect to one or more characteristics are increasingly recognized. The limited knowledge about these biologically heterogeneous lymphomas hampers their assignment to a known entity, raising incertitude about optimal treatment approaches. We therefore searched for discriminative, prognostic, and predictive factors for their better characterization. We analyzed 242 cytogenetically defined aggressive mature BCL for differential protein expression. Marker selection was based on recent gene-expression profile studies. Predictive models for diagnosis were established and validated by a different set of lymphomas. CSE1L- and inhibitor of DNA binding-3 (ID3)-overexpression was associated with the diagnosis of BL and signal transduction and transcription-3 (STAT3) with DLBCL (P<0.001 for all markers). All three markers were associated with patient outcome in DLBCL. A new algorithm discriminating BL from DLBCL emerged, including the expression of CSE1L, STAT3, and MYC translocation. This 'new classifier' enabled the identification of patients with intermediate BL/DLBCL who benefited from intensive chemotherapy regimens. The proposed algorithm, which is based on markers with reliable staining properties for routine diagnostics, represents a novel valid tool in separating BL from DLBCL. Most interestingly, it allows segregating intermediate BL/DLBCL into groups with different treatment requirements.

A coumarin-based “turn-on” fluorescent sensor for the determination of Al3+: single crystal X-ray structure and cell staining properties

An efficient Al(3+) receptor, 6-(2-hydroxybenzylideneamino)-2H-chromen-2-one (HBC), has been synthesized by condensing salicylaldehyde with 6-aminocoumarin. The molecular structure of HBC has been determined by a single crystal X-ray analysis. It was established that in the presence of Al(3+), HBC shows 25 fold enhancement of fluorescence intensity which might be attributed to the chelation-enhanced fluorescence (CHEF) process. HBC binds Al(NO3)3 in a 1 : 1 stoichiometry with a binding constant (K) of 7.9 × 10(4) M(-1). Fe(3+) and Mn(2+) quench the emission intensity of the [HBC + Al(3+)] system to an insignificant extent at a concentration 10 times higher compared to that of Al(3+). HBC is highly efficient in the detection of intracellular Al(3+) under a fluorescence microscope.

Enhanced tissue differentiation in the developing mouse brain using magnetic resonance micro‐histology

Worldwide efforts to understand developmental processes demand new high-resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that MRI contrast agents can highlight specific tissue structures in ex vivo adult mouse brains. MR imaging of mouse embryos is currently limited by a lack of tissue staining capabilities that would provide the flexibility and specificity offered by histological stains conventionally used for mouse embryo phenotyping. The MRI staining properties of two readily available contrast agents, Mn-DPDP and Gd-DTPA, were investigated in mid-gestation mouse embryos. Brain tissue substructures not normally visible using MRI were detected. Mn-DPDP and Gd-DTPA provided spatially distinct tissue staining patterns. An initial assessment indicated that these agents utilized independent contrast enhancement mechanisms. Mn-DPDP was identified as a potential MRI contrast agent for enhancement of mouse embryonic cellular density and enabled identification of regions containing populations of neural stem and progenitor cells within the intact embryo brain. Different contrast agents may be used to provide tissue-specific contrast enhancement, suggesting that a host of specialized MRI stains may be available for probing the developing mouse brain and investigating developmental and disease mechanisms.

Prostatic stromal sarcoma with neuroectodermal differentiation

Prostatic stromal sarcoma is a fairly rare tumor that constitutes approximately 0.1–0.2% of all prostatic cancers. Detailed characteristics of the tumor are still unclear due to its rarity.We describe a case of prostatic stromal sarcoma in a 63 year-old man who suffered from urinary obstructive symptoms. Palliative transuterine resection was performed and the preliminary histopathological diagnosis was neuroendocrine carcinoma. After chemotherapy, total pelvic exenteration was performed. Histopathologically, the tumor was composed of monotonously proliferating small to medium-sized round cells, which existed in compact islands with loose or dense fibrovascular networks. Immunohistochemically, the tumor cells were widely positive for vimentin, CD56, CD99 and focally positive for synaptophysin, CD10, progesterone receptor, desmin and CD34, but negative for EMA, cytokeratin, estrogen receptor, S-100 and myoglobin. Most of the previously reported tumors exhibited positive stainability for CD10 and progesterone receptor. In addition to these markers, expressions of CD56, CD99 and synaptophysin were characteristically detected in our case. To the best of our knowledge, we present the first case of prostatic stromal sarcoma with characteristic immunohistochemical staining properties. Although the biological characteristics of this rare tumor have not yet been elucidated, these findings suggest prostatic stromal sarcoma can potentially show neuroectodermal differentiation.Virtual slideThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7291874028051262

Cerebrospinal fluid shunt infection due to Gemella haemolysans

Gemella haemolysans has long been considered a commensal in the human upper respiratory tract. Commensals are natural inhabitants on or within another organism, deriving benefit without harming or benefiting the host. Opportunistic infection of the CNS by the species is exceedingly rare. In the present case, a 16-year-old boy was admitted with a ventriculoperitoneal shunt infection, which was confirmed to be due to G. haemolysans. Following antibiotic treatment, removal of the old shunt, and delayed insertion of a new shunt, the patient made a full neurological recovery. To the authors' knowledge, this is the eighth case of CNS infection with G. haemolysans. Although prosthesis-related infections have been reported in other systems, this is the first case of CNS infection by the bacterium associated with an implant. Previous reported cases of CNS infection by G. haemolysans are reviewed. Due to the variable Gram staining property of the organism, the difficulty in diagnosing G. haemolysans infection is emphasized.

The use of fluorescent indoline dyes for side population analysis

Dye efflux assay evaluated by flow cytometry is useful for stem cell studies. The side population (SP) cells, characterized by the capacity to efflux Hoechst 33342 dye, have been shown to be enriched for hematopoietic stem cells (HSCs) in bone marrow. In addition, SP cells are isolated from various tissues and cell lines, and are also potential candidates for cancer stem cells. However, ultra violet (UV) light, which is not common for every flow cytometer, is required to excite Hoechst 33342. Here we showed that a fluorescent indoline dye ZMB793 can be excited by 488-nm laser, equipped in almost all the modern flow cytometers, and ZMB793-excluding cells showed SP phenotype. HSCs were exclusively enriched in the ZMB793-excluding cells, while ZMB793 was localized in cytosol of bone marrow lineage cells. The efflux of ZMB793 dye was mediated by ATP binding cassette (ABC) transporter Abcg2. Moreover, staining properties were affected by the side-chain structure of the dyes. These data indicate that the fluorescent dye ZMB793 could be used for the SP cell analysis.

Monotypic gland-cell regions on the body surface of two species of Arion: ultrastructure and lectin-binding properties

Distinct areas of the body surface of Arion (Arion) rufus (Linnaeus, 1758) and A. (A.) vulgaris Moquin-Tandon, 1855 contain only one type of glandular cell. The specific monotypic glandular cells that are characteristic for each of these areas differ with respect to staining properties, the structure of glycans (binding of different lectins) and in their ultrastructure, particularly in the appearance of the endoplasmic reticulum. Four such glandular areas can be defined. (1) The protein gland of the anterior margin of the foot probably produces the proteins responsible for changing the trail mucus produced from other glands to strong glue-mucus. Histochemical reactions point to the presence of neutral mucosubstances, probably mucoproteins. The strongest binding of applied lectins were those specific for N-acetyl-α-d-galactosaminyl residues, specific for β-linked terminal galactose, and specific for galactose β1-3N-acetylgalactosamine. A characteristic feature of the ultrastructure of the gland cells is tightly arranged membranes of the endoplasmic reticulum as typically found in protein secreting cells. (2) The suprapedal gland consists of a flat anterior part (located in the bottom of a funnel-shaped cavity between the ventral surface of the head and the opposite dorsal surface of the anterior foot) and a posterior tubular part on the dorsal side of the foot musculature (opening by an excretory duct into the funnel-shaped cavity). Histochemical reactions indicate the presence of acid glycosaminoglycans of high negative charge density. Binding of lectins with all secretion stages indicate the presence of the terminal sequence Galα-1-3Galβ1-R. As a special feature of the ultrastructure of the gland cells, their endoplasmic reticulum contains densely packed tubules protruding at right angle from its membrane into its lumen. (3) On the ventral surface of the head, a monotypic glandular region extends into the roof of the funnel-shaped recess. Histochemical reactions indicate the presence of neutral mucosubstances. The strongest bindings of the applied lectins with the secretion were specific for N-acetyl-α-d-galactosaminyl residues and for β-linked terminal galactose. (4) Another monotypic glandular region is situated in the ventral surface of the anterior part of the mantle. Histochemical reactions indicate acid mucosubstances with high negative charge density. None of the applied lectins reacted with cells of that region. Each of the first three glandular regions has an extension into the peripodial groove on either side of the foot sole; the ‘peripodial gland’ of authors is therefore a compound structure. The branches of the protein gland extend as far back as the level of the pneumostome, while the branches of the suprapedal gland and of the glandular region of the ventral surface of the head extend further, to the corners of the base of the triangular caudal gland. The trail mucus is produced mainly by the suprapedal gland that extends with its branches into the peripodial groove. Monotypic gland cell regions are possible candidates as sources of mucus of known cellular origin for biochemical analyses.

Perspectives on the history of research on starch Part IV: On the visualization of granule architecture

AbstractStarch has been used over several millennia for a number of different applications. However, research on understanding this substance only spans about three centuries starting with Leeuwenhoek who observed it in 1716. This story of discovery of the molecular structure and architectural makeup of starch is chronicled in a series of six essays of which this is the fourth with a focus on visualizing the granule, its development and architecture. Since the starch granule was visualized initially, researchers focused on the content and composition of the granule and also tried to understand what they saw by way of layers, polarization, and staining properties using a microscope. Two schools of thought prevailed in the 1800s with regards to granule development. Confusion prevailed with respect to terminologies and composition through this period. The first model of the crystallized granule was proposed in the late 19th century and it was not until mid‐20th century that the blocklet and crystalline arrangement of starch was understood. However, the crystallinity and helical complex between AM and iodine continued to obscure the source of starch crystallinity during the second half of the 20th century. magnified imagePlease read the Editorial for more details.

Digital analysis of staining properties of clear aesthetic brackets

To analyse the staining properties of clear orthodontic brackets using a digital analysis. In vitro, laboratory study A total of 500 tooth-coloured brackets from 10 brands (five ceramic and five plastic) were investigated. The cumulative discolouring effect of staining agents (tea, coffee, curry and red wine) were analysed at two consumption levels: light and heavy, based on a 6-month period of exposure. Study group brackets were immersed in the agents consecutively at 37°C. The control group was only exposed to artificial saliva. Samples were analysed digitally to obtain L*, a* and b* (lightness, red-green and yellow-blue) colour readings. Using these values, total colour change (ΔE*) at each level was also calculated. Three-way analysis of variance (ANOVA) test was used for statistical comparisons. L* and b* colour parameters showed significant differences (P<0·001) between different bracket groups, consumption levels and the type of exposure. However, the a* value only differed between bracket groups (P<0·001). According to the ΔE* values, ceramic brackets had less colour change than plastic brackets at the end of phase 1 for both the study and control groups. However, as consumption time increased, the rate of colour change decreased for the plastic brackets. In general, ceramic brackets demonstrated much more resistance to staining agents than plastic brackets. Both plastic and ceramic brackets showed changes in colour when exposed to heavy consumption of staining agents, with plastic brackets being the most affected.

Kola nut (Cola acuminata) extract as a substitute to histological tissue stain eosin.

The application of natural dyes for staining of various biological tissues from an alternative source will decrease the expense for purchasing the synthetic dye and reduce their effects on human and environment. Therefore, the objective of this study was to investigate the extraction of natural dye from Cola nut ( Cola acuminata ) using various solvents and its staining property on the rat tissues. The cola nut was pulvized using pestle and mortar, 5gram was used to make 5% of aqueous extract. The rat tissues were processed for paraffin embedding technique and sectioned at 5 μm thicknesses. The sections were stained with haematoxylin and the extracts as secondary stain. The results showed that the natural extract from Cola acuminata stained the cytoplasm of various tissues with yellowish-brown colouration. This finding suggests that Cola acuminata can be used as an alternative dye for histological staining.