Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA.

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

Morphobiological aspects of Rhodnius brethesi Matta, 1919 (Hemiptera:Reduviidae) from the Upper and Middle Negro River, Amazon region of Brazil: I - scanning electron microscopy

The occurrence of autochthonous cases of Chagas disease in the Amazon region of Brazil over recent decades has motivated an intensification of studies in this area. Different species of triatomines have been identified, and ten of these have be proven to be carriers of the parasite Trypanosoma cruzi or " cruzi-like " parasites. Studies conducted in the municipalities of Santa Isabel do Rio Negro and Barcelos, located on the Upper and Middle of the Negro River, microregion of Negro River, state of Amazonas have confirmed not only that Rhodnius brethesi is present in the palm tree Leopoldinia piassaba, but also that this insect was recognized by palm fiber collectors. A morphological study of eyes, inter-ocular and inter-ocellar regions, antennae, buccula, labrum, rostrum, stridulatory sulcus and feet, including the apex of the tibia, spongy fossette and ctenidium was conducted by scanning electron microscopy. The buccula and the stridulatory sulcus presented notable differences in specimens of different genera and also of different species. These data make it possible to suggest that the details presented in these structures can be included as diagnostic characteristics to be used in new dichotomous keys, thereby contributing towards studies of taxonomy and systematics and furnishing backing for comparative analysis of specimens collected from different localities.

Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Immunocytochemical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. II. Electron microscopic study.

Following a former immunohistochemical study in the rat brain [Arluison, M., Quignon, M., Nguyen, P., Thorens, B., Leloup, C., Penicaud, L. Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. I. Immunohistochemical study. J. Chem. Neuroanat., in press], we have analyzed the ultrastructural localization of GLUT2 in representative and/or critical areas of the forebrain and hindbrain. In agreement with previous results, we observe few oligodendrocyte and astrocyte cell bodies discretely labeled for GLUT2 in large myelinated fibre bundles and most brain areas examined, whereas the reactive glial processes are more numerous and often localized in the vicinity of nerve terminals and/or dendrites or dendritic spines forming synaptic contacts. Only some of them appear closely bound to unlabeled nerve cell bodies and dendrites. Furthermore, the nerve cell bodies prominently immunostained for GLUT2 are scarce in the brain nuclei examined, whereas the labeled dendrites and dendritic spines are relatively numerous and frequently engaged in synaptic junctions. In conformity with the observation of GLUT2-immunoreactive rings at the periphery of numerous nerve cell bodies in various brain areas (see previous paper), we report here that some neuronal perikarya of the dorsal endopiriform nucleus/perirhinal cortex exhibit some patches of immunostaining just below the plasma membrane. However, the presence of many GLUT2-immunoreactive nerve terminals and/or astrocyte processes, some of them being occasionally attached to nerve cell bodies and dendrites, could also explain the pericellular labeling observed. The results here reported support the idea that GLUT2 may be expressed by some cerebral neurones possibly involved in glucose sensing, as previously discussed. However, it is also possible that this transporter participate in the regulation of neurotransmitter release and, perhaps, in the release of glucose by glial cells.

Ascending aorta measurements as assessed by ECG-gated multi-detector computed tomography : a pilot study to etablish normative values for transcatheters therapies

Rapport de synthèse : Mesures de l'aorte ascendante par scanner synchronisé au rythme cardiaque: une étude pilote pour établir des valeurs normatives dans le cadre des futures thérapies par transcathéter. Objectif : L'objectif de cette étude est d'établir les valeurs morphométriques normatives de l'aorte ascendante à l'aide de l'angiographie par scanner synchronisé au rythme cardiaque, afin d'aider au développement des futurs traitements par transcathéter. Matériels et méthodes : Chez soixante-dix-sept patients (âgé de 22 à 83 ans, âge moyen: 54,7 ans), une angiographie par scanner synchronisé au rythme cardiaque a été réalisée pour évaluation des vaisseaux coronaires. Les examens ont été revus afin d'étudier l'anatomie de la chambre de chasse du ventricule gauche jusqu'au tronc brachio-céphalique droit. A l'aide de programmes de reconstructions multiplanaires et de segmentation automatique, différents diamètres et longueurs considérés comme importants pour les futurs traitements par transcathéter ont été mesurés. Les valeurs sont exprimées en moyennes, médianes, maximums, minimums, écart-types et en coefficients de variation. Les variations de diamètre de l'aorte ascendante durant le cycle cardiaque ont été aussi considérées. Résultats : Le diamètre moyen de la chambre de chasse du ventricule gauche était de 20.3+/-3.4 mm. Au niveau du sinus coronaire de l'aorte, il était de 34.2+/-4.1 mm et au niveau de la jonction sinotubulaire il était de 29.7+/-3.4 mm. Le diamètre moyen de l'aorte ascendante était de 32.7+/-3.8 mm. Le coefficient de variation de ces mesures variait de 12 à 17%. La distance moyenne entre l'insertion proximale des valvules aortiques et le départ du tronc brachio-céphalique droit était de 92.6+/-11.8 mm. La distance moyenne entre l'insertion proximale des valvules aortiques et l'origine de l'artère coronaire proximale était de 12.1+/-3.7 mm avec un coefficient de variation de 31%. La distance moyenne entre les deux ostia coronaires était de 7.2+/-3.1 mm avec un coefficient de variation de 43%. La longueur moyenne du petit arc de l'aorte ascendante entre l'artère coronaire gauche et le tronc brachio-céphalique droit était de 52.9+/-9.5 mm. La longueur moyenne de la continuité fibreuse entre la valve aortique et la valvule mitrale antérieure était de 14.6+/-3.3 mm avec un coefficient de variation de 23%. L'aire moyenne de la valve aortique était de 582.0+/-131.9 mm2. La variation du diamètre antéro-postérieur et transverse de l'aorte ascendante était respectivement de 8.4% et de 7.3%. Conclusion Il existe d'importantes variations inter-individuelles dans les mesures de l'aorte ascendante avec cependant des variations intra-individuelles faibles durant le cycle cardiaque. De ce fait, une approche personnalisée pour chaque patient est recommandée dans la confection des futures endoprothèses de l'aorte ascendante. Le scanner synchronisé au rythme cardiaque jouera un rôle prépondérant dans le bilan préthérapeutique. Abstract : The aim of this study was to provide an insight into normative values of the ascending aorta in regards to novel endovascular procedures using ECG-gated multi-detector CT angiography. Seventy-seven adult patients without ascending aortic abnormalities were evaluated. Measurements at relevant levels of the aortic root and ascending aorta were obtained. Diameter variations of the ascending aorta during cardiac cycle were also considered. Mean diameters (mm) were as follows: LV outflow tract 20.3+/-3.4, coronary sinus 34.2+/-4.1, sinotubular junction 29.7+-3.4 and mid ascending aorta 32.7+/-3.8 with coefficients of variation (CV) ranging from 12 to 17%. Mean distances (mm) were: from the plane passing through the proximal insertions of the aortic valve cusps to the right brachio-cephalic artery (BCA) 92.6111.8, from the plane passing through the proximal insertions of the aortic valve cusps to the proximal coronary ostium 12.1+/-3.7, and between both coronary ostia 7.2+/-3.1, minimal arc of the ascending aorta from left coronary ostium to right BCA 52.9 X9.5, and the fibrous continuity between the aortic valve and the anterior leaflet of the mitral valve 14.óf3.3, CV 13-43%. Mean aortic valve area was 582+-131.9 mm2. The variations of the antero-posterior and transverse diameters of the ascending aorta during the cardiac cycle were 8.4% and 7.3%, respectively. Results showed large inter-individual variations in diameters and distances but with limited intra-individual variations during the cardiac cycle. A personalized approach for planning endovascular devices must be considered.

A study of the scutellum in eight Chagas disease vector species from genus Triatoma (Hemiptera, Reduviidae) using optical and scanning electron microscopy

The aim of this study was to analyze the external morphology of the scutellum through optical microscopy and scanning electron microscopy (SEM) in male specimens of Triatoma costalimai, T. delpontei, T. eratyrusiformis, T. matogrossensis, T. infestans melanosoma, T. sherlocki, T. tibiamaculata, and T. vandae. A total of 30 photographs of the scutellum were made. Magnification varied from 50X to 750X. Regarding depth and forms of the central depression, the heart-shaped form was predominant, with some exceptions, so that this shape appears to be a common characteristic for species of genus Triatoma Laporte, 1832. In T. eratyrusiformis, a kind of sensillum with important taxonomic value was observed. The different sizes and shapes of the designs found on the posterior process of the scutellum were also of important taxonomic interest. The study of the scutellum based on SEM showed valuable characteristics, allowing the use of this structure to aid the diagnosis of triatomine species. Thus, more specimens in subsequent studies and analyses of morphometric parameters should contribute to agreement on phylogenetic aspects in this genus. A Key to eight species of Triatoma based on male scutellar morphology is presented.

Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy.

Cryo-electron microscopy of vitreous sections (CEMOVIS) has recently been shown to provide images of biological specimens with unprecedented quality and resolution. Cutting the sections remains however the major difficulty. Here, we examine the parameters influencing the quality of the sections and analyse the resulting artefacts. They are in particular: knife marks, compression, crevasses, and chatter. We propose a model taking into account the interplay between viscous flow and fracture. We confirm that crevasses are formed on only one side of the section, and define conditions by which they can be avoided. Chatter is an effect of irregular compression due to friction of the section of the knife edge and conditions to prevent this are also explored. In absence of crevasses and chatter, the bulk of the section is compressed approximately homogeneously. Within this approximation, it is possible to correct for compression by a simple linear transformation for the bulk of the section. A research program is proposed to test and refine our understanding of the sectioning process.

Electron microscopy of trypanosomes: a historical view

Since the discovery of the electron microscope and the development of the initial techniques for the processing of biological samples for electron microscopy, the protozoan Trypanosoma cruzi has been the subject of intense investigation. This review analyzes the results obtained by observation of whole trypanosomes as well as thin sections and replicas using several microscopic approaches. Micrographs detailing the appearance of T. cruzi using several methods illustrate the evolution of electron microscopic techniques as well as its contribution to understanding the structural organization of the protozoan.

Comparison of multislice computerized tomography angiography and digital subtraction angiography in the postoperative evaluation of patients with clipped aneurysms.

OBJECT: In this study the accuracy of multislice computerized tomography (MSCT) angiography in the postoperative examination of clip-occluded intracranial aneurysms was compared with that of intraarterial digital subtraction (DS) angiography METHODS: Forty-nine consecutive patients with 60 clipped aneurysms (41 of which had ruptured) were studied with the aid of postoperative MSCT and DS angiography. Both types of radiological studies were reviewed independently by two observers to assess the quality of the images, the artifacts left by the clips, the completeness of aneurysm occlusion, the patency of the parent vessel, and the duration and cost of the examination. The quality of MSCT angiography was good in 42 patients (86%). Poor-quality MSCT angiograms (14%) were a result of the late acquisition of images in three patients and the presence of clip or motion artifacts in four. Occlusion of the aneurysm on good-quality MSCT angiograms was confirmed in all but two patients in whom a small (2-mm) remnant was confirmed on DS angiograms. In one patient, occlusion of a parent vessel was seen on DS angiograms but missed on MSCT angiograms. The sensitivity and specificity for detecting neck remnants on MSCT angiography were both 100%, and the sensitivity and specificity for evaluating vessel patency were 80 and 100%, respectively (95% confidence interval 29.2-100%). Interobserver agreements were 0.765 and 0.86, respectively. The mean duration of the examination was 13 minutes for MSCT angiography and 75 minutes for DS angiography (p < 0.05). Multislice CT angiography was highly cost effective (p < 0.01). CONCLUSIONS: Current-generation MSCT angiography is an accurate noninvasive tool used for assessment of clipped aneurysms in the anterior circulation. Its high sensitivity and low cost warrant its use for postoperative routine control examinations following clip placement on an aneurysm. Digital subtraction angiography must be performed if the interpretation of MSCT angiograms is doubtful or if the aneurysm is located in the posterior circulation.

Morphological study of the eggs and nymphs of Triatoma dimidiata (Latreille, 1811) observed by light and scanning electron microscopy (Hemiptera: Reduviidae: Triatominae)

Eggs and nymphs of Triatoma dimidiata were described using both light and scanning electron microscopy. The egg body and operculum have an exochorion formed by irregular juxtaposed polygonal cells; these cells are without sculpture and the majority of them are hexagonal in shape. The five instars of T. dimidiatacan be distinguished from each other by characteristics of the pre, meso and metanotum. The number of setiferous tubercles increases progressively among instars. The sulcus stridulatorium of 1st instar nymphs is amorphous, showing median parallel grooves; from the 2nd instar on the sulcus is, progressively, elongate, deep and posteriorly pointed with stretched parallel grooves. All instars have a trichobothrium on the apical 1/3 of segment II of the antenna. The opening of the Brindley's gland is on the mesopleura. Fifth instar nymphs have an apical ctenidium on the ventral surface of the fore tibia. Dorsal glabrous patches are found on the lateral 1/3 of abdomen. Bright oval patches are found on the ventral median line of the abdomen, from segment IV-VI; 1st instar nymphs lack these patches. Abdominal dorsal plates are present from the 1st-5th instars; the 1st instar also contains a rectangular plate in segment IX. From the 2nd instar on, variably-shaped plates are present on segments VII to IX. Morphometric data were also obtained and proved to be useful for distinguishing T. dimidiata instars.

Evaluation of monoenergetic imaging to reduce metallic instrumentation artifacts in computed tomography of the cervical spine.

OBJECT Monoenergetic imaging with dual-energy CT has been proposed to reduce metallic artifacts in comparison with conventional polychromatic CT. The purpose of this study is to systematically evaluate and define the optimal dual-energy CT imaging parameters for specific cervical spinal implant alloy compositions. METHODS Spinal fixation rods of cobalt-chromium or titanium alloy inserted into the cervical spine section of an Alderson Rando anthropomorphic phantom were imaged ex vivo with fast-kilovoltage switching CT at 80 and 140 peak kV. The collimation width and field of view were varied between 20 and 40 mm and medium to large, respectively. Extrapolated monoenergetic images were generated at 70, 90, 110, and 130 kiloelectron volts (keV). The standard deviation of voxel intensities along a circular line profile around the spine was used as an index of the magnitude of metallic artifact. RESULTS The metallic artifact was more conspicuous around the fixation rods made of cobalt-chromium than those of titanium alloy. The magnitude of metallic artifact seen with titanium fixation rods was minimized at monoenergies of 90 keV and higher, using a collimation width of 20 mm and large field of view. The magnitude of metallic artifact with cobalt-chromium fixation rods was minimized at monoenergies of 110 keV and higher; collimation width or field of view had no effect. CONCLUSIONS Optimization of acquisition settings used with monoenergetic CT studies might yield reduced metallic artifacts.

Helical tomotherapy (HT) for the treatment of anal canal cancer: preliminary clinical results, and dosimetric comparison between HT and intensity-modulated or 3D conformal radiotherapy

Background: To report a single-center experience in 19 patients (pts) with anal canal cancer treated with helical tomotherapy (HT) and concurrent chemotherapy, and compare the dosimetric results with fixed-field intensitymodulated radiotherapy (IMRT) and 3D conformal radiotherapy (3D RT). Materials and Methods: Between 2007 and 2008, 19 consecutive pts were treated with HT and concurrent CT for anal canal cancer. Median age was 59 years (range, 38−83), and female/male ratio was 14/5. The majority of the pts had T2 or T3 tumours (68.4%), and 52.6% had positive lymph nodes. In all 19 pts, pelvic and inguinal nodes, and tumour irradiation was given using HT upto a median dose of 36 Gy (1.8 Gy/fr) followed by a 1-week gap. A boost dose of 23.4 Gy (1.8 Gy/fr) was delivered to the tumour and involved nodes using 3DRT (n = 12), HT (n = 6), or IMRT (n = 1). Simultaneous integrated boost was used in none of the pts. All but one patient with a T1N0 tumour received concomitant mitomycin/5- fluorouracil (n = 12) or mitomycin/capecitabin (n = 7) CT. Toxicity was scored according to the Common Terminology Criteria for Adverse Events (NCICTCAE v3.0). HT plans and treatments were generated using Tomotherapy, Inc., software and hardware; and 3D or IMRT boost plans with the CMS treatment planning system (TPS), using 6−18 MV photons from a Siemens Primus accelerator. For dosimetric comparison, computed tomography data sets of 10 pts were imported into the TPS, and 3D and 5-field step-andshoot IMRT plans were generated for each case. Plans were optimized with the aim of assessing organs at risk (OAR) and healthy-tissue sparing while enforcing highly conformal target coverage, and evaluated by dose-volume histograms (DVH) of planning target volumes (PTV) and OAR. Results: With a median follow-up of 13 months (range, 3−18), all pts are alive and well; except one patient developing local recurrence at 12 months. No patient developed grade 3 or more acute toxicity. No unplanned treatment interruption was necessary because of toxicity. With 360-degree-of-freedom beam projection, HT showed an advantage over 3D or IMRT plans in terms of dose conformity around the PTV, and dose gradients were steeper outside the PTV, resulting in reduced doses to OARs. Using HT, acute toxicity was acceptable, and seemed to be better than historical standards. Conclusion: We conclude that HT combined with concurrent chemotherapy for anal canal cancer is effective and tolerable. Compared to 3DRT or 5-field IMRT, there is better conformity around the PTV, and OAR sparing.

Stiffness tomography exploration of living and fixed macrophages.

Stiffness tomography is a new atomic force microscopy imaging technique that allows highlighting structures located underneath the surface of the sample. In this imaging mode, such structures are identified by investigating their mechanical properties. We present here, for the first time, a description of the use of this technique to acquire detailed stiffness maps of fixed and living macrophages. Indeed, the mechanical properties of several macrophages were studied through stiffness tomography imaging, allowing some insight of the structures lying below the cell's surface. Through these investigations, we were able to evidence the presence and properties of stiff column-like features located underneath the cell membrane. To our knowledge, this is the first evidence of the presence, underneath the cell membrane, of such stiff features, which are in dimension and form compatible with phagosomes. Moreover, by exposing the cells to cytochalasin, we were able to study the induced modifications, obtaining an indication of the location and mechanical properties of the actin cytoskeleton. Copyright © 2012 John Wiley & Sons, Ltd.

Light and electron microscopy of Myxobolus sciades n. sp. (Myxozoa), a parasite of the gills of the Brazilian fish Sciades herzbergii (Block, 1794) (Teleostei: Ariidae)

A myxosporean parasite in the gill lamellae of the freshwater teleost fish, Sciades herzbergii (Ariidae) (Block, 1794), from the Poti River (Northeast of Brazil) was described by light and electron microscopy studies. Polysporic histozoic cyst-like plasmodia containing several life-cycle stages, including mature spores, were observed. The spores were pyriform and uninucleate, measuring 9.15 ± 0.39 μm (n = 50) long, 4.36 ± 0.23 μm (n = 25) wide and 2.61 ± 0.31 μm (n = 25) thick. Elongated pyriform polar capsules (PC) were of equal size (4.44 ± 0.41 μm long and 1.41 ± 0.42 μm in diameter) and each contained a polar filament with 9-10 coils obliquely arranged in relation to the axis of PC. The PC wall was composed of two layers of different electron densities. Histological analysis revealed the close contact of the cyst-like plasmodia with the basal portion of the epithelial gill layer, which exhibited some alterations in the capillary vessels. Based on the morphological and ultrastructural differences, the similarity of the spore features to those of the genus Myxobolus and the specificity of this host to previously described species, we describe a new species named Myxobolus sciades n. sp. in this study.