Color Doppler sonography with contrast in the differentiation of ovarian tumors

The objective of this study was to differentiate benign ovarian tumors from malignant ones before surgery using color and pulsed Doppler sonography, and to compare results obtained before and after use of contrast medium, thereby verifying whether contrast results in an improvement in the diagnostic sensitivity. METHODS: Sixty two women (mean age 49.9 years) with ovarian tumors were studied, 45 with benign and 17 with malignant tumors. All women underwent a transvaginal color Doppler ultrasonographic exam. A study of the arterial vascular flow was made in all tumor areas, as well as an impedance evaluation of arterial vascular flow using the resistance index. RESULT: Localization of the vessels in the tumor revealed a greater proportion of malignant tumors with detectable internal vascular flows (64%) than benign tumors with such flows (22%). There was a considerable overlap of these findings. The use of contrast identified a greater number of vessels with confirmation in the totality of tumors, but did not improve the Doppler capacity in tumoral differentiation. Malignant tumors presented lower values of resistance index than the benign ones, whether or not contrast was used. The cutoff value for resistance index that better maximized the Doppler sensitivity and specificity was 0.55. Through this value, an increase of the sensitivity after contrast use was obtained, varying from 47% to 82%, while specificity remained statistically unchanged. CONCLUSION: Although the injection of a microbubble agent improved the sensitivity of the method detecting vascularization of tumors, a positive finding for vascularization by this method was not clinically useful in the differentiation of benign and malignant ovarian tumors.

Characterization of microbial populations in a wastewater treatment plant focusing on Staphylococcus aureus and Pseudomonas aeruginosa

Dissertação de mestrado em Bioengenharia

Intraseasonal variation in immune defence, body mass and hematocrit in adult house martins Delichon urbica

The intensity of parasite infections often increases during the reproductive season of the host as a result of parasite reproduction, increased parasite transmission and increased host susceptibility. We report within-individual variation in immune parameters, hematocrit and body mass in adult house martins Delichon urbica rearing nestlings in nests experimentally infested with house martin bugs Oeciacus hirundinis and birds rearing nestlings in initially parasite-free nests. From first to second broods body mass and hematocrit of breeding adult house martins decreased. In contrast leucocytes and immunoglobulins became more abundant. When their nests were infested with ectoparasites adults lost more weight compared with birds raising nestlings in nests treated with pyrethrin, whereas the decrease in hematocrit was more pronounced during infection with blood parasites. Neither experimental infestation with house martin bugs nor blood parasites had a significant effect on the amount of immune defences.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell

Selective disruption of Tcf7l2 in the pancreatic β cell impairs secretory function and lowers β cell mass.

Type 2 diabetes (T2D) is characterized by β cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired β cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the β cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in β cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual β cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in β cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of β cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Depression with atypical features and increase in obesity, body mass index, waist circumference, and fat mass: a prospective, population-based study.

IMPORTANCE: Depression and obesity are 2 prevalent disorders that have been repeatedly shown to be associated. However, the mechanisms and temporal sequence underlying this association are poorly understood. OBJECTIVE: To determine whether the subtypes of major depressive disorder (MDD; melancholic, atypical, combined, or unspecified) are predictive of adiposity in terms of the incidence of obesity and changes in body mass index (calculated as weight in kilograms divided by height in meters squared), waist circumference, and fat mass. DESIGN, SETTING, AND PARTICIPANTS: This prospective population-based cohort study, CoLaus (Cohorte Lausannoise)/PsyCoLaus (Psychiatric arm of the CoLaus Study), with 5.5 years of follow-up included 3054 randomly selected residents (mean age, 49.7 years; 53.1% were women) of the city of Lausanne, Switzerland (according to the civil register), aged 35 to 66 years in 2003, who accepted the physical and psychiatric baseline and physical follow-up evaluations. EXPOSURES: Depression subtypes according to the DSM-IV. Diagnostic criteria at baseline and follow-up, as well as sociodemographic characteristics, lifestyle (alcohol and tobacco use and physical activity), and medication, were elicited using the semistructured Diagnostic Interview for Genetic Studies. MAIN OUTCOMES AND MEASURES: Changes in body mass index, waist circumference, and fat mass during the follow-up period, in percentage of the baseline value, and the incidence of obesity during the follow-up period among nonobese participants at baseline. Weight, height, waist circumference, and body fat (bioimpedance) were measured at baseline and follow-up by trained field interviewers. RESULTS: Only participants with the atypical subtype of MDD at baseline revealed a higher increase in adiposity during follow-up than participants without MDD. The associations between this MDD subtype and body mass index (β = 3.19; 95% CI, 1.50-4.88), incidence of obesity (odds ratio, 3.75; 95% CI, 1.24-11.35), waist circumference in both sexes (β = 2.44; 95% CI, 0.21-4.66), and fat mass in men (β = 16.36; 95% CI, 4.81-27.92) remained significant after adjustments for a wide range of possible cofounding. CONCLUSIONS AND RELEVANCE: The atypical subtype of MDD is a strong predictor of obesity. This emphasizes the need to identify individuals with this subtype of MDD in both clinical and research settings. Therapeutic measures to diminish the consequences of increased appetite during depressive episodes with atypical features are advocated.

Specimen storage in transport medium and detection of group B streptococci by culture

Recovery of group B streptococci (GBS) was assessed in 1,204 vaginorectal swabs stored in Amies transport medium at 4 or 21°C for 1 to 4 days either by direct inoculation onto Granada agar (GA) or by culture in blood agar (BA) and GA after a selective broth enrichment (SBE) step. Following storage at 4°C, GBS detection in GA was not affected after 72 h by either direct inoculation or SBE; however, GBS were not detected after SBE in the BA subculture in some samples after 48 h of storage and in GA after 96 h. After storage at 21°C, loss of GBS-positive results was significant after 48 h by direct inoculation in GA and after 96 h by SBE and BA subculture; some GBS-positive samples were not detected after 24 h of storage followed by SBE and BA subculture or after 48 h of storage followed by SBE and GA subculture. Storage of swabs in transport medium, even at 4°C, produced after 24 h an underestimation of the intensity of GBS colonization in most specimens. These data indicate that viability of GBS is not fully preserved by storage of vaginorectal swabs in Amies transport medium, mainly if they are not stored under refrigeration

Compositional evolution with mass transfer in closed systems

Evolution of compositions in time, space, temperature or other covariates is frequentin practice. For instance, the radioactive decomposition of a sample changes its composition with time. Some of the involved isotopes decompose into other isotopes of thesample, thus producing a transfer of mass from some components to other ones, butpreserving the total mass present in the system. This evolution is traditionally modelledas a system of ordinary di erential equations of the mass of each component. However,this kind of evolution can be decomposed into a compositional change, expressed interms of simplicial derivatives, and a mass evolution (constant in this example). A rst result is that the simplicial system of di erential equations is non-linear, despiteof some subcompositions behaving linearly.The goal is to study the characteristics of such simplicial systems of di erential equa-tions such as linearity and stability. This is performed extracting the compositional differential equations from the mass equations. Then, simplicial derivatives are expressedin coordinates of the simplex, thus reducing the problem to the standard theory ofsystems of di erential equations, including stability. The characterisation of stabilityof these non-linear systems relays on the linearisation of the system of di erential equations at the stationary point, if any. The eigenvelues of the linearised matrix and theassociated behaviour of the orbits are the main tools. For a three component system,these orbits can be plotted both in coordinates of the simplex or in a ternary diagram.A characterisation of processes with transfer of mass in closed systems in terms of stability is thus concluded. Two examples are presented for illustration, one of them is aradioactive decay

Body girth as an alternative to body mass for establishing condition indexes in field studies: a validation in the king penguin.

Body mass and body condition are often tightly linked to animal health and fitness in the wild and thus are key measures for ecophysiologists and behavioral ecologists. In some animals, such as large seabird species, obtaining indexes of structural size is relatively easy, whereas measuring body mass under specific field circumstances may be more of a challenge. Here, we suggest an alternative, easily measurable, and reliable surrogate of body mass in field studies, that is, body girth. Using 234 free-living king penguins (Aptenodytes patagonicus) at various stages of molt and breeding, we measured body girth under the flippers, body mass, and bill and flipper length. We found that body girth was strongly and positively related to body mass in both molting (R(2) = 0.91) and breeding (R(2) = 0.73) birds, with the mean error around our predictions being 6.4%. Body girth appeared to be a reliable proxy measure of body mass because the relationship did not vary according to year and experimenter, bird sex, or stage within breeding groups. Body girth was, however, a weak proxy of body mass in birds at the end of molt, probably because most of those birds had reached a critical depletion of energy stores. Body condition indexes established from ordinary least squares regressions of either body girth or body mass on structural size were highly correlated (r(s) = 0.91), suggesting that body girth was as good as body mass in establishing body condition indexes in king penguins. Body girth may prove a useful proxy to body mass for estimating body condition in field investigations and could likely provide similar information in other penguins and large animals that may be complicated to weigh in the wild.

Modified Corneal Collagen Crosslinking (CXL) Reduces Corneal Edema and Diurnal Visual Fluctuations in Fuchs' Dystrophy

Purpose: Crosslinking of corneal collagen with riboflavin and ultraviolet-A irradiation (CXL) induces crosslinks within and between collagen fibers. CXL increases corneal biomechanical and biochemical stability and is currently used clinically to treat keratectasia. CXL also significantly reduces the stromal swelling capacity. We investigated whether a modified CXL treatment protocol would be beneficial in early Fuchs' dystrophy with various degrees of corneal edema and diurnal variations in visual acuity. Methods: CXL was performed as published previously with the following modification: in cases where the stroma was thicker than 450 µm after abrasion and 30 minutes of instillation of isoosmolar riboflavin solution, glycerol 70% solution was applied every 5 seconds for two minutes, and central corneal thickness (CCT) was measured using ultrasound pachymetry. Glycerol 70% solution was administered repeatedly until the target corneal thickness of 370-430 µm was reached. During irradiation, CCT was monitored by ultrasound pachymetry every five minutes and glycerol 70% solution was applied, if necessary. Results: Three eyes in two patients were treated using the modified CXL protocol. Representative case: a 50-year-old woman with Fuchs' dystrophy and a history of 3 years of diurnal visual fluctuations was referred to us in March 2008. Preoperative best spectacle-corrected visual acuity (BSCVA) was 20/50. We performed modified CXL in the left eye. At one month after CXL, Scheimpflug analysis of CCT showed a reduction of more than 100 µm, and the Corneal Thickness Spatial Profile (CTSP) and Percentage of Increase in Thickness (PIT) showed a regularization of the "flattening" typical for Fuchs' dystrophy. Accordingly, diurnal analysis of corneal thickness showed a distinct postoperative reduction in CCT at all time points measured. At one month after CXL, the patient reported a reduction of diurnal visual fluctuations and we measured an increase in BSCVA to 20/32. The patient showed stable topographical and visual acuity at the three months follow-up. Conclusions: We saw a distinct reduction in CCT, an improvement of the corneal thickness spatial profile (CTSP) and an increase in BSCVA at one month after treatment, which remained stable at the three months follow-up. Patients with early Fuchs' dystrophy and disturbing diurnal visual fluctuations represent a novel application for CXL. Although CXL may not prevent the outcome of the dystrophy, it may increase the patients' visual comfort until keratoplasty becomes necessary.

Conditional Involvement of CONSTITUTIVE PHOTOMORPHOGENIC1 in the Degradation of Phytochrome A.

All higher plants possess multiple phytochrome photoreceptors, with phytochrome A (phyA) being light labile and other members of the family being relatively light stable (phyB-phyE in Arabidopsis [Arabidopsis thaliana]). phyA also differs from other members of the family because it enables plants to deetiolate in far-red light-rich environments typical of dense vegetational cover. Later in development, phyA counteracts the shade avoidance syndrome. Light-induced degradation of phyA favors the establishment of a robust shade avoidance syndrome and was proposed to be important for phyA-mediated deetiolation in far-red light. phyA is ubiquitylated and targeted for proteasome-mediated degradation in response to light. Cullin1 and the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) have been implicated in this process. Here, we systematically analyze the requirement of cullins in this process and show that only CULLIN1 plays an important role in light-induced phyA degradation. In addition, the role of COP1 in this process is conditional and depends on the presence of metabolizable sugar in the growth medium. COP1 acts with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins. Unexpectedly, the light-induced decline of phyA levels is reduced in spa mutants irrespective of the growth medium, suggesting a COP1-independent role for SPA proteins.

Canine distemper virus : persistence and pathology in the central nervous system

Résumé : Le virus de la maladie de Carré (en anglais: canine distemper virus, CDV) qui est pathogène pour les chiens et autres carnivores, est très semblable au virus de la rougeole humaine (en anglais MV). Ces deux virus font partie du genre des Morbillivirus qui appartient à la famille des Paramyxoviridae. Ils induisent des complications dans le système nerveux central (SNC). Au stade précoce et aigu de l'infection du SNC, le CDV induit une démyélinisation (1). Ce stade évolue dans certains cas vers une infection chronique avec progression de la démyélinisation. Pendant le stade précoce, qui suit en général de trois semaines les premiers symptômes, le processus de démyélinisation est associé à la réplication du virus et n'est pas considéré comme inflammatoire (1). Par contre, au stade chronique, la progression des plaques de démyélinisation semble être plutôt liée à des processus immunogènes caractéristiques (2), retrouvés également dans la sclérose en plaques (SEP) chez les humains. Pour cette raison, le CDV est considéré comme un modèle pour la SEP humaine et aussi pour l'étude des maladies et complications induites par les Morbillivirus en général (3). Dans notre laboratoire, nous avons utilisé la souche A75/17-CDV, qui est considérée comme le modèle des souches neurovirulentes de CDV. Nous avons cherché en premier lieu à établir un système robuste pour infecter des cultures neuronales avec le CDV. Nous avons choisi les cultures primaires de l'hippocampe du nouveau-né de rat (4), que nous avons ensuite infecté avec une version modifiée du A75/17, appelée rgA75/17-V (5). Dans ces cultures, nous avons prouvé que le CDV infecte des neurones et des astrocytes. Malgré une infection qui se diffuse lentement entre les cellules, cette infection cause une mort massive aussi bien des neurones infectés que non infectés. En parallèle, les astrocytes perdent leur morphologie de type étoilé pour un type polygonal. Finalment, nous avons trouvé une augmentation importante de la concentration en glutamate dans le milieu de culture, qui laisse présumer une sécrétion de glutamate par les cultures infectées (6). Nous avons ensuite étudié le mécanisme des effets cytopathiques induits par le CDV. Nous avons d'abord démontré que les glycoprotéines de surface F et H du CDV s'accumulent massivement dans le réticulum endoplasmique (RE). Cette accumulation déclenche un stress du RE, qui est caractérisé par une forte expression du facteur de transcription proapoptotique CHOP/GADD 153 et de le la calreticuline (CRT). La CRT est une protéine chaperonne localisée dans le RE et impliquée dans l'homéostasie du calcium (Ca2+) et dans le repliement des protéines. En transfectant des cellules de Vero avec des plasmides codant pour plusieurs mutants de la glycoprotéine F de CDV, nous avons démontré une corrélation entre l'accumulation des protéines virales dans le RE et l'augmentation de l'expression de CRT, le stress du RE et la perte de l'homéostasie du Ca2+. Nous avons obtenu des résultats semblables avec des cultures de cellules primaires de cerveau de rat. Ces résultats suggèrent que la CRT joue un rôle crucial dans les phénomènes neurodégénératifs pendant l'infection du SNC, notamment par le relazgage du glutamate via le Ca2+. De manière intéressante, nous démontrons également que l'infection de CDV induit une fragmentation atypique de la CRT. Cette fragmentation induit une re-localisation et une exposition sélective de fragments amino-terminaux de la CRT, connus pour êtres fortement immunogènes à la surface des cellules infectées et non infectées. A partir de ce résultat et des résultats précédents, nous proposons le mécanisme suivant: après l'infection par le CDV, la rétention dans le RE des protéines F et H provoque un stress du RE et une perte de l'homéostasie du Ca2+. Ceci induit la libération du glutamate, qui cause une dégénération rapide du SNC (sur plusieurs jours ou semaines) correspondant à la phase aiguë de la maladie chez le chien. En revanche, les fragments amino-terminaux de la CRT libérés à la surface des cellules infectées peuvent avoir un rôle important dans l'établissement d'une démyélinisation d'origine immunogène, typique de la phase chronique de l'infection de CDV. Summary : The dog pathogen canine distemper virus (CDV), closely related to the human pathogen measles virus (MV), belongs to the Morbillivirus genus of the Paramyxoviridae family. Both CDV and NIV induce complications in the central nervous system (CNS). In the acute early stage of the infection in CNS, the CDV infection induces demyelination. This stage is sometimes followed by a late persistent stage of infection with a progression of the demyelinating lesions (1). The acute early stage occurs around three weeks after the infection and demyelinating processes are associated with active virus replication and are not associated to inflammation (1). In contrast during late persistent stage, the demyelination plaque progression seems to be mainly due to an immunopathological process (2), which characteristics are shared in many aspects with the human disease multiple sclerosis (MS). For these reasons, CDV is considered as a model for human multiple sclerosis, as well as for the study of Morbillivirus-mediated pathogenesis (3). In our laboratory, we used the A75/17-CDV strain that is considered to be the prototype of neurovirulent CDV strain. We first sought to establish a well characterized and robust model for CDV infection of a neuronal culture. We chose primary cultures from newborn rat hippocampes (4) that we infected with a modified version of A75/17, called rgA75/17-V (5). In these cultures, we showed that CDV infects both neurons and astrocytes. While the infection spreads only slowly to neighbouring cells, it causes a massive death of neurons, which includes also non-infected neurons. In parallel, astrocytes undergo morphological changes from the stellate type to the polygonal type. The pharmacological blocking of the glutamate receptors revealed an implication of glutamatergic signalling in the virus-mediated cytopathic effect. Finally, we found a drastic increase concentration of glutamate in the culture medium, suggesting that glutamate was released from the cultured cells (6). We further studied the mechanism of the CDV-induced cytopathic effects. We first demonstrated that the CDV surface glycoprotein F and H markedly accumulate in the endoplasmic reticulum (ER). This accumulation triggers an ER stress, which is characterized by increased expression of the proapoptotic transcription factor CHOP/GADD 153 and calreticulin (CRT). CRT is an ER resident chaperon involved in the Ca2+ homeostasis and in the response to misfolded proteins. Transfections of Vero cells with plasmids encoding various CDV glycoprotein mutants reveal a correlation between accumulation of viral proteins in the ER, CRT overexpression, ER stress and alteration of ER Ca2+ homeostasis. Importantly, similar results are also obtained in primary cell cultures from rat brain. These results suggest that CRT plays a crucial role in CNS infection, particularly due to CRT involvement in Ca2+ mediated glutamate releases, and subsequent neurodegenerative disorders. Very intriguingly, we also demonstrated that CDV infection induces an atypical CRT fragmentation, with relocalisation and selective exposure of the highly immunogenic CRT N-terminal fragments at the surface of infected and neighbouring non-infected cells. Altogether our results combined with previous findings suggest the following scenario. After CDV infection, F and H retention alter Ca2+ homeostasis, and induce glutamate release, which in turn causes rapid CNS degeneration (within days or a week) corresponding to the acute phase of the disease in dogs. In contrast, the CRT N-terminal fragments released at the surface of infected cells may rather have an important role in the establishment of the autoimmune demyelination in the late stage of CDV infection.

Optical nebulosities associated with IRAS sources in dense cloud cores

I, H¿ and [SII] CCD images of the regions around 4 young IRAS sources embedded in the dense molecular cloud cores CB 6, CB 39, AFGL 5142, and L 1251 are presented. Reflection nebulosities are found in all 4 regions. Herbig-Haro objects are detected in AFGL 5142 and L 1251. In both cases, the HH objects are new discoveries.