Two bifunctional desferrioxamine chelators for bioorthogonal labeling of biovectors with zirconium-89.

We report two bifunctional chelators, DFO-Cys and DFO-CBT, to label biovectors with zirconium-89 according to the 2-cyanobenzothiazole/1,2-aminothiol cycloaddition. Their features are high labeling yields, rapid and efficient bioconjugation, metabolically stable luciferin-based end products, and applicability to orthogonal two-step labeling of sensitive biomolecules.

Generation of patient-specific induced pluripotent stem cell lines from one patient with Jervell and Lange-Nielsen syndrome, one with type 1 long QT syndrome and two healthy relatives.

Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.

C-reactive protein in Brazilian adolescents: distribution and association with metabolic syndrome in ERICA survey.

BACKGROUND/OBJECTIVES: C-reactive protein (CRP) is a marker of inflammation that has been shown to be predictive of cardiovascular diseases in adults. To evaluate the distribution of CRP as well as its association with metabolic syndrome and its components. SUBJECTS/METHODS: This is a cross-sectional study on adolescents aged 12-17, participants in the Study of Cardiovascular Risk in Adolescents (ERICA). Anthropometric, biochemical and blood pressure data were collected from 6316 adolescents, selected from a random sample of students in the cities of Brasilia, Fortaleza, João Pessoa, Manaus, Porto Alegre and Rio de Janeiro. Metabolic syndrome was defined by the criteria proposed by International Diabetes Federation for adolescent. Poisson regression model with robust variance, taking into consideration the study's complex sampling design, was used to determine multivariate-adjusted prevalence rate ratios expressing the relationship of metabolic syndrome with CRP. RESULTS: In adolescents with metabolic syndrome, CRP concentrations were five times higher (1.01 mg/l; interquartile range (IQR): 0.54-3.47) compared with those without metabolic syndrome (0.19 mg/l; IQR: 0.10-0.78). In multivariate Poisson regression analysis adjusted by sex, age and skin color, the prevalence of elevated CRP (>3.0 mg/l) was almost three times higher in adolescents with metabolic syndrome than in those without this condition (prevalence ratio (PR): 2.9; 95%CI: 2.0-4.3; P<0.001). Of the metabolic syndrome components, elevated waist circumference, low high-density lipoprotein-cholesterol and high triglycerides were significantly related to CRP in a graded (dose-response) manner. CONCLUSIONS: The association of CRP with metabolic syndrome and its components suggests that inflammation may be useful in assessing cardiovascular risk in adolescents.

Nuclear status of immature and mature stallion spermatozoa.

'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol-disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn(2+) content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization.

A tunable diode laser system for ammonia flux measurements over multiple plots.

This paper reports on the field testing of a tunable diode laser trace gas analyzer system for micrometeorological monitoring of ammonia fluxes. This system uses infrared absorption spectroscopy to measure atmospheric ammonia concentrations and the fluxgradient method to relate the measured concentration gradient to a flux of ammonia. For the field tests, we monitored ammonia fuxes over three plots receiving different manure applications. Each plot was sampled for 15 or 30 min of each hour, producing a high-temporal resolution data set. Analysis of the system response showed that ammonia adsorption to the tubing walls was greatly reduced by the system design and did not interfere with the flux measurement.

Morphologic comparisons among equine endometrium categories I, II, and III, using light and transmission electron microscopy.

OBJECTIVE: To evaluate whether the pathologic changes observed by light microscopy in endometrium of categories II and III were reflected by cellular changes and to describe differences in the endometrial cell ultrastructure during estrus and diestrus. ANIMALS: 18 healthy mares. PROCEDURE: Endometrial tissues biopsied during the physiologic breeding season were categorized, using light microscopy, and were studied, using transmission electron microscopy (TEM). RESULTS: Using TEM, glycogen granules were associated with giant mitochondria for all endometrial types during diestrus. Development of rough endoplasmic reticulum (RER) and Golgi apparatus suggested protein synthesis in the endometrial glands during diestrus. TEM did not reveal major ultrastructural differences, between endometrium of categories I and II. This was unlike differences identified by light microscopy. The most extensive pathologic changes were seen in category-III tissue (TEM and light microscopy). Category-III endometria had a large number of light cells with more degenerative structures and fewer organelles, and lacked cilia in the lumen of the glands. This tissue had extensive fibrotic tissue in the lamina propria and many inflammatory cells in most tissue layers. CONCLUSIONS: The severe ultrastructural changes may be one of the many factors decreasing the fertility of mares with category-III, compared with category-1 and -2, endometrium.

Microvascular development and growth of uterine tissue during the estrous cycle in mares.

OBJECTIVE: To document uterine growth and microvascular development in the endometrium of uteri with differing degrees of fibrosis as well as uterine growth throughout the estrous cycle of mares. ANIMALS: 30 mares. PROCEDURE: Uterine tissue was obtained during the breeding season from a slaughter facility. Stage of estrous cycle of the mares was assessed on the basis of ovarian structures and plasma progesterone concentrations. Endometrium was characterized by use of light microscopy, and blood vessel walls were marked by histochemical techniques. Microvascular development was evaluated by a computerized image analysis system. Growth of uterine tissue was based on cellular content of DNA and RNA, RNA:DNA, and protein:DNA. RESULTS: Significant differences in vascular density were not observed in the endometrium of uteri obtained from mares euthanatized during the follicular or luteal phase of the estrous cycle, regardless of whether endometrial classification of degree of fibrosis was considered. There was a 3-fold increase in amount of DNA and RNA of endometrial cells in the follicular phase when compared to myometrium. Hypertrophy of endometrial tissue during the luteal phase was reflected by a significant increase in cell protein content and protein:DNA. CONCLUSIONS AND CLINICAL RELEVANCE: Endometrial growth of vascular tissues during the estrous cycle may be coordinated with development of nonvascular tissue. Estrogen and progesterone may play a role in regulation of uterine growth and angiogenesis.

[Transparieto-hepatic cholangiography with the Chiba needle. Experience in 100 cases]. / Colangiografia transparieto-hepática com agulha de Chiba. Experiência em 100 casos.

One-hundred cases submitted to percutaneous transhepatic cholangiography (PTC) with Chiba's needle were studied. Success rate was 93%, with 100% success in patients with dilated ducts and 78.8% in those with normal ducts. One false-positive result was noticed due to air injection into bile duct in the beginning of the procedure. Complications were seen in 8 cases, without fatalities ascribed to the PTC itself, nor emergency laparotomy was necessary in these cases.

Thiol-disulfide proteins of stallion epididymal spermatozoa.

Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididymal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two-dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal maturation process. The majority of the oxidized thiol sperm proteins identified correspond to structural molecules of the flagellum (as the outer dense fiber-1 protein - ODF1), followed by glycolytic enzymes (as glyceraldehyde-3-phosphate dehydrogenase spermatogenic), antioxidant protectors (as glutathione S-transferase and phospholipid hydroperoxide glutathione peroxidase - PHGPx). The magnitude of the thiol oxidation differs between proteins, and was more drastic in polypeptides with molecular weights of up to 33kDa, identified as ODF1 and PHGPx. A kinase anchor protein, a voltage-dependent anion channel protein and a zona pellucida-binding protein were also found in the polypeptide samples that contained oxidized SH groups. These proteins may be modified or controlled by the mechanisms involved in the cysteine-redox changes, corroborating the belief that a correct degree of protein oxidation is required for the stabilization of sperm structure, protection against oxidative damage, induction of progressive sperm motility and fertilization.

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells.

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10(-7)m) for 24 h. The concentrations of PGE(2) and PGF(2α) in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE(2) and PGF(2α) release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

mRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis.

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F(2α) synthase, PGFS; and prostaglandin E(2) synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE(2) and PGF(2α) in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF(2α) concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E(2) concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.

PP147. Prevalence of hypertensive syndromes according to pregnancy age.

INTRODUCTION: The decision of a woman to get pregnant at later age of her reproductive cycle has be a phenomenon around the world. Epidemiology data show frequent increase of clinical complications in direct proportion in advanced age motherhood, hypertensive disturbances being more prevalent. OBJECTIVES: Analyse the prevalence of hypertensive syndromes in pre-determined age groups at Hospital Guilherme Álvaro in Santos, São Paulo, Brazil showing the different segments in each one. METHODS: From data collected in the outpatient department of Hospital Guilherme Álvaro of High Risk Pre-natal between 04/06/2008 and 30/05/2011, a prevalence transversal study was carried out where data were obtained from 628 patients aged between 16 and 46years. Procedures of homogeny analysts were set out, always collecting data such as age and disorder for high risk gestation. According to age, patients were divided into groups: precocious (up to 19years old), middle age (between 20 and 34) and late pregnancies (over 35). RESULTS: In the precocious pregnancies, clinical illnesses/no hypertension (31%) were observed in first place, 25% (8) twin pregnancy in second place, 19% (6) showed hypertensive disturbances. Concerning pregnancies between 20 and 34years old, 36% (144 patients) showed hypertensive syndromes, 23% (92 patients) showed endocrine disturbances, 22% (90 patients) showed clinical illnesses/no hypertension, and 9% twin pregnancy. Regarding late pregnancies, the most frequent disturbance was isolated hypertensive syndromes: 44% (88 patients) in first place, only endocrine disturbances, 24% (47 patients) in second place followed by association between hypertensive syndromes and endocrinopathy with 13% (26 patients). CONCLUSION: About precocious pregnancies, greater prevalence showed clinical illnesses/no hypertension, whereas middle age and late pregnancies showed greater hypertensive syndrome prevalence, results, which are compatible with other studies, have been observed that due to advance of age, hypertensive syndromes are more frequent. For late pregnancies, the prevalence of clinical illnesses/no hypertension was a lower percentage regarding the other two groups: precocious pregnancies (31%=10 women), middle age pregnancies (22%=90 women), and late pregnancies (2%=7 women). Considering the fact that the occurrence of pregnancy is more and more late in life, it can be concluded that the professionals must be prepared to attend pregnancies on women with hypertensive disturbances and their eventual complications.

Giardia spp. e Cryptosporidium spp. em água de manancial superficial de abastecimento contaminada por dejetos humano e animal / Giardia spp. and Cryptosporidium spp. in a fresh water supply source contaminated with human and animal excreta and waste water

Estudou-se ocorrência de cistos de Giardia spp. e oocistos de Cryptosporidium spp. - (oo)cistos - na bacia hidrográfica do Ribeirão São Bartolomeu, localizada no município de Viçosa, MG. O estudo incluiu as populações humana e animal existentes na área da bacia, bem como no efluente de uma estação de tratamento de esgoto (ETE) e de duas instalações para suínos. Os resultados indicam presença de (oo)cistos no manancial (médias geométricas: 3,92 e 3,62 (oo)cistos/l para Giardia spp. e Cryptosporidium spp., respectivamente). Propriedades com exploração bovina foram positivas ao longo de todo período (prevalência média de propriedades positivas 36,4 por cento para Giardia spp. e 18,0 por cento para Cryptosporidium spp.). O efluente da ETE apresentou elevada concentração de cistos de Giardia spp. (média geométrica na ordem de 10(4)/l), mas não foram encontrados oocistos de Cryptosporidium spp. A ocorrência de (oo)cistos apresentou comportamento sazonal, sendo que os valores médios de (oo)cistos e de pluviosidade do trimestre de coleta revelaram bom ajuste de correlação (R²=98,3 por cento; P=0,0087 para Giardia spp. e R²=91,8 por cento; P=0,0421 para Cryptosporidium spp.). A significativa ocorrência de (oo)cistos no manancial sugere que a forma de ocupação do solo interfere na qualidade parasitológica da água bruta. Os resultados apontam para a importância de adoção de medidas preventivas, como proteção de áreas de mananciais, objetivando reduzir riscos de transmissão de protozoários via água de consumo humano.

The occurrence of Giardia spp. cysts and Cryptosporidium spp. oocysts - (oo)cysts - was studied in a watershed (São Bartolomeu Stream-SB), at Viçosa city, MG. In the studied area, characterization of animal farms and identification of sewage and animal wastes discharges were undertaken. In addition, a sewage treatment plant (STP) and effluents of two swine farms were sampled. The results presented protozoan contamination (geometric averages: 3.92 and 3.62 (oo)cysts/l for Giardia spp. and Cryptosporidium spp., respectively). Positive cattle farms were detected during all the period, and the correspondent mean prevalences were: 36.4 percent for Giardia spp. and 18.0 percent for Cryptosporidium spp. STP effluent presented high concentration of Giardia spp. (geometric average of cysts in the order of 10(4)/l). However, Cryptosporidium spp. oocysts were not detected. The results showed that (oo)cysts concentration were influenced by climate factors. Precipitation and (oo)cysts concentration data were well correlated (R²=98.3 percent; P=0.0087 for Giardia spp. and R²=91.8 percent; P=0.0421 for Cryptosporidium spp.). Cysts and oocysts levels were remarkably high in the raw water source, pointing out the role of land use in an unprotected watershed as a source of protozoa. The results demonstrate the importance of preventive measures rather than relying on, sometimes unreliable, corrective measures such as water treatment, in order to minimize human health risks.