The Over-Irradiation Metabolite Derivative, 24-Hydroxylumister-ol3, Reduces UV-Induced Damage in Skin.

The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce "over-irradiation products" such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.

Classic transcrural celiac plexus block simulated on 100 computed tomography images.

STUDY OBJECTIVE: To evaluate intra-abdominal needle trajectories of the classic transcrural celiac plexus block (tCPB) with a simulation technique. DESIGN: Classic tCPB technique cited from 10 latest authoritative textbooks were simulated on abdominal computed tomography images retrospectively. SETTING: University-affiliated community hospital. MEASUREMENTS: One hundred axial computed tomography images across the celiac trunk were retrieved. Three lines simulating classic tCPB were executed on each image. The organs traversed by each line were noted and analyzed. The frequencies of organ traverse were compared with the incidences described in the literature. MAIN RESULTS: All 3 lines traversed various organs with different frequencies. The right side line frequently traversed the right kidney (36%). The left side line always traversed the aorta (100%). The modified line on the right side frequently traversed the inferior vena cava (32%). The highest kidney traverse percentage on both sides and the highest aorta traverse percentage on the right side were observed in pancreatic cancer patients. CONCLUSIONS: Despite uncommon clinical complications, classic tCPB needle placement frequently traversed through several key organs in this simulation series. Organ penetrations could be avoided by needle trajectory adjustment.

The accuracy of chromosomal microarray testing for identification of embryonic mosaicism in human blastocysts.

BACKGROUND: Most previous studies of chromosomal mosaicism in IVF embryos were performed by fluorescence in situ hybridization (FISH) methods. While there are reports implicating chromosome aneuploidy in implantation failure following transfer and pregnancy loss by spontaneous miscarriage, the significance of mosaicism for the developmental potential of growing embryos is unknown. However, the low prevalence of chromosomal mosaicism in chorionic villus sampling and amniotic fluid specimens suggests the presence of selection against mosaic embryos for implantation and early pregnancy. The absence of evidence for selective allocation of abnormal cells to the trophectoderm (TE) of mosaic blastocysts permits these cells to be a good proxy for embryonic mosaicism detection by chromosomal microarrays (CMA). The purpose of this study was to establish the limits of detection and the prevalence of chromosome mosaicism in day 5/6 human embryos using CMA with TE biopsies. RESULTS: From reconstitution experiments we established log2 ratio thresholds for mosaicism detection. These studies indicated that chromosomal mosaicism at levels as low as between 25-37% can be consistently identified. Follow-up studies by FISH on non-transferred abnormal embryos confirmed the diagnostic accuracy of CMA testing. The number of cells in a TE biopsy can influence mosaicism detection. CONCLUSIONS: Chromosomal microarrays can detect mosaicism in TE biopsies when present at levels as low as between 25-37% and the prevalence of day 5/6 blastocysts which were mosaic and had no other abnormalities reached 15% among a cohort of 551 embryos examined. Validated protocols for establishing detection thresholds for mosaicism are important to reduce the likelihood of transferring abnormal embryos.

Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential.

Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.

1α,25-Dihydroxyvitamin D3 reduces several types of UV-induced DNA damage and contributes to photoprotection.

Vitamin D production requires UVB. In turn, we have shown that vitamin D compounds reduce UV-induced damage, including inflammation, sunburn, thymine dimers, the most frequent type of cyclobutane pyrimidine dimer, immunosuppression, and photocarcinogenesis. Our previous studies have shown most of the photoprotective effects by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) occurred through the nongenomic pathway because similar protection was seen with an analog, 1α,25-dihydroxylumistrol3 (JN), which has little ability to alter gene expression and also because a nongenomic antagonist of 1,25(OH)2D3 abolished protection. In the current study, we tested whether this photoprotective effect would extend to other types of DNA damage, and whether this could be demonstrated in human ex vivo skin, as this model would be suited to pre-clinical testing of topical formulations for photoprotection. In particular, using skin explants, we examined a time course for thymine dimers (TDs), the most abundant DNA photolesion, as well as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a mutagenic DNA base lesion arising from UV-induced oxidative stress, and 8-nitroguanosine (8-NG). Nitric oxide products, known markers for chronic inflammation and carcinogenesis, are also induced by UV. This study showed that 1,25(OH)2D3 significantly reduced TD and 8-NG as early as 30min post UV, and 8-oxodG at 3h post UV, confirming the photoprotective effect of 1,25(OH)2D3 against DNA photoproducts in human skin explants. At least in part, the mechanism of photoprotection by 1,25(OH)2D3 is likely to be through the reduction of reactive nitrogen species and the subsequent reduction in oxidative and nitrosative damage. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Microvascular anastomosis using the vascular closure device in free flap reconstructive surgery: A 13-year experience.

The achievement of patency of the microvascular anastomosis in free flap surgery is dependent on a number of factors, central to which is atraumatic handling of the vessel lumen, and intimal apposition. Initial laboratory studies demonstrating the superiority of the non-penetrating vascular closure staple (VCS - Anastoclip ®) were followed by our report in 1999 on a series of free flaps. There is still a paucity of data in the literature on the use of non-penetrating devices for microvascular anastomosis, and our review gives evidence to support the routine use of the VCS in microsurgical free flap surgery. We now report on its successful use over a thirteen year period in 819 free flap reconstructions. Our data indicates the VCS device to be as effective as sutured anastomoses in free tissue transfer surgery. There is also statistically significant data (Barnard's Exact Test) to demonstrate a higher vascular patency rate of the VCS device over sutured anastomoses when sub group analysis is performed. 'Take-back' revision rates were lower amongst flaps that employed VCS use. For arterial anastomoses, this equated to 3/654(0.05%) vs 4/170(2.4%) with hand-sewn anastomoses (p = 0.02). Similarly, for venous anastomoses the 'take-back' revision rate was 7/661(1.1%) vs 8/165(4.8%) with hand-sewn anastomoses (p = 0.003). Furthermore, the major advantage of the VCS is reduction in anastomosis time, from approximately 25 min per anastomosis for sutures to between five and 10 min for staples.

Protocols associated with no mortality in 100 consecutive Fontan procedures.

OBJECTIVES: Results of Fontan's procedure have improved considerably, but perioperative mortality still occurs, attributed to ventricular dysfunction, stroke, arrhythmia, thromboembolism, and multi-organ dysfunction. Our protocols of operative and intensive care unit management address these potential issues, and have been associated with zero mortality, even with many high-risk candidates. METHODS: From 1996 to 2006, all Fontan patients were managed as follows: operative strategy based on aortic and single atrial cannulation, cooling on full-flow bypass, and hypothermic circulatory arrest to create the Fontan pathway. No direct caval cannulation. Use of central venous lines was completely avoided. Fresh whole blood was used for pump prime and for volume restoration. Inotropic and vasodilator therapy was continued for at least 48 h. Aspirin was used exclusively as anti-thrombotic therapy. Postoperative pleural drainage was accomplished with small pigtail catheters. The usual Fontan pathway was by lateral atrial tunnel (84), with extra-cardiac conduit when dictated by anatomy (16). RESULTS: One hundred Fontan operations were performed with no mortality. All patients were extubated by postoperative day 1. Hospital stay was 10+/-5 days. Complications were: bleeding (1), reintubation (1), emergent fenestration closure (1), pericardial effusion (4), and seizures (1). Risk factors included Fontan connection to one lung (3), diminutive pulmonary arteries (PAs) and unifocalized major aortopulmonary collateral arteries (MAPCAs) (1), discontinuous PAs (3), right ventricle dependent coronaries (3), neonatal pulmonary venous obstruction (3), Trisomy 21 (1), preoperative pacemaker dependence (2), and heterotaxy (10). No candidate was excluded. CONCLUSIONS: While many surgeons try to avoid bypass or aortic clamping when performing Fontan operations, the strategies we have employed facilitate safe accomplishment of Fontan's operation in diverse anatomic groups with multiple risk factors, with avoidance of operative mortality in 100 consecutive cases.

Pott's puffy tumor: Sonographic diagnosis.

We report a case of a child with Pott's puffy tumor (PPT) in which ultrasonography contributed to making the diagnosis. The clinical presentation and laboratory investigations were inconclusive, and the diagnosis of PPT was made by ultrasonography and subsequently confirms CT scan and MRI.

Non-disclosing preimplantation genetic diagnosis for Huntington disease.

OBJECTIVES: Individuals at risk for Huntington disease face difficult decisions regarding their reproductive options. Most do not wish to pass on the gene for Huntington disease to their children, but may not be prepared themselves to undergo presymptomatic testing and learn their genetic status. For these reasons, many at-risk individuals with a family history of HD would choose a method of genetic diagnosis that would assure them that they can have children unaffected with HD without revealing their own genetic status (non-disclosing). We have shown that, with a carefully designed and executed programme of non-disclosing preimplantation genetic testing, one can successfully assist at-risk couples to have their own biological children who are free from Huntington disease, without forcing parents to confront knowledge of their own genetic status. METHODS: Couples where one partner was at 50% risk for Huntington disease underwent in vitro fertilization with preimplantation embryo biopsy and molecular analysis for Huntington disease where appropriate. RESULTS: After extensive counselling and informed consent, 10 couples underwent 13 in vitro fertilization and two frozen embryo transfer cycles in a programme for non-disclosing preimplantation genetic diagnosis for Huntington disease. In 11 cycles, embryos determined to be free of Huntington disease were transferred, resulting in five clinical pregnancies. One set of twins and three singleton pregnancies have delivered. One pregnancy resulted in a first-trimester loss. CONCLUSIONS: The option of non-disclosing preimplantation genetic diagnosis should be reviewed, along with other relevant medical options, when counselling at-risk Huntington disease families.

Principios y aplicaciones de la reacción en cadena de la polimerasa / Principle and application of the chain reaction of the polymerase

El descubrimiento de la reacción en cadena de la polimerasa (PCR), técnica para la amplificación de ácidos nucleicos, ha tenido un enorme impacto sobre áreas diversas, tanto de la investigación básica como de la investigación clínica. Desde su descubrimiento en 1985, los informes sobre una gran variedad de aplicaciones de la PCR han recibido mucha atención en la literatura médica y científica. Esta tecnología ha demostrado tener una gran aplicabilidad para el diagnóstico de enfermedades humanas, incluyendo áreas tan diversas como enfermedades infecciosas, desórdenes genéticos y cáncer. Este artículo presenta una amplia revisión de los principios de la PCR, que incluyen conceptos genéricos que deben manejarse cuando se use o se diseñe un ensayo basado en la PCR. También se discute la aplicación de tales ensayos para el diagnóstico de desórdenes genéticos y para el estudio de enfermedades infecciosas. En los últimos 10 años ha aumentado, de manera notable, la aplicación de herramientas de la biología molecular para el diagnóstico de las enfermedades humanas. Los ensayos con sondas de ADN están, ahora, disponibles comercialmente para la detección e identificación de una gran variedad de patógenos humanos, así como para el diagnóstico de desórdenes genéticos humanos. Una manifestación del rápido crecimiento de la tecnología del ADN, ha sido el desarrollo de técnicas para amplificar secuencias específicas de ácidos nucleicos. En la literatura, han sido descritos muchos métodos, que se enumeran en la tabla i. Una comparación de estos métodos ha sido el tema de una revisión frecuente. Entre ellos se destacan la reacción en cadena de la polimerasa, como la de mayor impacto, tanto como herramienta de investigación como de diagnóstico. El objetivo de este artículo es presentar una breve revisión de los principios y aplicaciones de la amplificación de PCR, que incluyen una discusión de la selección correcta de las secuencias en blanco, el diseño de los "primer" y los medios necesarios para llevar a cabo la técnica

Principios e aplicaçoes da reaçao da cadeia de polimerase / Principles and applications of the polymerase chain reaction

A invençao da tecnica da reaçao da cadeia de polimerase(PCR) para a amplificaçao de acido nucleico tem tido um impacto maior sobre diversas areas tanto da pesquisa basica como da clinica. Desde seu inicio em 1985, comunicaçoes sobre uma grande variedade de aplicaçoes da PCR foram recebidas com grande atençao na literatura cientifica e medica. Esta tecnologia tem mostrado grande aplicabilidade ao diagnostico de doenças humanas, incluindo areas tao diversas como doenças infecciosas, desordens geneticas e cancer. O presente artigo apresenta uma ampla revisao dos principios da PCR incluindo consideraçoes geneticas que devem ser observadas no uso ou no planejamento de ensaios baseados na PCR. A aplicaçao destes ensaios com base na PCR para o diagnostico de desordens geneticas e para triagem de doenças infecciosas sao tambem discutidas