HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Impact of Caffeine Ingestion on the Driving Performance of Anesthesiology Residents After 6 Consecutive Overnight Work Shifts.

BACKGROUND: Residency training in anesthesiology involves care of hospitalized patients and necessitates overnight work, resulting in altered sleep patterns and sleep deprivation. Caffeine consumption is commonly used to improve alertness when fatigued after overnight work, in preparation for the commute home. METHODS: We studied the impact of drinking a caffeinated energy drink (160 mg of caffeine) on driving performance in a high-fidelity, virtual reality driving simulator (Virginia Driving Safety Laboratory using the Driver Guidance System) in anesthesiology resident physicians immediately after 6 consecutive night-float shifts. Twenty-six residents participated and were randomized to either consume a caffeinated or noncaffeinated energy drink 60 minutes before the driving simulation session. After a subsequent week of night-float work, residents performed the same driving session (in a crossover fashion) with the opposite intervention. Psychomotor vigilance task (PVT) testing was used to evaluate reaction time and lapses in attention. RESULTS: After 6 consecutive night-float shifts, anesthesiology residents who consumed a caffeinated energy drink had increased variability in driving for throttle, steering, and speed during the first 10 minutes of open-road driving but proceeded to demonstrate improved driving performance with fewer obstacle collisions (epoch 2: 0.65 vs 0.87; epoch 3: 0.47 vs 0.95; P = .03) in the final 30 minutes of driving as compared to driving performance after consumption of a noncaffeinated energy drink. Improved driving performance was most apparent during the last 30 minutes of the simulated drive in the caffeinated condition. Mean reaction time between the caffeine and noncaffeine states differed significantly (278.9 ± 29.1 vs 294.0 ± 36.3 milliseconds; P = .021), while the number of major lapses (0.09 ± 0.43 vs 0.27 ± 0.55; P = .257) and minor lapses (1.05 ± 1.39 vs 2.05 ± 3.06; P = .197) was not significantly different. CONCLUSIONS: After consuming a caffeinated energy drink on conclusion of 6 shifts of night-float work, anesthesiology residents had improved control of driving performance variables in a high-fidelity driving simulator, including a significant reduction in collisions as well as slightly faster reaction times.

Feasibility of Spinal Anesthesia Placement Using Automated Interpretation of Lumbar Ultrasound Images: A Prospective Randomized Controlled Trial.

BACKGROUND: This study evaluated the efficacy of spinal anesthesia administration by resident physicians when using an ultrasound system with automated neuraxial landmark detection capabilities. METHODS: 150 patients were enrolled in this trial. Anesthesiology residents placed spinals in subjects undergoing scheduled cesarean delivery using one of three techniques to identify neuraxial landmarks: palpation, ultrasound, or combined palpation and ultrasound. Ultrasound was performed using a handheld system that automatically identified neuraxial landmarks (e.g. midline, intervertebral spaces). All residents watched a 10-minute video and received 20 minutes of hands-on training prior to participating in the study. First insertion success rate was the primary end point. RESULTS: Among all patients, use of ultrasound resulted in a 11% greater first-insertion success rate (RR: 1.11 [0.85-1.47], p=0.431), a 15% reduction in needle insertions (RR: 0.85, p=0.052), and a 26% decrease in needle passes (RR: 0.74, p=0.070). In obese patients of BMI ≥ 30 kg/m2, use of ultrasound resulted in 26% greater first-insertion success rates (RR: 1.26, p=0.187), a 21% decrease in needle insertions (RR: 0.79, p=0.025), a 38% decrease in needle passes (RR: 0.62, p=0.030), and a 75% decrease in patients reporting neutral or low patient satisfaction with anesthesia administration (RR: 0.25, p=0.004). DISCUSSION: Resident anesthesiologists competently utilized the ultrasound system after receiving minimal training. Technical endpoints and patient satisfaction trended towards improvement when ultrasound was used prior to spinal placement, with stronger trends observed in obese patients. Additional study is required to fully characterize the impact of the ultrasound system on clinical efficacy.

Haptoglobin 2-2 Phenotype Is Associated With Increased Acute Kidney Injury After Elective Cardiac Surgery in Patients With Diabetes Mellitus.

BACKGROUND: Recent studies reported an association between the 2-2 phenotype of haptoglobin (Hp 2-2) and increased cardiorenal morbidity in nonsurgical diabetic patients. Our goal was to determine whether the Hp 2-2 phenotype was associated with acute kidney injury (AKI) after elective cardiac surgery in patients with diabetes mellitus. METHODS AND RESULTS: We prospectively enrolled 99 diabetic patients requiring elective cardiac surgery with cardiopulmonary bypass. Haptoglobin phenotypes were determined by gel electrophoresis. Cell-free hemoglobin, haptoglobin, and total serum bilirubin were quantified as hemolysis markers. The primary outcome was postoperative AKI, as defined by the Acute Kidney Injury Network classification. The incidence of AKI was significantly higher in Hp 2-2 patients compared with patients without this phenotype (non-Hp-2-2; 55.6% versus 27%, P<0.01). The need for renal replacement therapy was also significantly higher in the Hp 2-2 group (5 patients versus 1 patient, P=0.02). Thirty-day mortality (3 versus 0 patients, P=0.04) and 1-year mortality (5 versus 0 patients, P<0.01) were also significantly higher in patients with the Hp 2-2 phenotype. In multivariable analysis, Hp 2-2 was an independent predictor of postoperative AKI (P=0.01; odds ratio: 4.17; 95% confidence interval, 1.35-12.48). CONCLUSIONS: Hp 2-2 phenotype is an independent predictor of postoperative AKI and is associated with decreased short and long-term survival after cardiac surgery in patients with diabetes mellitus.

Perioperative statin use is associated with decreased incidence of primary graft dysfunction after lung transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is a major cause of early morbidity and mortality after lung transplantation. Statins reduce the risk of chronic rejection after lung transplantation, but their effects on PGD are unknown. We hypothesized that perioperative statin therapy decreases the risk for PGD after lung transplantation. METHODS: We retrospectively reviewed records of all patients undergoing lung transplantation between January 1999 and December 2014 at the University of Virginia Health System. The primary outcome was PGD (grades 1-3). Secondary outcomes included grade 3 PGD, length of intensive care unit and hospital stay, and mortality. RESULTS: Of 266 patients who met final inclusion criteria, 138 (52%) were diagnosed with PGD. In-hospital mortality among patients with PGD was 6.5%. There were no deaths in patients without PGD (p < 0.001). PGD was diagnosed in 24 patients taking statins (34.8%) and in 114 patients (57.9%) who did not take statins (p = 0.001). After propensity score adjustments, perioperative statin use was independently associated with a reduced risk for PGD (odds ratio [OR] 0.41, 95% confidence interval [CI] 0.20-0.84, p = 0.015) and reduced risk to develop grade 3 PGD (OR 0.42, 95% CI 0.18-0.94, p = 0.036). Other risk factors associated with PGD included intraoperative use of cardiopulmonary bypass (OR 3.74, 95% CI 1.75-8.02, p = 0.001) and positive donor smoking status (OR 2.27, 95% CI 1.18-4.35, p = 0.014). CONCLUSIONS: The results demonstrate that perioperative use of statins is independently associated with reduced risk for PGD after lung transplantation.

Imaging Performance of a Handheld Ultrasound System With Real-Time Computer-Aided Detection of Lumbar Spine Anatomy: A Feasibility Study.

OBJECTIVES: The aim of this study was to evaluate the imaging performance of a handheld ultrasound system and the accuracy of an automated lumbar spine computer-aided detection (CAD) algorithm in the spines of human subjects. MATERIALS AND METHODS: This study was approved by the institutional review board of the University of Virginia. The authors designed a handheld ultrasound system with enhanced bone image quality and fully automated CAD of lumbar spine anatomy. The imaging performance was evaluated by imaging the lumbar spines of 68 volunteers with body mass index between 18.5 and 48 kg/m. The accuracy, sensitivity, and specificity of the lumbar spine CAD algorithm were assessed by comparing the algorithm's results to ground-truth segmentations of neuraxial anatomy provided by radiologists. RESULTS: The lumbar spine CAD algorithm detected the epidural space with a sensitivity of 94.2% (95% confidence interval [CI], 85.1%-98.1%) and a specificity of 85.5% (95% CI, 81.7%-88.6%) and measured its depth with an error of approximately ±0.5 cm compared with measurements obtained manually from the 2-dimensional ultrasound images. The spine midline was detected with a sensitivity of 93.9% (95% CI, 85.8%-97.7%) and specificity of 91.3% (95% CI, 83.6%-96.9%), and its lateral position within the ultrasound image was measured with an error of approximately ±0.3 cm. The bone enhancement imaging mode produced images with 5.1- to 10-fold enhanced bone contrast when compared with a comparable handheld ultrasound imaging system. CONCLUSIONS: The results of this study demonstrate the feasibility of CAD for assisting with real-time interpretation of ultrasound images of the lumbar spine at the bedside.

Driving Performance of Residents after Six Consecutive Overnight Work Shifts.

BACKGROUND: Residency training requires work in clinical settings for extended periods of time, resulting in altered sleep patterns, sleep deprivation, and potentially deleterious effects on safe performance of daily activities, including driving a motor vehicle. METHODS: Twenty-nine anesthesiology resident physicians in postgraduate year 2 to 4 drove for 55 min in the Virginia Driving Safety Laboratory using the Driver Guidance System (MBFARR, LLC, USA). Two driving simulator sessions were conducted, one experimental session immediately after the final shift of six consecutive night shifts and one control session at the beginning of a normal day shift (not after call). Both sessions were conducted at 8:00 AM. Psychomotor vigilance task testing was employed to evaluate reaction time and lapses in attention. RESULTS: After six consecutive night shifts, residents experienced significantly impaired control of all the driving variables including speed, lane position, throttle, and steering. They were also more likely to be involved in collisions. After six consecutive night shifts, residents had a significant increase in reaction times (281.1 vs. 298.5 ms; P = 0.001) and had a significant increase in the number of both minor (0.85 vs. 1.88; P = 0.01) and major lapses (0.00 vs. 0.31; P = 0.008) in attention. CONCLUSIONS: Resident physicians have greater difficulty controlling speed and driving performance in the driving simulator after six consecutive night shifts. Reaction times are also increased with emphasis on increases in minor and major lapses in attention after six consecutive night shifts.

Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

The redundancy of the mammalian heterochromatic compartment.

Two chromatin compartments are present in most mammalian cells; the first contains primarily euchromatic, early replicating chromatin and the second, primarily late-replicating heterochromatin, which is the subject of this review. Heterochromatin is concentrated in three intranuclear regions: the nuclear periphery, the perinucleolar space and in pericentromeric bodies. We review recent evidence demonstrating that the heterochromatic compartment is critically involved in global nuclear organization and the maintenance of genome stability, and discuss models regarding how this compartment is formed and maintained. We also evaluate our understanding of how heterochromatic sequences (herein named heterochromatic associated regions (HADs)) might be tethered within these regions and review experiments that reveal the stochastic nature of individual HAD positioning within the compartment. These investigations suggest a substantial level of functional redundancy within the heterochromatic compartment.

Wash interacts with lamin and affects global nuclear organization.

The cytoplasmic functions of Wiskott-Aldrich syndrome family (WAS) proteins are well established and include roles in cytoskeleton reorganization and membrane-cytoskeletal interactions important for membrane/vesicle trafficking, morphogenesis, immune response, and signal transduction. Misregulation of these proteins is associated with immune deficiency and metastasis [1-4]. Cytoplasmic WAS proteins act as effectors of Rho family GTPases and polymerize branched actin through the Arp2/3 complex [1, 5]. Previously, we identified Drosophila washout (wash) as a new member of the WAS family with essential cytoplasmic roles in early development [6, 7]. Studies in mammalian cells and Dictyostelium suggest that WASH functions primarily in a multiprotein complex that regulates endosome shape and trafficking in an Arp2/3-dependent manner [8-11]. However, roles for classically cytoplasmic proteins in the nucleus are beginning to emerge, in particular, as participants in the regulation of gene expression [12, 13]. Here, we show that Drosophila Wash is present in the nucleus, where it plays a key role in global nuclear organization. wash mutant and knockdown nuclei disrupt subnuclear structures/organelles and exhibit the abnormal wrinkled morphology reminiscent of those observed in diverse laminopathies [14-16]. We find that nuclear Wash interacts with B-type Lamin (Lamin Dm0), and, like Lamin, Wash associates with constitutive heterochromatin. Wash knockdown increases chromatin accessibility of repressive compartments and results in a global redistribution of repressive histone modifications. Thus, our results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.