Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We use CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment reveals that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We used CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment revealed that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Professional Advancement and Performance of Amateur Baseball Players Selected in the Major League Baseball Draft With Previous Anterior Cruciate Ligament Reconstruction.

BACKGROUND: The effect that an anterior cruciate ligament injury requiring reconstructive surgery has on the professional advancement and performance of amateur baseball players selected in the Major League Baseball draft is not known. Return to sports after anterior cruciate ligament reconstruction (ACLR) in professional athletes has been shown to be high, but mixed results with regard to performance and return to preinjury level have been demonstrated in other sports. PURPOSE: To (1) investigate the highest level of professional advancement among Major League Baseball draftees with a history of ACLR before entering the draft, (2) examine how much time these players spent on the disabled list (DL) and determine if it was related to the knee, and (3) compare the batting and pitching performance of these players against healthy matched controls. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Predraft medical records of all players selected in the Major League Baseball draft from 2004 to 2008 were reviewed. Players with a documented anterior cruciate ligament injury treated with ACLR before the draft were included. For each study player, 3 controls were identified. Data were accumulated from the time when players were drafted through the 2015 season. Outcome variables included highest professional level of advancement, DL time, and batting and pitching performance. RESULTS: Forty draftees with a history of ACLR (22 pitchers, 18 position players) were identified and matched to 120 controls (66 pitchers, 54 position players). The difference in the highest level of professional advancement between the groups was not statistically significant (P = .488). The mean total number of times and the mean total number of days on the DL were similar between the groups (1.83 vs 1.47, P = .297; 121.54 vs 109.62, P = .955); however, the mean number of times on the DL because of a knee injury was significantly different (0.28 vs 0.11, P = .004), as was the mean number of days on the DL because of a knee injury (17.36 vs 7.72, P = .009). Among pitchers, there were no differences in performance. Similarly, there were no differences among position players in batting performance. CONCLUSION: There was no difference between draftees with a history of ACLR and their controls in terms of advancement from the minor to the major leagues. Additionally, pitching and batting performance were similar. Although the 2 groups spent similar time on the DL, the ACLR group spent more time on the DL because of a knee injury than the control group.

Analytical control of process impurities in Pazopanib hydrochloride by impurity fate mapping.

Understanding the origin and fate of organic impurities within the manufacturing process along with a good control strategy is an integral part of the quality control of drug substance. Following the underlying principles of quality by design (QbD), a systematic approach to analytical control of process impurities by impurity fate mapping (IFM) has been developed and applied to the investigation and control of impurities in the manufacturing process of Pazopanib hydrochloride, an anticancer drug approved recently by the U.S. FDA. This approach requires an aggressive chemical and analytical search for potential impurities in the starting materials, intermediates and drug substance, and experimental studies to track their fate through the manufacturing process in order to understand the process capability for rejecting such impurities. Comprehensive IFM can provide elements of control strategies for impurities. This paper highlights the critical roles that analytical sciences play in the IFM process and impurity control. The application of various analytical techniques (HPLC, LC-MS, NMR, etc.) and development of sensitive and selective methods for impurity detection, identification, separation and quantification are highlighted with illustrative examples. As an essential part of the entire control strategy for Pazopanib hydrochloride, analytical control of impurities with 'meaningful' specifications and the 'right' analytical methods is addressed. In particular, IFM provides scientific justification that can allow for control of process impurities up-stream at the starting materials or intermediates whenever possible.

Testing of a new T-wave subtraction algorithm as an aid to localizing ectopic atrial beats.

BACKGROUND: Identifying the timing and morphology of an ectopic P wave from the surface electrogram can aid in the diagnosis and localization of atrial arrhythmias. Given the relatively short coupling interval of atrial ectopic beats, the P wave is often obscured by the larger amplitude QRS-T wave complex. A method to uncover such "buried" P waves using a standard 12-lead surface ECG would be clinically useful and could potentially be a noninvasive guide to catheter ablation of focal atrial tachycardia. METHODS: We developed an automated computerized program (BARD DUO LAB SYSTEM trade mark ) designed to subtract the QRS-T wave complex from the surface electrogram and uncover a previously obscured P wave. The purpose of the present study was to validate this program. The surface ECG from 21 patients undergoing atrial pacing during electrophysiologic study (group I) and 10 patients with atrial tachycardia (group II) were analyzed and the derived P-wave morphology assessed using correlation waveform analysis (CWA) and visual grading by three reviewers. RESULTS: The algorithm successfully uncovered the P wave in each surface ECG. For the 21 patients in group I, average CWA comparing the derived P wave with the previous paced P wave was 83%. Average CWA for group II was 82%. Visual grading of the match between derived P waves and paced P waves revealed a 21/21 match in group I patients and a 12/12 match in 9/10 of group II patients. CONCLUSIONS: An ectopic atrial P wave obscured by a coincident QRS-T wave complex can be accurately uncovered using this new algorithm. Addition of this technique to existing methods may improve the diagnosis of atrial arrhythmias and aid in the localization and ablation of ectopic atrial foci.

Unexplained pulmonary hypertension is associated with systolic arterial hypertension in patients undergoing routine Doppler echocardiography.

STUDY OBJECTIVE: To determine the validity of the association between systemic hypertension (HTN) and unexplained pulmonary hypertension (PHTN) as identified with Doppler echocardiography. METHODS: All patients with a reported systolic pulmonary artery pressure (SPAP) on routine Doppler echocardiography from our 1997 echocardiographic database were identified. Exclusions included all diseases known to be associated with PHTN. Of 1,174 patients, 503 had PHTN (defined as a SPAP of >/= 40 mm Hg), of whom 42 (8.4%) had unexplained PHTN. These PHTN patients were matched for age (mean [+/- SD] age, 70 +/- 11 years) with 84 randomly selected patients from the same database who had normal SPAP values and no diseases associated with PHTN. RESULTS: The mean SPAP of those patients with unexplained PHTN was 48 +/- 9 mm Hg vs 31 +/- 5 mm Hg for those without unexplained PHTN. HTN was more prevalent in those with PHTN (98% vs 72%, respectively; p = 0.0008). Patients with unexplained PHTN had significantly higher mean systolic BP, as routinely measured at the end of the echo (154 +/- 26 vs 138 +/- 21 mm Hg, respectively; p = 0.0006), but they did not differ in diastolic BP (80 +/- 14 vs 78 +/- 11 mm Hg, respectively; p = 0.39). PHTN patients and control subjects did not differ with respect to gender (women, 74% vs 70%, respectively), race (white, 64% vs 65%, respectively), body mass index (30 +/- 8 vs 28 +/- 8 kg/m(2), respectively), or left ventricular ejection fraction (64 +/- 6% vs 63 +/- 7%, respectively). When only those with known HTN were considered, PHTN patients still had higher systolic arterial BP (155 +/- 25 vs 143 +/- 21 mm Hg, respectively; p = 0.013) and tended to be on more BP medications (1.6 +/- 1.1 vs 1.2 +/- 0.9, respectively; p = 0.09). CONCLUSIONS: Unexplained PHTN occurs mostly in the elderly, is associated with systolic HTN, and those hypertensive patients with concomitant PHTN have higher systolic arterial pressures.