Comparison of gene expression in pre-implantation bovine embryos either injected or transfected with a siRNA targeted against E-cadherin

The ability to create transgenic livestock is a tremendous benefit in scientific research for many disciplines including functional genomics, pharmaceutical synthesis and development of enhanced production animals. Transgenes can either be stably or transiently expressed to alter gene function and obtain a specifically engineered phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and skill associated with both methods, a preliminary investigation exploring alternative delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel and that, although bovine zygotes are not susceptible to chemical transfection, the trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA (Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1 Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts transfected at a ratio of 9:6 with GeneJammer® truly experience gene knock down after siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P < 0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than the transfected blastocysts (P < 0.05). From these data, it can be concluded that although siRNA injection may be capable of knocking down gene expression for the first 7 days of embryonic development, it does not persist to the hatching stage; however, blastocysts transfected at day 7 do express altered gene expression in the trophectoderm which can continue through embryonic hatching events

A population-based study of dystrophin mutations in Canada

Introduction: We carried out a population-based study of dystrophin mutations in patients followed by members of the Canadian Paediatric Neuromuscular Group (CPNG) over a ten-year period. Objectives: We aimed to describe the changes in diagnostic testing for dystrophinopathy and to determine the frequency of dystrophin mutations from 2000 to 2009. Methods: De-identified data containing the clinical phenotypes, diagnostic methods, and mutational reports from dystrophinopathy patients followed by CPNG centres from January 2000 to December 2009 were analyzed using descriptive statistics. Results: 773 patients had a confirmed diagnosis of dystrophinopathy based on genetic testing (97%), muscle biopsy (2%), or family history (1%). 573 (74%) had complete deletion/duplication analysis of all 79 exons or whole gene sequencing, resulting in 366 (64%) deletions, 64 (11%) duplications, and 143 (25%) point mutations. The percentage of patients who were diagnosed using currently accepted genetic testing methods varied across Canada, with a mean of 63% (SD 23). 246 (43%) mutations involved exons 45 to 53. The top ten deletions (n=147, 26%) were exons 45-47, 45-48, 45, 45-50, 45-55, 51, 45-49, 45-52, 49-50, and 46-47. 169 (29%) mutations involved exons 2 to 20. The most common duplications (n=29, 5.1%) were exons 2, 2-7, 2-17, 3-7, 8-11, 10, 10-11, and 12. Conclusion: This is the most comprehensive report of dystrophin mutations in Canada. Consensus guidelines regarding the diagnostic approach to dystrophinopathy will hopefully reduce the geographical variation in mutation detection rates in the coming decade

A mind free to wander: neural and computational constraints on spontaneous thought

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Pharmacokinetics, safety and tolerability of olaparib and temozolomide for recurrent glioblastoma: results of the phase I OPARATIC trial

Background: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide chemotherapy in pre-clinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side-effects of temozolomide. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib, and assess the safety and tolerability of its combination with temozolomide. Methods: Pre-clinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose temozolomide in a 3+3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumour core and tumour margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results: Olaparib was a substrate for multi-drug resistance protein-1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients, median 496nM) and 21/21 tumor margin specimens (9 patients, median 512.3nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150mg (3 days/week) with TMZ 75mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression-free at 6 months. Olaparib radiosensitized six glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusions: Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better pre-clinical models

Human Hendra Virus Encephalitis Associated with Equine Outbreak, Australia, 2008

Emergence of this virus is a serious medical, veterinary, and public health challenge

Decidual natural killer cell receptor expression is altered in pregnancies with impaired vascular remodeling and a higher risk of pre-eclampsia.

During pregnancy, a specialized type of NK cell accumulates in the lining of the uterus (decidua) and interacts with semiallogeneic fetal trophoblast cells. dNK cells are functionally and phenotypically distinct from PB NK and are implicated in regulation of trophoblast transformation of the uterine spiral arteries, which if inadequately performed, can result in pregnancy disorders. Here, we have used uterine artery Doppler RI in the first trimester of pregnancy as a proxy measure of the extent of transformation of the spiral arteries to identify pregnancies with a high RI, indicative of impaired spiral artery remodeling. We have used flow cytometry to examine dNK cells isolated from these pregnancies compared with those from pregnancies with a normal RI. We report a reduction in the proportion of dNK cells from high RI pregnancies expressing KIR2DL/S1,3,5 and LILRB1, receptors for HLA-C and HLA-G on trophoblast. Decreased LILRB1 expression in the decidua was examined by receptor blocking in trophoblast coculture and altered dNK expression of the cytokines CXCL10 and TNF-α, which regulate trophoblast behavior. These results indicate that dNK cells from high RI pregnancies may display altered interactions with trophoblast via decreased expression of HLA-binding cell-surface receptors, impacting on successful transformation of the uterus for pregnancy

First narrow-band search for continuous gravitational waves from known pulsars in advanced detector data

Spinning neutron stars asymmetric with respect to their rotation axis are potential sources of continuous gravitational waves for ground-based interferometric detectors. In the case of known pulsars a fully coherent search, based on matched filtering, which uses the position and rotational parameters obtained from electromagnetic observations, can be carried out. Matched filtering maximizes the signalto- noise (SNR) ratio, but a large sensitivity loss is expected in case of even a very small mismatch between the assumed and the true signal parameters. For this reason, narrow-band analysis methods have been developed, allowing a fully coherent search for gravitational waves from known pulsars over a fraction of a hertz and several spin-down values. In this paper we describe a narrow-band search of 11 pulsars using data from Advanced LIGO’s first observing run. Although we have found several initial outliers, further studies show no significant evidence for the presence of a gravitational wave signal. Finally, we have placed upper limits on the signal strain amplitude lower than the spin-down limit for 5 of the 11 targets over the bands searched; in the case of J1813-1749 the spin-down limit has been beaten for the first time. For an additional 3 targets, the median upper limit across the search bands is below the spin-down limit. This is the most sensitive narrow-band search for continuous gravitational waves carried out so far

Neighborhood socioeconomic status, Medicaid coverage and medical management of myocardial infarction: Atherosclerosis risk in communities (ARIC) community surveillance

<p>Abstract</p> <p>Background</p> <p>Pharmacologic treatments are efficacious in reducing post-myocardial infarction (MI) morbidity and mortality. The potential influence of socioeconomic factors on the receipt of pharmacologic therapy has not been systematically examined, even though healthcare utilization likely influences morbidity and mortality post-MI. This study aims to investigate the association between socioeconomic factors and receipt of evidence-based treatments post-MI in a community surveillance setting.</p> <p>Methods</p> <p>We evaluated the association of census tract-level neighborhood household income (nINC) and Medicaid coverage with pharmacologic treatments (aspirin, beta [β]-blockers and angiotensin converting enzyme [ACE] inhibitors; optimal therapy, defined as receipt of two or more treatments) received during hospitalization or at discharge among 9,608 MI events in the ARIC community surveillance study (1993-2002). Prevalence ratios (PR, 95% CI), adjusted for the clustering of hospitalized MI events within census tracts and within patients, were estimated using Poisson regression.</p> <p>Results</p> <p>Seventy-eight percent of patients received optimal therapy. Low nINC was associated with a lower likelihood of receiving β-blockers (0.93, 0.87-0.98) and a higher likelihood of receiving ACE inhibitors (1.13, 1.04-1.22), compared to high nINC. Patients with Medicaid coverage were less likely to receive aspirin (0.92, 0.87-0.98), compared to patients without Medicaid coverage. These findings were independent of other key covariates.</p> <p>Conclusions</p> <p>nINC and Medicaid coverage may be two of several socioeconomic factors influencing the complexities of medical care practice patterns.</p

Sensitivity of the Advanced LIGO detectors at the beginning of gravitational wave astronomy

The Laser Interferometer Gravitational Wave Observatory (LIGO) consists of two widely separated 4 km laser interferometers designed to detect gravitational waves from distant astrophysical sources in the frequency range from 10 Hz to 10 kHz. The first observation run of the Advanced LIGO detectors started in September 2015 and ended in January 2016. A strain sensitivity of better than 10−23/Hz−−−√ was achieved around 100 Hz. Understanding both the fundamental and the technical noise sources was critical for increasing the astrophysical strain sensitivity. The average distance at which coalescing binary black hole systems with individual masses of 30  M⊙ could be detected above a signal-to-noise ratio (SNR) of 8 was 1.3 Gpc, and the range for binary neutron star inspirals was about 75 Mpc. With respect to the initial detectors, the observable volume of the Universe increased by a factor 69 and 43, respectively. These improvements helped Advanced LIGO to detect the gravitational wave signal from the binary black hole coalescence, known as GW150914