Development of a microwave-based extraction for forensic biological samples

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples

Extent of Left Ventricular Scar Predicts Outcomes in Ischemic Cardiomyopathy Patients With Significantly Reduced Systolic Function A Delayed Hyperenhancement Cardiac Magnetic Resonance Study

ObjectivesThe objective of the study was to determine whether the extent of left ventricular scar, measured with delayed hyperenhancement cardiac magnetic resonance (DHE-CMR), predicts survival in patients with ischemic cardiomyopathy (ICM) and severely reduced left ventricular ejection fraction (LVEF).BackgroundPatients with ICM and reduced LVEF have poor survival. Such patients have a high myocardial scar burden. CMR is highly accurate in delineation of myocardial scar.MethodsWe studied 349 patients (76% men) with severe ICM (≥70% disease in ≥1 epicardial coronary, and mean LVEF of 24%) that underwent DHE-CMR (Siemens 1.5-T scanner, Erlangen, Germany), between 2003 and 2006. Scar (quantified as percentage of myocardium) was defined on DHE-MR images as an intensity >2 standard deviations above the viable myocardium. Transmurality score was semiquantitatively recorded in a 17-segment model as: 0 = no scar, 1 = 1% to 25% scar, 2 = 26% to 50%, 3 = 51% to 75%, and 4 = >75%. The LVEF, demographic data, risk factors, need for cardiac transplantation (CTx), and all-cause mortality were recorded.ResultsThe mean age and follow-up were 65 ± 11 years and 2.6 ± 1.2 years (median 2.4 years [1.1, 3.5]), respectively. There were 56 events (51 deaths and 5 CTx). Mean scar percentage and transmurality score were higher in patients with events versus those without (39 ± 22 vs. 30 ± 20, p = 0.003, and 9.7 ± 5 vs. 7.8 ± 5, p = 0.004). On Cox proportional hazard survival analysis, quantified scar was greater than the median (30% of total myocardium), and female gender predicted events (relative risk 1.75 [95% Confidence Interval: 1.02 to 3.03] and relative risk 1.83 [95% Confidence Interval: 1.06 to 3.16], respectively, both p = 0.03).ConclusionsIn patients with ICM and severely reduced LVEF, a greater extent of myocardial scar, delineated by DHE-CMR is associated with increased mortality or the need for cardiac transplantation, potentially aiding further risk-stratification

Feasibility of Patient Reporting of Symptomatic Adverse Events via the Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events (PROCTCAE) in a Chemoradiotherapy Cooperative Group Multicenter Clinical Trial

Purpose—To assess the feasibility of measuring symptomatic adverse events (AEs) in a multicenter clinical trial using the National Cancer Institute’s Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE). Methods and Materials—Patients enrolled in Trial XXXX (XXXX) were asked to self-report 53 PRO-CTCAE items representing 30 symptomatic AEs at 6 time points (baseline; weekly x4 during treatment; 12-weeks post-treatment). Reporting was conducted via wireless tablet computers in clinic waiting areas. Compliance was defined as the proportion of visits when an expected PRO-CTCAE assessment was completed. Results—Among 226 study sites participating in Trial XXXX, 100% completed 35-minute PROCTCAE training for clinical research associates (CRAs); 80 sites enrolled patients of which 34 (43%) required tablet computers to be provided. All 152 patients in Trial XXXX agreed to selfreport using the PRO-CTCAE (median age 66; 47% female; 84% white). Median time for CRAs to learn the system was 60 minutes (range 30–240), and median time for CRAs to teach a patient to self-report was 10 minutes (range 2–60). Compliance was high, particularly during active treatment when patients self-reported at 86% of expected time points, although compliance was lower post-treatment (72%). Common reasons for non-compliance were institutional errors such as forgetting to provide computers to participants; patients missing clinic visits; internet connectivity; and patients feeling “too sick”. Conclusions—Most patients enrolled in a multicenter chemoradiotherapy trial were willing and able to self-report symptomatic adverse events at visits using tablet computers. Minimal effort was required by local site staff to support this system. The observed causes of missing data may be obviated by allowing patients to self-report electronically between-visits, and by employing central compliance monitoring. These approaches are being incorporated into ongoing studies

Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent

The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.National Institutes of Health (U.S.) (grant no. P30-ES002109)National Institutes of Health (U.S.) (grant no. GM65337)National Institutes of Health (U.S.) (grant no. GM65337-03S2)National Institutes of Health (U.S.) (grant no. CA055042)National Institutes of Health (U.S.) (grant no. CA092584)Repligen Corporation (KIICR Graduate Fellowship