Structural differences between "dark" and "bright" isolated human osteonic lamellae

This investigation presents new insights into the structure of human secondary lamellae. Lamellar specimens that appear dark and bright on alternate osteon transverse sections under circularly polarizing light were isolated using a new technique, and examined by polarizing light microscopy, synchrotron X-ray diffraction, and confocal microscopy. A distribution of unidirectional collagen bundles and of two overlapping oblique bundles appears on circularly polarizing light microscopy images in relation to the angle between the specimen and the crossed Nicols' planes. The unidirectional collagen bundles observed at 45 degrees run parallel to the osteon axis in the dark lamellar specimens and perpendicular to it in the bright ones. Small and wide-angle micro-focus X-ray diffraction indicates that the dark lamellae are structurally quite homogeneous, with collagen fibers and apatite crystals preferentially oriented parallel to the osteon axis. Bright lamellar specimens exhibit different orientation patterns with the dominant ones bidirectional at +/-45 degrees with respect to the osteon axis. Accordingly, confocal microscopy evidences the presence of longitudinal bundles in dark lamellar specimens and oblique bundles in the bright ones. Radial bundles are evidenced in both lamellar types. The alternate osteon structure is described by a rather continuous multidirectional pattern, in which dark and bright lamellae display different mechanical and possibly biological functions.     

A new convenient technique for making cell blocks

Cell block sections serve as an important diagnostic annex for cytological smears, liquid-based SurePath cytology and the Liquid-based Thin-prep Cytology Test (TCT). A variety of methods for the preparation of cell blocks are described in the literature and the techniques in cell blocks are in continuous improvement. A new technique for making cell blocks was introduced in the present study. We first used pregelatinized starch as the frame for the cell block, which is a really simple and economic method, because it can be carried out at room temperature without additional special instruments. We have performed hematoxylin and eosin (HE) staining, immunohistochemistry analysis and fluorescence in situ hybridization (FISH) in the cell block sections in 122 cytological specimens. The results demonstrated in this article show that pregelatinized starch is a useful frame for cell blocks. The pregelatinized starch can effectively collect even a few cells with powerful adhesiveness. Therefore, this new technique for making cell blocks is especially useful for cytologic samples with low cellularity, such as cerebrospinal fluid specimens.     

Biochemical Inhibition of Positional Signalling in the Avian Limb Bud

A region at the posterior margin of the developing avian limb bud, the zone of polarizing activity, appears to be responsible for signalling positional information along the limb antero-posterior axis. The mechanism of signalling is unknown and, unfortunately, no subcellular preparation from the polarizing region has shown polarizing activity in vivo. We have performed a series of experiments in which isolated polarizing regions were. treated with chemical agents or inhibitors prior to being grafted into anterior sites on host limb buds. A previous paper described the effects of some metabolic and biosynthetic inhibitors [5]. This paper describes the results of treatments with agents that primarily affect cell or cell surface integrity, or intracellular small molecules such as those involved in sulphydryl or cation balance. Chick and quail polarizing regions are compared, and a disaggregated cell assay is used to analyze inhibition. Drugs affecting cytostructure (colchicine, vinblastine, and cytochalasin B) inhibited the activity of polarizing regions, but did not affect the activity of treated cell suspensions, thus their action seemed dependent on retention of the agent by tissue. Inhibition observed with metabolic and biosynthetic inhibitors did not appear to involve drug retention. Agents interfering with cell surface integrity (meta-periodate, endoglycosidases, and concanavalin A) did not greatly interfere with polarizing activity at concentrations where they effectively eliminated cell spreading. Aldehyde fixatives or Triton X-100 abolished polarizing activity. Ouabain had little effect on positional signalling, but valinomycin abolished activity.     

Supramolecular ordering of DNA in the cholesteric liquid crystalline phase: an ultrastructural study.

Aqueous solutions of 146-base pair DNA fragments form a cholesteric liquid crystalline phase in the range of about 160-290 mg/ml. We present a structural analysis of this phase by comparing the data obtained from polarizing and electron microscopy. This phase shows multiple aspects or "textures" which are presented and interpreted. They mainly depend on the orientation of the structure relative to the observation plane and on the nature, distribution, and amount of defects present in the phase. These defects are then analyzed with the two methods, and the molecular orientations can be defined precisely in their core. The biological interest of such structural analyses is discussed in relation with the understanding of chromatin structure and function.     

Detection of Para-Aminosalicylic Acid (PAS) in Urine: A Simple Spot Test

A simple spot test for the detection of PAS in urine has been described and its sensitivity compared with that of other methods such as the Ehrlich''s reagent and the ferric chloride tests. In patients receiving 4 g. PAS the three methods gave similar results in urine specimens collected within eight hours. The new test is an inexpensive micro method which can easily be performed on a large scale. There is no reaction with sulfonamide or salicylic acid derivatives.     

Preparatory methods for DNA hydrolysis, cytochemistry, immunocytochemistry and ploidy analysis. Their application to automated and routine diagnostic cytopathology

A review is presented of some methods used to prepare cytologic specimens for analytical and/or automated studies, with the steps of the procedures detailed in appendices. The preparation of the cell monolayers required for optimal automated cell image analysis and classification, e.g., by the Cytoscan 110, is discussed, as is the preparation of poly-L-lysine-coated slides used in the production of monolayered specimens. These monolayers, which can be prepared from a variety of specimens, are also useful for cytochemical and immunocytochemical studies and DNA ploidy analysis. For DNA analysis, a modified gallocyanin chrome alum staining procedure is described as a stoichiometric alternative to the time-consuming Feulgen reaction. The hydrolysis technique required by the latter method is also detailed. The freeze-fracturing technique for the enhancement of monoclonal antibody immunocytochemical staining of detectable antigens is described, along with an indirect immunoalkaline phosphatase staining method. The use of enzyme cytochemical reactions for glucose 6 phosphate dehydrogenase and lysosomal naphthylamidase is also presented.     

Cholesteric organization of DNA in the stallion sperm head

The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chromatin structure of these spermatozoa is that of a cholesteric liquid crystal. This structure resembles that of a plywood, made of superimposed layers of parallel filaments, but instead of having a right angle between two successive layers, there is a progressive rotation and similar orientation occurs at each 180 degrees rotation. The apparent lamellae result from cleavages due to freeze-fracture between levels of parallel filament orientation. The thickness of lamellae corresponds therefore to the half helicoidal pitch of the cholesteric liquid crystal. This model is consistent with our observations by polarizing microscopy. The lamellation is not visible in thin sections of stallion spermatozoa. There are however biochemical methods to decondense chromatin and we are able to observe this lamellation in sections normal to the flattening plane of sperm heads. The methods used classically to decondense the sperm chromatin lead to extremely varied aspects which are discussed, some of them being closely related to the structure of cholesteric liquid crystals.     

Oligonucleotide indexing of DNA barcodes: identification of tuna and other scombrid species in food products

Background  

DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species.     

Histological and histochemical examination of American Indian scalps, mummies, and a shrunken head

The histological and histochemical detail remaining in Jivaro shrunken heads, North American Indian scalps, and mummified remains from Peru, Chile, and the American southwest, is remarkable when these specimens are rehydrated and fixed by Sandison's methods. Special techniques and stains used frequently in the histochemistry of the skin brought out details which were less evident or not apparent in the routine hematoxylin and eosin preparations. The polarizing microscope was particularly useful in demonstrating keratin and collagen fibers. Use of the silver stain for melanin not only aided in demonstrating hairs, but also outlined the pattern of epidermal detail by supranuclear accumulation and melanin dust in the stratum corneum. The hematoxylin-phloxine-alcian blue orange G stain for prekeratin, keratin, and mucin showed long persistence of the prekeratin and keratin staining properties. The stain identified strands as epidermis where they would otherwise have been overlooked. The Luxol Fast Blue stain, birefringence, and appearance with phase contrast microscopy are promising means for evaluating the degradation of skin collagen under conditions of dry preservation. The first loss of histological information occurs in extravascular cells of the dermis; mast cells, eosinophils, and other perivascular cells could not be identified in any of the specimens. Fine elastic fibers near the epidermis disappear before the larger, mid-dermal elastic fibers. Some of the disease processes which could be diagnosed in very old mummified skin are discussed in terms of the remaining structures.     

Vacuum injection--a new indirect fixing method for the demonstration of initial lymphatics in biopsy specimens of human organs

With the indirect vacuum injection method (IVIM) at a negative pressure of 30 kPa, it is possible to demonstrate the initial lymphatics (IL) by scanning electron microscopy even in biopsy specimens of various organs (minimum size 0.5 X 0.5 cm2). IL can also be dilated and fixed using the indirect perfusor injection method (IPIM), but only in larger specimens (minimum size 2.0 X 2.0 cm2). Only dilated IL can be examined by scanning electron microscopy, and only the IL of the ampulla of the Fallopian tube can be reliably dilated during immersion fixation. The quality of fixation on use of the vacuum technique conforms to the standard achieved with other methods. No one has yet succeeded in demonstrating the IL in biopsy specimens smaller than mentioned above using any of the methods described.     

The in vitro maintenance of the limb-bud apical ridge by cell-free preparations

A factor(s) that has properties similar to previously described limb-bud polarizing activity and ectodermal ridge maintenance activity can be detected in cell-free preparations of posterior, but not anterior, halves of 4-day chick embryo limb buds. The apparent size of the factor differs depending upon the method of isolation. Homogenization in isotonic saline results in a particulate active component, whereas homogenization in hypertonic saline results in a soluble active component that is nondialyzable. When culture medium is conditioned by incubating several pieces of polarizing tissue in it for 24 hr, a dialyzable, active component is found in the conditioned medium.     

Separation of two species standing as Helophorus aquaticus (L.) (Coleoptera, Hydrophilidae) by banded chromosome analysis

Abstract. Methods are described for making chromosome preparations from developing embryos of Helophorus , for producing C- and G-banding, and for staining the nucleolus organizer with silver. These methods are used to compare the karyotypes of two species currently included in H.aquaticus (L.). It is shown that these species differ because of reciprocal translocations between some chromosomes, and that they would therefore be unable to produce fertile hybrids. Morphological differences in the male and female genitalia are described, and the range of aedeagal variation shown by each species is established by reference to chromosome preparations from testis. Reference to the relevant type specimens shows that the two species are H.aequalis Thomson and H.aquaticus (L.). The latter is not a British species. Differences in the egg cocoons and third instar larvae are described. The present distributions and Pleistocene histories of the two species are described.     

Sampling methods for phlebotomine sandflies

A review is presented of methods for sampling phlebotomine sandflies (Diptera: Psychodidae). Among approximately 500 species of Phlebotominae so far described, mostly in the New World genus Lutzomyia and the Old World genus Phlebotomus, about 10% are known vectors of Leishmania parasites or other pathogens. Despite being small and fragile, sandflies have a wide geographical range with species occupying a considerable diversity of ecotopes and habitats, from deserts to humid forests, so that suitable methods for collecting them are influenced by environmental conditions where they are sought. Because immature phlebotomines occupy obscure terrestrial habitats, it is difficult to find their breeding sites. Therefore, most trapping methods and sampling procedures focus on sandfly adults, whether resting or active. The diurnal resting sites of adult sandflies include tree holes, buttress roots, rock crevices, houses, animal shelters and burrows, from which they may be aspirated directly or trapped after being disturbed. Sandflies can be collected during their periods of activity by interception traps, or by using attractants such as bait animals, CO2 or light. The method of trapping used should: (a) be suited to the habitat and area to be surveyed, (b) take into account the segment of the sandfly population to be sampled (species, sex and reproduction condition) and (c) yield specimens of appropriate condition for the study objectives (e.g. identification of species present, population genetics or vector implication). Methods for preservation and transportation of sandflies to the laboratory also depend on the objectives of a particular study and are described accordingly.     

Linear discrete models with different time scales

Aggregation of variables allows to approximate a large scale dynamical system (the micro-system) involving many variables into a reduced system (the macro-system) described by a few number of global variables. Approximate aggregation can be performed when different time scales are involved in the dynamics of the micro-system. Perturbation methods enable to approximate the large micro-system by a macro-system going on at a slow time scale. Aggregation has been performed for systems of ordinary differential equations in which time is a continuous variable. In this contribution, we extend aggregation methods to time-discrete models of population dynamics. Time discrete micro-models with two time scales are presented. We use perturbation methods to obtain a slow macro-model. The asymptotic behaviours of the micro and macro-systems are characterized by the main eigenvalues and the associated eigenvectors. We compare the asymptotic behaviours of both systems which are shown to be similar to a certain order.     

Estimating biomarker-based HIV incidence using prevalence data in high risk groups with missing outcomes

The novel two-step serologic sensitive/less sensitive testing algorithm for detecting recent HIV seroconversion (STARHS) provides a simple and practical method to estimate HIV-1 incidence using cross-sectional HIV seroprevalence data. STARHS has been used increasingly in epidemiologic studies. However, the uncertainty of incidence estimates using this algorithm has not been well described, especially for high risk groups or when missing data is present because a fraction of sensitive enzyme immunoassay (EIA) positive specimens are not tested by the less sensitive EIA. Ad hoc methods used in practice provide incorrect confidence limits and thus may jeopardize statistical inference. In this report, we propose maximum likelihood and Bayesian methods for correctly estimating the uncertainty in incidence estimates obtained using prevalence data with a fraction missing, and extend the methods to regression settings. Using a study of injection drug users participating in a drug detoxification program in New York city as an example, we demonstrated the impact of underestimating the uncertainty in incidence estimates using ad hoc methods. Our methods can be applied to estimate the incidence of other diseases from prevalence data using similar testing algorithms when missing data is present.     

Fine needle aspiration biopsy and immunostaining findings in an aggressive inflammatory myofibroblastic tumor of the lung: a case report

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) can vary from benign pseudosarcomatous tumors to low grade sarcomas. To date, fine needle aspiration (FNA) findings of lung IMTs, especially in the aggressive form, have not been fully described. Here we present FNA biopsy findings in conjunction with immunohistochemical studies in a case of primary and recurrent pulmonary IMT. CASE: A 22-year-old man first presented with a left lung mass and 4.5 years later with a recurrent mass. Preoperative computed tomography-guided FNA was performed on both tumors. FNA cytologic smears of both specimens consisted of scant, distorted spindle cells suggestive of a spindle cell lesion but were insufficient for further classification. Needle core biopsies as well as touch imprints were performed during the FNA procedures. The imprints revealed abundant, well-preserved spindle cells with mild to moderate atypia and intermixed lymphocytes and plasma cells. The spindle cells in both specimens were immunoreactive for vimentin and smooth muscle actin and were negative for pancytokeratin, desmin, CD34 and c-kit. Thirty percent of the tumor cells were positive for p53. The findings were compatible with those of IMT. Histologic examination of the surgically resected initial and recurrent masses confirmed the diagnosis of lMT. CONCLUSION: The cytologic findings of pulmonary IMT in FNA specimens are suggestive of, although not specific for, IMT. Immunohistochemical studies can assist in the diagnosis by excluding other spindle cell lesions. Cytologic atypia and p53 immunoreactivity may be indicators of aggressive IMTs.     

Estimation of food consumption from field observations of fish feeding cycles

The available methods of estimating food consumption by fish require that experiments be performed on confined animals and that experimental results may be validly applied to free fish. A method is described by which food consumption in periodically feeding fish may be estimated without performing laboratory experiments. A relatively simple input–output model of stomach contents is fitted to the observed time trajectory of stomach fullness, and food consumption is calculated from the estimated model parameters. Feeding is considered to be restricted to a distinct feeding period, and the rate of feeding during that period can be either constant or linearly decreased with the quantity of food already present in the stomach.
The method is applied to three examples. The model appears robust, and generally provides very similar food consumption estimates to those obtained from methods requiring an independent estimate of gastric evacuation rate. However, the described method is sensitive to violation of the assumption that feeding occurs only during a discrete feeding period.     

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.     

Preparation of cells from paraffin-embedded tissue for cytometry and cytomorphologic evaluation

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells.