Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

<p>Abstract</p> <p>Background</p> <p>High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.</p> <p>Results</p> <p>We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.</p> <p>Conclusions</p> <p>Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.</p

Multidisciplinary approach to treatment of a Multiple Sclerosis Exacerbation in a 10 day Inpatient Rehabilitation Setting

Background/purpose: Multiple Sclerosis (MS) is a central nervous disorder that has a prevalence of 3.2 per 100,000 individuals in the United States and leads to persistent disability in 80% of these individuals. MS typically results in an inability to perform activities of daily living and instrumental activities of daily living. Research has shown that physical and occupational therapy are beneficial treatments but limited research has determined their impact in a shortened multidisciplinary approach in the inpatient rehab setting. Case Description: The patient was a 49 year old male with secondary progressive MS that presented with generalized weakness, decreased coordination and decreased endurance. The symptoms resulted in decreased ability to perform transfers and ambulation both independently and safely. Outcomes: This patient completed 9 days of therapy that involved occupational, physical and speech therapy over a 10 day inpatient stay. Patient showed significant increases in transfers and ambulation demonstrated by improvements in the Functional Independent Measure (FIM). There were no differences seen in fatigue when utilizing the Fatigue Severity Scale. Discussion: The increases seen in the FIM indicate an improved ability for the subject to be independent and participate as an active member of his family. Although, the multidisciplinary approach in a shortened duration did not have a significant impact on perceived fatigue in this case study, further research will be required to determine if the improvements seen in the inpatient rehab setting correlate in the home and are maintained in the long term

Hemodynamic Assessment of the Pathological Left Ventricle Function under Rest and Exercise Conditions

Purpose: The analysis of pathological human left ventricular hemodynamics using high-resolved image-based blood flow simulations shows a major potential for examining mitral valve insufficiency (MI) under exercise conditions. Since capturing and simulating the patient-specific movement of the left ventricle (LV) during rest and exercise is challenging, this study aims to propose a workflow to analyze the hemodynamics within the pathologically moving LV. Methods: Patient-specific ultrasound (US) data of ten patients with MI in different stages were captured with three-dimensional real-time echocardiography. US measurements were performed while patients were resting and while doing handgrip exercise (2–4 min work). Patient-specific hemodynamic simulations were carried out based on the captured ventricular wall movement. Velocity and kinetic energy were analyzed for rest and exercise and for the different MI stages. Results: The results reveal a dependency of the kinetic energy over time in the ventricular volume curves. Concerning the comparison between rest and exercise, the left ventricular function reveals lower systolic kinetic energy under exercise (kinetic energy normalized by EDV; mean ± standard deviation: rest = 0.16 ± 0.14; exercise = 0.06 ± 0.05; p-value = 0.04). Comparing patients with non-limiting (MI I) and mild/moderate (MI II/III) MI, lower velocities (mean ± standard deviation: non-limiting = 0.10 ± 0.03; mild/moderate = 0.06 ± 0.02; p-value = 0.01) and lower diastolic kinetic energy (kinetic energy normalized by EDV; mean ± standard deviation: non-limiting = 0.45 ± 0.30; mild/moderate = 0.20 ± 0.19; p-value = 0.03) were found for the latter. Conclusion: With the proposed workflow, the hemodynamics within LVs with MI can be analyzed under rest and exercise. The results reveal the importance of the patient-specific wall movement when analyzing intraventricular hemodynamics. These findings can be further used within patient-specific simulations, based on varying the imaging and segmentation methods

Value of progression of coronary artery calcification for risk prediction of coronary and cardiovascular events

Background: Computed tomography (CT) allows estimation of coronary artery calcium (CAC) progression. We evaluated several progression algorithms in our unselected, population-based cohort for risk prediction of coronary and cardiovascular events. Methods: In 3281 participants (45–74 years of age), free from cardiovascular disease until the second visit, risk factors, and CTs at baseline (b) and after a mean of 5.1 years (5y) were measured. Hard coronary and cardiovascular events, and total cardiovascular events including revascularization, as well, were recorded during a follow-up time of 7.8±2.2 years after the second CT. The added predictive value of 10 CAC progression algorithms on top of risk factors including baseline CAC was evaluated by using survival analysis, C-statistics, net reclassification improvement, and integrated discrimination index. A subgroup analysis of risk in CAC categories was performed. Results: We observed 85 (2.6%) hard coronary, 161 (4.9%) hard cardiovascular, and 241 (7.3%) total cardiovascular events. Absolute CAC progression was higher with versus without subsequent coronary events (median, 115 [Q1–Q3, 23–360] versus 8 [0–83], P<0.0001; similar for hard/total cardiovascular events). Some progression algorithms added to the predictive value of baseline CT and risk assessment in terms of C-statistic or integrated discrimination index, especially for total cardiovascular events. However, CAC progression did not improve models including CAC5y and 5-year risk factors. An excellent prognosis was found for 921 participants with double-zero CACb=CAC5y=0 (10-year coronary and hard/total cardiovascular risk: 1.4%, 2.0%, and 2.8%), which was for participants with incident CAC 1.8%, 3.8%, and 6.6%, respectively. When CACb progressed from 1 to 399 to CAC5y≥400, coronary and total cardiovascular risk were nearly 2-fold in comparison with subjects who remained below CAC5y=400. Participants with CACb≥400 had high rates of hard coronary and hard/total cardiovascular events (10-year risk: 12.0%, 13.5%, and 30.9%, respectively). Conclusions: CAC progression is associated with coronary and cardiovascular event rates, but adds only weakly to risk prediction. What counts is the most recent CAC value and risk factor assessment. Therefore, a repeat scan >5 years after the first scan may be of additional value, except when a double-zero CT scan is present or when the subjects are already at high risk