HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development.We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the samewas detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos. © 2013 Society for Reproduction and Fertility.

Comparison Between Diuretic Urography (IVP) and Diuretic Renography for Diagnosis of Ureteropelvic Junction Obstruction in Children

Background: Ureteropelvic junction obstruction (UPJO) is one of the most common causes of urinary tract obstruction in children. Several methods are used to diagnose upper urinary tract obstruction including renal ultrasonography (US), intravenous pyelogram (IVP), diuretic renography (DR), magnetic resonance urography (MRU) and antegrade or retrograde pyelography. Nowadays it is suggested to use diuretic renography as the best method for diagnosing of UPJO. There is no comparative study between IVP and DR scan for diagnosis of UPJO in children. Objectives: The aim of the present study was to compare IVP with furosemide injection and diuretic renography in diagnosis of clinically significant UPJO. Patients and Methods: This was a cross sectional study performed in 153 UPJO suspected children (121 boys, 32 girls) based on US findings in cases presented with urinary tract infection (UTI), prenatal hydronephrosis, abdominal/flank pain, abdominal mass and hematuria. Renal ultrasound was used as an initial screening tool for detection of urinary tract abnormality. Vesicoureteral reflux (VUR) was ruled out by voiding cystourethrography (VCUG). Serum creatinin, blood urea nitrogen, urinalysis and urine culture was screened in all cases. IVP with furosemide and DR were performed as soon as possible after the mentioned workup. Results: During a five year period, 46 out of 153 patients were diagnosed as UPJO based on diuretic renography: the age ranged from 4 months to 13 years (mean: 3.1 ± 0.78 years). There was a significant higher (76%) proportion of UPJO in the boys and in the left side (78%). The sensitivity of IVP with furosemide injection in diagnosis of UPJO was 91.3% whereas DR was accepted as standard for diagnostic procedure in diagnosis of UPJO. Conclusions: Although DR is accepted as the best method for diagnosis of UPJO, we found a small sensitivity difference between IVP and DR in kidneys with normal or near normal function. In many settings such as small cities lacking facilities for advanced isotope imaging technology, use of IVP with diuretic maybe an acceptable procedure for diagnosis of UPJO.

The optimum concentration and timing of melatonin supplementation during IVP to improve bovine embryo development.

2015

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Effect of follicular wave synchronization on in vitro embryo production in heifers

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150 mu g D-cloprostenol. On Day 7, all follicles >3 mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14 d. Each experiment had three treatment groups. In Experiment I (n = 12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28 d. Heifers from Group 1X-EB also received 2 mg EB i.m. immediately after each OPU session. In Experiment 2 (n = 11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5 mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28 d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos. (C) 2009 Elsevier B.V. All rights reserved.

Implicit vector equilibrium problems with applications

Let X and Y be Hausdorff topological vector spaces, K a nonempty, closed, and convex subset of X, C: K--> 2(Y) a point-to-set mapping such that for any x is an element of K, C(x) is a pointed, closed, and convex cone in Y and int C(x) not equal 0. Given a mapping g : K --> K and a vector valued bifunction f : K x K - Y, we consider the implicit vector equilibrium problem (IVEP) of finding x* is an element of K such that f (g(x*), y) is not an element of - int C(x) for all y is an element of K. This problem generalizes the (scalar) implicit equilibrium problem and implicit variational inequality problem. We propose the dual of the implicit vector equilibrium problem (DIVEP) and establish the equivalence between (IVEP) and (DIVEP) under certain assumptions. Also, we give characterizations of the set of solutions for (IVP) in case of nonmonotonicity, weak C-pseudomonotonicity, C-pseudomonotonicity, and strict C-pseudomonotonicity, respectively. Under these assumptions, we conclude that the sets of solutions are nonempty, closed, and convex. Finally, we give some applications of (IVEP) to vector variational inequality problems and vector optimization problems. (C) 2003 Elsevier Science Ltd. All rights reserved.

Árvores frutíferas nos quintais urbanos de Boa Vista, Roraima, Amazônia brasileira

O objetivo do trabalho foi estimar a riqueza e a diversidade das espécies de árvores frutíferas cultivadas nos quintais caseiros da cidade de Boa Vista, Roraima, bem como determinar quais são as espécies cultivadas preferencialmente pela população urbana local. Os levantamentos foram realizados em dois bairros surgidos com a expansão da cidade em 1982: (1) BEst - Bairro dos Estados (Zona Norte) e (2) BAsa - Bairro Asa Branca (Zona Oeste). Foram observados 722 quintais no BEst (06 a 22.03.2004) e 339 no BAsa (07.04 a 01.07.2004). Trinta e seis espécies (19 famílias botânicas) foram encontradas no BEst e 37 (20 famílias) no BAsa, configurando um total de 43 espécies (20 famílias) observadas. Deste total, 30 espécies (69,8%) de 19 famílias (95%) ocorreram em ambos os bairros, sugerindo preferências frutíferas comuns. Os três maiores índices de valor de preferência (IVP) foram coincidentes e registrados para coco (Cocos nucifera L. - BEst: 19,4% e BAsa: 20,5%), manga (Mangifera indica L. - BEst: 14,9% e BAsa: 22,5%) e jambo (Syzygium malaccence (L.) Merr. & L.M. Perry - BEst: 10,5% e BAsa: 10,1%), todos de origem externa à Amazônia, mas que congregaram conjuntamente 44,9% (BEst) e 53,0% (BAsa) de IVP. Estes resultados sugerem que o cultivo de árvores frutíferas em quintais caseiros de Boa Vista segue um padrão que concentra a escolha em poucas espécies, não-originárias da Amazônia, mas tradicionalmente consagradas por seu êxito na produção de frutos.

Utilidade do Ultrassom intracardíaco no isolamento de veias pulmonares usando cateter-balão a laser

FUNDAMENTO: O isolamento das veias pulmonares (IVP) tem sido usado como endpoint para a ablação da fibrilação atrial (FA) com cateter balão. OBJETIVO: Determinar a utilidade do ultrassom intracardíaco (USIC) para guiar o IVP, usando cateter balão a laser. MÉTODOS: 59 VP foram ablacionadas em 27 cães. Imagens de Doppler foram usadas para identificar os vazamentos do fluxo sanguíneo entre a VP e o balão. Após cada liberação de energia, o cateter de mapeamento circular foi reposicionado para verificar se o isolamento tinha sido obtido. A posição de vazamento foi então correlacionada com a posição do gap no estudo patológico. A análise de regressão logística multivariada foi realizada. RESULTADOS: Cinquenta e nove VP foram submetidas à ablação. O tempo médio de energia liberada foi de 279±177 seg., o diâmetro médio do balão era de 23±3 mm, e o comprimento médio do balão era 25±4 mm. O isolamento completo foi obtido em 38/59 (64%), e foi significantemente mais comum sem vazamento: [30/38 (79%) versus 8/23 (35%), p<0,001]. Isso foi independente do tempo de aplicação (302±223 seg. vs. 266±148 sec., p=ns), potência (3,5 W/cm, 4,5 W/cm, e 5,5 W/cm), diâmetro do balão (24± 3 mm vs. 22± 3 mm, p= ns) e comprimento (27±4 mm vs. 24±4 mm, p=ns). O valor preditivo positivo para previsão de isolamento completo foi de 65% e o valor preditivo negativo foi 83%. CONCLUSÃO: Um vazamento identificável entre a VP e o dispositivo de ablação por cateter-balão observado no USIC é preditor de menor taxa de isolamento de VP mais baixas. O USIC pode ser útil para detectar vazamentos a fim de evitar o uso ineficaz de aplicação energia durante a ablação circunferencial da VP. Isto também pode ser útil quando outras energias são utilizadas.

Índices de onda P na predição de recorrência de fibrilação atrial após isolamento da veia pulmonar

FUNDAMENTO: Índices de ondas P são marcadores interessantes para prever recorrências de fibrilação atrial (FA) pós ablação. OBJETIVO: Esse estudo avalia o valor dos índices de onda P para prever recorrências após isolamento da veia pulmonar (IVP) em pacientes com fibrilação atrial paroxística. MÉTODOS: Foram selecionados 198 pacientes (57 ± 8 anos, 150 homens) com FA paroxística sintomática refratária a medicamentos submetidos ao IVP em nosso hospital. Um eletrocardiograma de 12 derivações foi utilizado para medir a duração da onda P na derivação II, a força terminal de P (FTP) na derivação V1, o eixo e a dispersão da onda P. RESULTADOS: No acompanhamento de 9 ± 3 meses, as recorrências ocorreram em 60 (30,3%) pacientes. Os pacientes que apresentaram recorrência de FA tiveram maior duração média de onda P (122,9 ± 10,3 versus 104,3 ± 14,2 ms, p < 0,001), maior dispersão da onda P (40,7 ± 1,7 ms vs 36,6 ± 3,2 ms, p < 0,001). A duração da onda P > 125 ms apresenta 60% de sensibilidade, especificidade de 90%, valor preditivo positivo (VPP) de 72% e valor preditivo negativo (VPN) de 83,7%, enquanto a dispersão da onda P > 40 ms tem 78% de sensibilidade, 67% de especificidade, PPV 51% e VPN de 87,6%. 48/66 (72,7%) dos pacientes com FTP < -0,04 mm/segundo vs 12/132 (9%) com FTP > -0,04 mm/segundo tiveram recorrência de FA (p < 0,001). O eixo da onda P não diferiu entre os dois grupos. Na análise multivariada, os índices da onda P não foram independentes do tamanho do átrio esquerdo e da idade. CONCLUSÕES: A duração da onda P > 125 ms, a dispersão da onda P > 40 ms e FTP em V1 < -0,04 mm/sec são bons preditores clínicos das recorrências de FA pós IVP em pacientes com fibrilação atrial paroxística; contudo, eles não foram independentes do tamanho do átrio esquerdo e da idade.

Imaging spectrum of the operated bladder : P41

Multiple complex procedures involving the bladder have been developed by surgeons in both children and adults, with their own advantages and drawbacks. A greater knowledge of the anatomic changes induced by each category of procedure is mandatory in the follow-up of patients and detection of early and late postoperative complications. A retrospective review of 50 pediatric and adult patients referred or treated in our institution is presented. The combined use of plain films, IVP, MCUG, US, Doppler US, CT, 3DCT and MRI is described. The imaging features are compared with surgical and anatomopathological findings. The purpose of this pictorial essay is to present a wide spectrum of diagnostic imaging findings in children and adult patients with operated bladder. The imaging features of surgical procedures with preserved, partially replaced and totally replaced bladder are described, with emphasis on a systematic multimodality approach. Learning objectives: 1) To provide an in-depth review of bladder surgical procedures with their indications, anatomopathological and imaging features. 2) To describe optimal multimodality imaging techniques for evaluating the operated bladder.

In-Vessel Penetrator -käärmerobotin mallinnus ADAMS-ohjelmistolla

Työn tavoitteena oli muodostaa virtuaaliprototyyppi fuusioreaktorin huollossa käytettävästä IVP-robotista. Työssä mallinnettiin robotin mekaniikka joustavana sekä toimilaitteiden ja käyttöjen dynaamiset ominaisuudet valmistajien esitietojen ja mitoitustietojen perusteella. Käyttöjen ja mekaniikan mallit yhdistettiin ADAMS-ohjelmistossa. Mekaanisten joustojen mallinnuksessa sekä verifioinnissa käytettiin apuna ANSYS –ohjelmistoa. Virtuaaliprototyypin toimivuudesta varmistuttiin vertaamalla sitä robotin suunnittelutietoihin ja fyysiseen prototyyppiin. Robotin ohjauksessa käytettävän P-säätäjän vaikutusta tutkittiin eri vahvistuksen arvoilla sekä verrattiin mekaanisia vasteita fyysisen prototyypin dynaamisiin testeihin. Esimerkkinä robotin käyttäytymisestä todellisessa tilanteessa simuloitiin sen ajoa reaktoriin. Toteutetun simulointimallin todettiin vastaavan rakenteeltaan sekä siinä esiintyvien voimien osalta suunnitelmien mukaista konstruktiota. Käytetyillä parametreilla se toteutti hyvin robotille asetetut nopeusvaatimukset.