Uniform Band Intensities in Fluorescent Dye Terminator Sequencing

Abstract

The use of Cyanine dye (Cy^TM 5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase^TM DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.     

Effects of sodium nitroprusside in aortic stenosis associated with severe heart failure: pressure-volume loop analysis using a numerical model

In the recently published clinical study [Use of Nitroprusside in Left Ventricular Dysfunction and Obstructive Aortic Valve Disease (UNLOAD)], sodium nitroprusside (SNP) improved cardiac function in patients with severe aortic stenosis (AS) and left ventricular (LV) systolic dysfunction. We explored the possible mechanisms of these findings using a series of numerical simulations. A closed-loop lumped parameters model that consists of 24 differential equations relating pressure and flow throughout the circulation was used to analyze the effects of varying hemodynamic conditions in AS. Hemodynamic data from UNLOAD study subjects were used to construct the initial simulation. Systemic vascular resistance (SVR), heart rate, and aortic valve area were directly entered into the model while end-systolic and end-diastolic pressure-volume (P-V) relationships were adjusted using previously published data to match modeled and observed end-systolic and end-diastolic pressures and volumes. Initial simulation of SNP treatment by a reduction of SVR was not adequate. To obtain realistic model hemodynamics that reliably reproduce SNP treatment effects, we performed a series of simulations while simultaneously changing end-systolic elastance (E(es)), end-systolic volume at zero pressure (V(0)), and diastolic P-V shift. Our data indicate that either an E(es) increase or V(0) decrease is necessary to obtain realistic model hemodynamics. In five patients, we corroborated our findings by using the model to duplicate individual P-V loops obtained before and during SNP treatment. In conclusion, using a numerical model, we identified ventricular function parameters that are responsible for improved hemodynamics during SNP infusion in AS with LV dysfunction.     

Cross‐talk between MAP kinase pathways is involved in IGF‐independent,IGFBP‐6‐induced Rh30 rhabdomyosarcoma cell migration

Insulin‐like growth factor binding protein‐6 (IGFBP‐6) inhibits the tumorigenic properties of IGF‐II‐dependent cancer cells by directly inhibiting IGF‐II actions. However, in some cases, IGFBP‐6 is associated with increased cancer cell tumorigenicity, which is unlikely to be due to IGF‐II inhibition. The mechanisms underlying the contradictory actions of IGFBP‐6 remain unclear. We recently generated an IGFBP‐6 mutant that does not bind IGFs (mIGFBP‐6) to address this issue. Although RD rhabdomyosarcoma cells express IGF‐II, we previously showed that mIGFBP‐6 promoted migration through an IGF‐independent, p38‐dependent pathway. We further studied the role of MAP kinases in IGFBP‐6‐induced migration of Rh30 rhabdomyosarcoma cells, which also express IGF‐II. In these cells, mIGFBP‐6 induced chemotaxis rather than chemokinesis. Both wild‐type (wt) and mIGFBP‐6 transiently induced phosphorylation of ERK1/2 and JNK1, but not p38. Inhibition of ERK1/2 phosphorylation completely prevented mIGFBP‐6‐induced ERK1/2 activation and cell migration, whereas a JNK inhibitor partially prevented migration. Interestingly, p38 pathway inhibition completely prevented mIGFBP‐6‐induced ERK1/2 and JNK1 activation and migration despite mIGFBP‐6 not activating p38. Furthermore, blocking the ERK1/2 pathway also inhibited mIGFBP‐6‐induced JNK1 activation. In contrast, IGFBP‐6 had no effect on Akt phosphorylation and an Akt inhibitor had no effect on migration. These results indicate that IGFBP‐6 promotes Rh30 rhabdomyosarcoma chemotaxis in an IGF‐independent manner, and that MAPK signaling pathways and their cross‐talk play an important role in this process. Therefore, besides decreasing Rh30 cell proliferation by inhibiting IGF‐II, IGFBP‐6 promotes their migration via a distinct pathway. Understanding these disparate actions of IGFBP‐6 may lead to the development of novel cancer therapeutics. J. Cell. Physiol. 224: 636–643, 2010. © 2010 Wiley‐Liss, Inc.     

Seasonal variation and time trends of deaths from asthma in England and Wales 1960-82.

Monthly trends in deaths attributed to asthma in the 5-34 year age group were examined for England and Wales over the period 1960-82. Deaths were most frequent in the third quarter (July to September) and peaked in August. Analysis of the 1970-82 period showed that this variation was present in the three constituent age groups 5-14, 15-24, and 25-34 but was most pronounced in the 5-14 year olds.     

Comparative performance of pearl millet- and sorghum- based diets vs. wheat- and rice-based diets for trace metal bioavailability.

Pearl millet and sorghum offer a cheap source of energy compared to wheat and rice and are widely consumed by rural communities in many parts of the world. Due to the low consumption of vegetables and animal foods, millets also are the major suppliers of micronutrients especially for low-income groups. It is of prime importance to study how millets perform in terms of bioavailable contents of trace metals. Investigations were carried out using weanling mice which offer a model for the initial testing of bioavailability of trace metals before human trials. Four isocaloric diets differing only in the type of cereal, i.e. pearl millet, sorghum, wheat and rice, were prepared representing habitual dietary patterns observed by National Nutrition Monitoring Bureau (NNMB) of India. Mice were allocated randomly to 4 groups of 8 mice each, and housed individually in metal free metabolic cages. A fifth group of 8 mice fed a balanced synthetic diet served as control. All the groups were fed ad libitum. The absorption of zinc and iron averaged for 3 periods of 5 days each was significantly higher for the wheat and pearl millet group than for the other 2 experimental groups (p < 0.05), as were also the levels of liver zinc and iron. The weight gain was also highest (6.9 +/- 1.2 g) in the pearl millet group as compared to sorghum (1.58 +/- 0.59 g), wheat (1.66 +/- 1.27 g) and rice (-0.72 +/- 0.62 g) groups. The levels of liver copper were comparable in all the 5 groups. These results further confirm our earlier in vitro results indicating the superiority of pearl millet but not sorghum in bioavailability of zinc and iron.     

The discovery of novel calcium sensing receptor negative allosteric modulators

The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly improved potency over ronacaleret and have the potential as an oral, anabolic treatment for osteoporosis.     

Seasonal variation and time trends in childhood asthma in England and Wales 1975-81.

In England and Wales hospital admissions for childhood asthma almost trebled over the period 1975-81. This may have reflected a true increase in the incidence of acute asthma, a swing from primary to hospital care, or both. The trend was not due to a change in diagnostic fashion. Monthly admissions showed a pronounced seasonal variation with fewest admissions in winter, rising in spring and early summer to peak in the autumn. A deep admission trough was present in August. The monthly admission profile was very similar throughout England and Wales, suggesting that major "trigger" factors were responsible.     

Fluorescence resonance energy transfer dye nucleotide terminators: a new synthetic approach for high-throughout DNA sequencing

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.     

Child Mortality Estimation 2013: An Overview of Updates in Estimation Methods by the United Nations Inter-Agency Group for Child Mortality Estimation

Background

In September 2013, the United Nations Inter-agency Group for Child Mortality Estimation (UN IGME) published an update of the estimates of the under-five mortality rate (U5MR) and under-five deaths for all countries. Compared to the UN IGME estimates published in 2012, updated data inputs and a new method for estimating the U5MR were used.

Methods

We summarize the new U5MR estimation method, which is a Bayesian B-spline Bias-reduction model, and highlight differences with the previously used method. Differences in UN IGME U5MR estimates as published in 2012 and those published in 2013 are presented and decomposed into differences due to the updated database and differences due to the new estimation method to explain and motivate changes in estimates.

Findings

Compared to the previously used method, the new UN IGME estimation method is based on a different trend fitting method that can track (recent) changes in U5MR more closely. The new method provides U5MR estimates that account for data quality issues. Resulting differences in U5MR point estimates between the UN IGME 2012 and 2013 publications are small for the majority of countries but greater than 10 deaths per 1,000 live births for 33 countries in 2011 and 19 countries in 1990. These differences can be explained by the updated database used, the curve fitting method as well as accounting for data quality issues. Changes in the number of deaths were less than 10% on the global level and for the majority of MDG regions.

Conclusions

The 2013 UN IGME estimates provide the most recent assessment of levels and trends in U5MR based on all available data and an improved estimation method that allows for closer-to-real-time monitoring of changes in the U5MR and takes account of data quality issues.