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In brief The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Abstract Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor–driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain– and PX motif–containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum–induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.

Os tratamentos radioterapicos devem oferecer seguranca ao paciente. Porem, falhas podem ocorrer e desencadear exposicoes acidentais. Neste trabalho foi realizada analise de riscos prospectiva das etapas no processo de tratamento com quilovoltagem, utilizando a FMEA e o SEVRRA. Inicialmente foi elaborado o mapa de processos e os modos de falha envolvidos. Posteriormente foi aplicada a FMEA e, mediante resultados medios obtidos nesta analise, realizou-se outra analise com base no SEVRRA para comparar a efetividade dessas ferramentas. Entrevistas foram feitas com os profissionais envolvidos, levantamento dos modos de falhas, pontuacao dos riscos e levantamento das barreiras. A analise FMEA mostrou que 39% dos modos de falha apresentam (NPR)≥100, sendo este limite escolhido mais conservador. A analise SEVRRA demonstrou que 16% dos modos de falha apresentam riscos altos, 51% riscos moderados, 33% riscos baixos e nenhuma falha considerada de risco muito alto. Apesar da analise FMEA e SEVRRA ser diferente os riscos considerados altos e medios pelo SEVRRA estao contemplados pelo FMEA com NPR≥100 e, que apesar de haver alguns modos de falha de Risco Alto no SEVRRA, a frequencia em que eles ocorrem e baixa. Os metodos sao complementares e importantes para identificacao de possiveis falhas ou erros em Radioterapia.

Accurate chromosome segregation is critical for the formation of haploid gametes, and therefore healthy offspring. Errors in segregation result in aneuploidy, which increases exponentially with maternal age (Hassold and Hunt, 2001). Maternally aged mouse oocytes were recently found to exhibit premature sister chromatid separation due to reduced levels of Securin, an essential regulator of sister chromatid cohesion (Nabti et al, 2017). This landmark finding was facilitated by using chromosome spreads that allow for the visualization of meiotic processes, such as recombination, synapsis, crossing over, and cohesion. This powerful technique is easy to perform and analyze, and allows for the identification of markers for different processes, including DNA damage and repair. Fil: Bertucci, Micka C.. University of Chicago; Estados Unidos Fil: Das, Arunika. University of Chicago; Estados Unidos Fil: Fitzgerald, Harriet C.. University of Chicago; Estados Unidos Fil: Goszczynski, Daniel Estanislao. University of Chicago; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Em braquiterapia, o controle de qualidade é necessário para garantir a consistência entre a dose clínica prescrita para o tratamento e a dose real administrada ao paciente. Entre os procedimentos necessários na adoção de um programa de garantia de qualidade se enquadra a verificação do sistema de planejamento e processo de planejamento e controle de qualidade rotineiro. Visando os processos de verificação do sistema de planejamento, inicialmente, neste trabalho uma fonte de 192Ir GammaMed Plus foi caracterizada com base em parâmetros estabelecidos pelo Task Group 43 (TG-43) da AAPM. Para avaliação de seus parâmetros dosimétricos, a fonte foi simulada através do Método de Monte Carlo, com o código MCNP6 (Monte Carlo N-Particle). Os valores de distribuição de dose obtidos a partir da simulação foram comparados com os dados fornecidos pela literatura. As simulações serviram de base para o desenvolvimento de um software de código aberto, o BrachySure, que permite comparar a dose pontual calculada pelo sistema de planejamento e por Monte Carlo em um caso clínico real. Os resultados obtidos na caracterização da fonte, mostram uma ótima concordância com os dados bibliográficos, apresentando diferenças próximas ou inferiores à incerteza associada as simulações. O software BrachySure foi pré-validado e forneceu uma dupla verificação de dose pontual de maneira rápida e simples, além de contribuir para organização dos dados e registro de tratamentos. Intenta-se com os dados obtidos neste trabalho impulsionar o desenvolvimento de novas metodologias para uso na rotina clínica, que contribuam na incorporação de novas estimativas de doses com maior acurácia.

1. Cyclo-oxygenase (COX)-2 inhibitors and other non-steroidal anti-inflammatory drugs (NSAIDs) have been implicated in increased cardiovascular events. However, the direct effects of these drugs on cardiac function have not been explored extensively. Given the important role of the renin-angiotensin-aldosterone system (RAAS) in cardiac remodelling, we sought to determine the effect of COX-2 inhibitors and non-specific (NS-) NSAIDs on RAAS-induced cardiac hypertrophy and fibrosis in neonatal rat cardiac myocytes (NCM) and fibroblasts (NCF) isolated from 1-2-day-old Sprague-Dawley rat pups. 2. The NCM were pretreated for 2 h with COX-2 inhibitors (celecoxib or rofecoxib) or NS-NSAIDs (naproxen; all at 0.1-10 micromol/L) before being stimulated with 10 micromol/L aldosterone for 72 h or with 0.1 micromol/L angiotensin (Ang) II for 60 h. Hypertrophy of NCM was assessed by [3H]-leucine incorporation. 3. The NCF were pretreated with COX-2 inhibitors or naproxen as described for NCM before being stimulated with 0.1 micromol/L AngII for 48 h. Collagen synthesis was subsequently assayed by [3H]-proline incorporation. 4. Pooled cryopreserved male and female rat hepatocytes were treated with or without COX-2 inhibitors for 1 h before 1 nmol/L aldosterone ( approximately 540 pg/mL) was added to all wells. Cells were incubated for a further 60 min and culture media harvested by centrifugation. Human hepatic HepG2 cells were treated with compounds with or without serum starvation for 48 h. All cells were pretreated with COX-2 inhibitors for 2 h before the addition of aldosterone. Cell culture media were harvested after a further 3, 18, 24 or 48 h incubation. Aldosterone concentrations in the culture media were determined by enzyme immunoassay. 5. Aldosterone- and AngII-stimulated NCM hypertrophy was inhibited by celecoxib, but not by rofecoxib or naproxen. In NCF, AngII-stimulated collagen synthesis was inhibited by celecoxib and, to a lesser extent, by rofecoxib, whereas naproxen had no effect. The COX-2 inhibitors inhibited aldosterone uptake and/or metabolism by rat hepatocytes, but had no effect in human hepatic HepG2 cells. 6. These results demonstrate a potential antiremodelling effect of selective COX-2 inhibitors in the setting of RAAS stimulation in cardiac cells, whereas naproxen has no effect.

During the first 1.5 years of an ongoing successful mission, the Mars Global Surveyor Magnetometer and Electron Reflectometer (MAG/ER) has provided unprecedented information on different boundaries and regions of the Mars–solar wind interaction. This article discusses the magnetic structure and variability of two major plasma boundaries: the bow shock and the magnetic pileup boundary (MPB). MAG/ER observations conclude that the Martian bow shock is a fast bow shock, strongly affected by kinetic effects. The Martian bow shock position is highly variable on the daily basis, but it does not depend on the solar cycle phase. In addition, the bow shock position shows a slight asymmetry related to the convection electric field but no evidence for influence by crustal fields has been found. The MPB is a permanent, well-defined plasma boundary which is characterized by strong changes in the magnetic field and drastic changes in the dominant ion population. Studies on the magnetic structure of the MPB from MGS MAG/ER have led to the conclusion that this boundary is a well-defined current sheet whose normal vector agrees with the normal to the fit obtained from Mars Global Surveyor crossings. Similar boundaries have been observed around Venus and comets, suggesting that the MPB could be a common feature for all atmospheric unmagnetized bodies. At Mars, the MPB position varies on the daily basis and correlates with the bow shock position. On the other hand, the MPB position does not seem to be influenced by the solar cycle, although a small north–south asymmetry in its position suggests a weak influence by crustal magnetic fields.

The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

The magnetic pileup boundary (MPB) is a sharp, thin, and permanent plasma boundary reported, up to now, at comets and Mars, and located between the bow shock and the ionospheric boundary. The MPB separates the magnetosheath, a region with high wave activity, from the magnetic pileup region, where the interplanetary magnetic field piles up regularly in front of the planetary obstacle. We use magnetic field and electron plasma measurements from the MAG/ER experiment onboard the Mars Global Surveyor spacecraft to study two characteristic features of the MPB. The first feature is the sudden enhancement of the magnetic field line draping at the MPB. This new signature reveals that the MPB marks the entry into the veritable induced magnetosphere, where the magnetic field topology is more regular, in opposition to what is observed in the magnetosheath. Secondly, we study the properties and the occurrence of compressive, linearly polarized, low frequency waves, frequently observed on both sides of the boundary. An analysis of the correlation between the magnetic and the electron data reveals that on the upstream side the waves are mirror mode waves, while on the downstream side they are large amplitude, quasi-monochromatic fast magnetosonic waves. The presence of these features at other atmospheric, unmagnetized bodies can be used as indicators of the existence of a MPB.

High dose rate (HDR) brachytherapy (BT) used in treatments of gynecological cancer often results in high doses in the pelvic organs at risk (OARs) and the complications in the rectum are a serious concern. Dosimetry procedures in vivo can be used as an evaluation method of calculated dose in treatment planning. One dosimetric method is the use of alanine with electron spin resonance (ESR) that has been used in different clinical practices. The aim of this study was to indicate the dose level in the female rectum volume, using alanine dosimeters during 192 Ir HDR gynecological BT, for cervical cancer. Doses were compared with the values obtained using the computational treatment planning system based on two orthogonal radiographic images. Firstly a phantom study in water was performed, enabling the in vivo study. Ten patients had the dose in rectum measured, resulting from 10 points properly referred; variations found were in the range of +60% and −50% of the delivered doses compared to the treatment planning system. Differences between planned and measured doses can be mainly due to uncertainty of dosimeter position determination, averaging of dose points specified over the whole dosimeter position, uncontrolled changes in detector position during treatment due to rectum movement and to a simplified treatment system planning, that do not take into account the details of the patient anatomy and the difference among the tissues. Results show that improvements of the protocol treatment should be done to enhance the relation between treatment planning system and experimental results, nevertheless the dose at the OARs was lower than the recommended by the ICRU Report 38.

The photostability of melatonin, a hormone used as supplementary drug in the alleviation of jet-lag and other sleep disorders, was studied. The drug photodegradation at different pH values was monitored by HPLC methods. The main photoproduct was isolated and characterised on the basis of the NMR, FTIR, and mass spectra. A HPLC method, in combination with a post-column on-line photochemical derivatisation was developed for the selective and reliable quality control of commercially available melatonin containing products.

Microsporidiosis with concurrent megabacteriosis in budgerigar ( Melopsittacus undulatus) chicks contributed to significant economic loss in a commercial pet bird aviary in Mississippi. Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied. Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No. 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3). The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species. The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon hellem, an organism that has only been identified in humans. Encephalitozoon hellem causes keratoconjunctivitis and respiratory infections in humans with acquired immunodeficiency syndrome. This report presents the first confirmed case of microsporidiosis in budgerigars. The finding of E. hellem in pet birds may be important in elucidating the epidemiology of human infections with this organism.

The PI3K/Akt signaling pathway is frequently increased in many human cancers, including breast cancer. Recent studies have identified INPP4B, which inhibits PI3K signaling, as an emerging tumor suppressor in breast cancer. This short review discusses these issues and the possibility that INPP4B is an important regulator in many cancers.

A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.

Phosphoinositide signalling molecules interact with a plethora of effector proteins to regulate cell proliferation and survival, vesicular trafficking, metabolism, actin dynamics and many other cellular functions. The generation of specific phosphoinositide species is achieved by the activity of phosphoinositide kinases and phosphatases, which phosphorylate and dephosphorylate, respectively, the inositol headgroup of phosphoinositide molecules. The phosphoinositide phosphatases can be classified as 3-, 4- and 5-phosphatases based on their specificity for dephosphorylating phosphates from specific positions on the inositol head group. The SAC phosphatases show less specificity for the position of the phosphate on the inositol ring. The phosphoinositide phosphatases regulate PI3K/Akt signalling, insulin signalling, endocytosis, vesicle trafficking, cell migration, proliferation and apoptosis. Mouse knockout models of several of the phosphoinositide phosphatases have revealed significant physiological roles for these enzymes, including the regulation of embryonic development, fertility, neurological function, the immune system and insulin sensitivity. Importantly, several phosphoinositide phosphatases have been directly associated with a range of human diseases. Genetic mutations in the 5-phosphatase INPP5E are causative of the ciliopathy syndromes Joubert and MORM, and mutations in the 5-phosphatase OCRL result in Lowe's syndrome and Dent 2 disease. Additionally, polymorphisms in the 5-phosphatase SHIP2 confer diabetes susceptibility in specific populations, whereas reduced protein expression of SHIP1 is reported in several human leukaemias. The 4-phosphatase, INPP4B, has recently been identified as a tumour suppressor in human breast and prostate cancer. Mutations in one SAC phosphatase, SAC3/FIG4, results in the degenerative neuropathy, Charcot-Marie-Tooth disease. Indeed, an understanding of the precise functions of phosphoinositide phosphatases is not only important in the context of normal human physiology, but to reveal the mechanisms by which these enzyme families are implicated in an increasing repertoire of human diseases.

Phosphoinositide signalling molecules interact with a plethora of effector proteins to regulate cell proliferation and survival, vesicular trafficking, metabolism, actin dynamics and many other cellular functions. The generation of specific phosphoinositide species is achieved by the activity of phosphoinositide kinases and phosphatases, which phosphorylate and dephosphorylate, respectively, the inositol headgroup of phosphoinositide molecules. The phosphoinositide phosphatases can be classified as 3-, 4- and 5-phosphatases based on their specificity for dephosphorylating phosphates from specific positions on the inositol head group. The SAC phosphatases show less specificity for the position of the phosphate on the inositol ring. The phosphoinositide phosphatases regulate PI3K/Akt signalling, insulin signalling, endocytosis, vesicle trafficking, cell migration, proliferation and apoptosis. Mouse knockout models of several of the phosphoinositide phosphatases have revealed significant physiological roles for these enzymes, including the regulation of embryonic development, fertility, neurological function, the immune system and insulin sensitivity. Importantly, several phosphoinositide phosphatases have been directly associated with a range of human diseases. Genetic mutations in the 5-phosphatase INPP5E are causative of the ciliopathy syndromes Joubert and MORM, and mutations in the 5-phosphatase OCRL result in Lowe’s syndrome and Dent 2 disease. Additionally, polymorphisms in the 5-phosphatase SHIP2 confer diabetes susceptibility in specific populations, whereas reduced protein expression of SHIP1 is reported in several human leukaemias. The 4-phosphatase, INPP4B, has recently been identified as a tumour suppressor in human breast and prostate cancer. Mutations in one SAC phosphatase, SAC3/FIG4, results in the degenerative neuropathy, Charcot-Marie-Tooth disease. Indeed, an understanding of the precise functions of phosphoinositide phosphatases is not only important in the context of normal human physiology, but to reveal the mechanisms by which these enzyme families are implicated in an increasing repertoire of human diseases.

While color Doppler flow mapping has yielded a quick and relatively sensitive method for visualizing the turbulent jets generated in valvular insufficiency, quantification of the degree of valvular insufficiency has been limited by the dependence of visualization of turbulent jets on hemodynamic as well as instrument-related factors. Color Doppler flow imaging, however, does have the capability of reliably showing the spatial relations of laminar flows. An area where flow accelerates proximal to a regurgitant orifice is commonly visualized on the left ventricular side of a mitral regurgitant orifice, especially when imaging is performed with high gain and a low pulse repetition frequency. This area of flow convergence, where the flow stream narrows symmetrically, can be quantified because velocity and the flow cross-sectional area change in inverse proportion along streamlines centered at the orifice. In this study, a gravity-driven constant-flow system with five sharp-edged diaphragm orifices (ranging from 2.9 to 12 mm in diameter) was imaged both parallel and perpendicular to the direction of flow through the orifice. Color Doppler flow images were produced by zero shifting so that the abrupt change in display color occurred at different velocities. This "aliasing boundary" with a known velocity and a measurable radial distance from the center of the orifice was used to determine an isovelocity hemisphere such that flow rate through the orifice was calculated as 2 pi r2 x Vr, where r is the radial distance from the center of the orifice to the color change and Vr is the velocity at which the color change was noted. Using Vr values from 54 to 14 cm/sec obtained with a 3.75-MHz transducer and from 75 to 18 cm/sec obtained with a 2.5-MHz transducer, we calculated flow rates and found them to correlate with measured flow rates (r = 0.94-0.99). The slope of the regression line was closest to unity when the lowest Vr and the correspondingly largest r were used in the calculation. The flow rates estimated from color Doppler flow imaging could also be used in conjunction with continuous-wave Doppler measurements of the maximal velocity of flow through the orifice to calculate orifice areas (r = 0.75-0.96 correlation with measured areas).(ABSTRACT TRUNCATED AT 250 WORDS)

We describe the application of single-strand conformation polymorphism (SSCP) analysis to the speciation of human microsporidia after polymerase chain reaction (PCR) amplification with the panmicrosporidian primers PMP1 and PMP2. We compared the DNA extracted and amplified from different genotypes or isolates of Enterocytozoon bieneusi, Encephalitozoon cuniculi, E. hellem, and E. intestinalis plus an isolate of Vittaforma corneae. The PCR-SSCP, when performed at 20 degrees C, generated 2 bands in distinctive, reproducible patterns in polyacrylamide gels for each species of microsporidia tested, regardless of genotype or isolate. We found PCR-SSCP to be an easy and reproducible method for speciation of human microsporidia when the primer pair PMP1 and PMP2 is used.

The fatty acid-binding proteins (FABPs) are a class of low-molecular-mass proteins that bind fatty acids and are thought to be involved in their intracellular transport. FABPs have been isolated and studied from several tissues, but their precise function and mechanism of action are still not clear. Chicken liver (basic) fatty acid-binding protein (bFABP) was immobilised on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to bFABP. The retention and enantioselectivity of the new column for a large number of chiral drugs was investigated. The enantiomers of basic and neutral compounds were poorly retained and not resolved by the bFABP column. On the contrary the resolution of the enantiomers of some acidic compounds was obtained. Therefore the influence of the mobile phase pH and organic modifier on the chromatographic performance of acidic compounds was studied. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short-chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure-retention relationship correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.

HPLC on chiral stationary phases has been used for the enantioselective assay of propafenone (PPF), 5-hydroxypropafenone (PPF-50H) and N-despropylpropafenone (PPF-NOR) enantiomers. The results obtained on Chiralpak AD column showed that it is useful for the resolution of PPF and of its main metabolites, although the peaks obtained for PPF-NOR were not symmetrical under the conditions investigated. This column and circular dichroism-based detection system were used to determine the absolute configuration of the eluates. Furthermore, the influence of the mobile phase composition on the resolution of PPF and of its main metabolites was investigated on cellulose derivatives (Chiralcel OD-H and Chiralcel OD-R) and protein (Chiral AGP and Ultron ES-OVM)-based chiral stationary phases. The enantiomers of PPF were resolved on all the columns, except for the Ultron ES-OVM. This column, the Chiralpak AD and the Chiralcel OD-H columns were suitable for the resolution of the PPF-50H enantiomers. The PPF-NOR enantiomers were resolved on the Chiralpak AD, Chiral AGP and Chiralcel OD-R columns.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.

Derivatization of the free cys34 in human serum albumin (HSA) anchored to a silica matrix has been performed by in situ reaction with ethacrynic acid. This modification, which is reported to occur under physiological conditions, gives rise in practice to a new column with different binding properties with respect to the column based on the native protein. Significant differences were observed in the binding of drugs known to bind to site I, (R)-(S)-warfarin and phenylbutazone, and to site II, 1,4-benzodiazepin-2-ones and nonsteroidal anti-inflammatory agents. In particular, the chromatographic retentions markedly decreased for most of the drugs, and, in the case of chiral compounds, significant differences were often observed in the behavior of the two enantiomers, with higher values of enantioselectivity obtained for some of the examined compounds. Furthermore, the noncovalent binding of ethacrynic acid to the protein modifies the binding properties of the albumin.

A female AIDS patient, dying with widely disseminated Encephalitozoon cuniculi microsporidiosis, cytomegalovirus (CMV) disease, and Pneumocystis carinii infection, is described. Indirect immunofluorescent antibody staining studies and molecular analyses identified the microsporidian as the dog strain of E. cuniculi. Autopsy revealed necrotizing microsporidiosis of the adrenal glands and kidneys, with lesser involvement of the brain, heart, trachea, urinary bladder, spleen, and lymph nodes. Cellular targets included macrophages, epithelium, endothelium, and cardiac myocytes. Spore detection was enhanced by Gram-staining, polarization, and fluorescence chitin stains. Central nervous system microglial nodules were present and either contained microsporidia, CMV, or no identifiable pathogen. CMV disease was most severe in the central nervous system, trachea, adrenal glands, and colon, whereas the Pneumocystis carinii infection was focal in the lungs, lymph nodes, and spleen. This is the first demonstration of Encephalitozoon microsporidiosis of the brain, heart, and adrenal glands in a patient with AIDS. E. cuniculi should be included in the differential diagnosis of disseminated opportunistic infections in patients with AIDS.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.

SUMMARYMicrosporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals.Encephalitozoon cuniculiis probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8E. cuniculiisolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54–59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54–58 kDa as well as 28–40 kDa by Western blot immunodetection using murine antisera raised againstE. cuniculi, Encephalitozoon hellem,and theEncephalitozoon-like Septata intestinalis.Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the differentE. cuniculiisolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses.FokI digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5'-GTTT-3' was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3E. cuniculistrains which may become important in the epidemiology of humanE. cuniculiinfections. Furthermore, as additionalE. cuniculiisolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.

Surgical repair is the procedure of choice for atrial septal defect correction. Even though surgical mortality is low (1%), morbidity is significant (anesthesia, thoracotomy, cardiopulmonary bypass, longer hospitalization and intensive care unit monitoring). Transcatheter methods to occlude atrial septal defects have been in development during the last two decades. We report our experience in Italy with the Sideris buttoned double-disk device.From March 1992 to April 1993, 14 patients aged 20 months to 52 years, weight 10 to 82 kg, underwent transcatheter atrial septal defect occlusion with the Sideris buttoned device. The buttoned device is a miniaturized two disk device introduced through small venous sheaths (8-9 F). The diameter of atrial septal defects by echocardiography varied between 9 and 23 mm, while the balloon stretched diameter of the defects varied between 13 and 24 mm. The devices selected were 19 +/- 4 mm larger than the stretched diameter of the defect, but less than the total length of the septum (33-56 mm) by echo.Pulmonary-to-systemic flow ratio varied between 1.5 to 4.0. Mean pulmonary artery pressure varied between 10 and 24 mmHg (mean value 17 +/- 3.5 mm Hg). The relationship between different atrial septal defect measurements (echo, shunt flow, stretched diameter) was statistically analysed: transthoracic echo diameter had a good correlation with the balloon stretched diameter (r = 0.63, p0.001). The atrial septal defect was occluded in 12 patients (86% success rate). There was one early "unbuttoning" which was surgically corrected with success. The other patient underwent surgical correction 3 months later because of unsuitability for transcatheter closure. Minimal residual shunt detected by color flow mapping at 1 month follow-up was seen in 4 patients (33%). No complications occurred in any of our patients.This initial experience with the Sideris buttoned double-disk device demonstrated that transcatheter closure of atrial septal defect is feasible and effective. It can be accomplished through small introducing sheaths. Further clinical trials are justified. This method could become the procedure of choice for the correction of small ASDs.

Perinatal morbidity and mortality are significantly higher in pregnancies complicated by chronic hypoxia and intrauterine growth restriction (IUGR). Clinically, placental insufficiency and IUGR are strongly associated with a fetoplacental inflammatory response. To explore this further, hypoxia was induced in one fetus in twin-bearing pregnant sheep (n = 9) by performing single umbilical artery ligation (SUAL) at 110 days gestation. Five ewes were administered the anti-inflammatory drug sulfasalazine (SSZ) daily, beginning 24 h before surgery. Fetal blood gases and inflammatory markers were examined. In both SSZ- and placebo-treated ewes, SUAL fetuses were hypoxic and growth-restricted at 1 week (P

The continuity equation, applied to the flow convergence region (FCR), fournishes a simple alternative to calculate stenotic valve area. The flow rate in the FCR can be calculated by multiplying the hemispheric isovelocity surface area by the velocity of the isovelocity surface. Since according to the continuity principle the flow rate through any isovelocity surface equals the flow rate through the stenotic orifice, the stenotic orifice area can be calculated as: 2 pi r2Vr/Vm, where 2 pi r2 is the hemispheric isovelocity area, Vr is the velocity at the radial distance r from the orifice and Vm is the peak jet velocity. This study was designed to analyze the validity of application of the continuity equation to the FCR for estimating mitral orifice area by Dopler ultrasound. We studied 35 consecutive patients with rheumatic mitral stenosis. Three patients were excluded; the final study population consisted of 32 patients (8 men and 24 women; mean age 56 years). Nine patients were in normal sinus rhythm and 23 in atrial fibrillation. Doppler examination was performed from the apical approach within 24 hours of cardiac catheterization. On color Doppler image Vr was defined as the first aliasing limit (lowered to 38 cm/s to increase FCR r); r represented the maximal early diastolic distance between the first alias and the stenotic orifice in a direction parallel to that of the transducer; Vm was the early diastolic peak jet velocity by continuous wave Doppler.(ABSTRACT TRUNCATED AT 250 WORDS)