Methodologic Considerations for Collecting Patient-reported Outcomes from Unselected Surgical Patients.

BACKGROUND: The impact of surgery on health is only appreciated long after hospital discharge. Furthermore, patients' perceptions of postoperative health are not routinely ascertained. The authors instituted the Systematic Assessment and Targeted Improvement of Services Following Yearlong Surgical Outcomes Surveys (SATISFY-SOS) registry to evaluate patients' postoperative health based on patient-reported outcomes (PROs). METHODS: This article describes the methods of establishing the SATISFY-SOS registry from an unselected surgical population, combining perioperative PROs with information from electronic medical records. Patients enrolled during their preoperative visit were surveyed at enrollment, 30 days, and 1-yr postoperatively. Information on PROs, including quality of life, return to work, pain, functional status, medical complications, and cognition, was obtained from online, mail, or telephone surveys. RESULTS: Using structured query language, 44,081 patients were identified in the electronic medical records as having visited the Center for Preoperative Assessment and Planning for preoperative assessment between July 16, 2012, and June 15, 2014, and 20,719 patients (47%) consented to participate in SATISFY-SOS. Baseline characteristics and health status were similar between enrolled and not enrolled patients. The response rate for the 30-day survey was 62% (8% e-mail, 73% mail, and 19% telephone) and for the 1-yr survey was 71% (13% e-mail, 78% mail, and 8% telephone). CONCLUSIONS: SATISFY-SOS demonstrates the feasibility of establishing a PRO registry reflective of a busy preoperative assessment center population, without disrupting clinical workflow. Our experience suggests that patient engagement, including informed consent and multiple survey modalities, enhances PROs collection from a large cohort of unselected surgical patients. Initiatives like SATISFY-SOS could promote quality improvement, enable efficient perioperative research, and facilitate outcomes that matter to surgical patients.

Psychological sequelae of surgery in a prospective cohort of patients from three intraoperative awareness prevention trials.

BACKGROUND: Elective surgery can have long-term psychological sequelae, especially for patients who experience intraoperative awareness. However, risk factors, other than awareness, for symptoms of posttraumatic stress disorder (PTSD) after surgery are poorly defined, and practical screening methods have not been applied to a broad population of surgical patients. METHODS: The Psychological Sequelae of Surgery study was a prospective cohort study of patients previously enrolled in the United States and Canada in 3 trials for the prevention of intraoperative awareness. The 68 patients who experienced definite or possible awareness were matched with 418 patients who denied awareness based on age, sex, surgery type, and awareness risk. Participants completed the PTSD Checklist-Specific (PCL-S) and/or a modified Mini-International Neuropsychiatric Interview telephone assessment to identify symptoms of PTSD and symptom complexes consistent with a PTSD diagnosis. We then used structural equation modeling to produce a composite PTSD score and examined potential risk factors. RESULTS: One hundred forty patients were unreachable; of those contacted, 303 (88%) participated a median of 2 years postoperatively. Forty-four of the 219 patients (20.1%) who completed the PCL-S exceeded the civilian screening cutoff score for PTSD symptoms resulting from their surgery (15 of 35 [43%] with awareness and 29 of 184 [16%] without). Nineteen patients (8.7%; 5 of 35 [14%] with awareness and 14 of 184 [7.6%] without) both exceeded the cutoff and endorsed a breadth of symptoms consistent with the Diagnostic and Statistical Manual Fourth Edition diagnosis of PTSD attributable to their surgery. Factors independently associated with PTSD symptoms were poor social support, previous PTSD symptoms, previous mental health treatment, dissociation related to surgery, perceiving that one's life was threatened during surgery, and intraoperative awareness (all P ≤ 0.017). Perioperative dissociation was identified as a potential mediator for perioperative PTSD symptoms. CONCLUSIONS: Events in the perioperative period can precipitate psychological symptoms consistent with subsyndromal and syndromal PTSD. We not only confirmed the high rate of postoperative PTSD in awareness patients but also identified a significant rate in matched nonawareness controls. Screening surgical patients, especially those with potentially mediating risk factors such as intraoperative awareness or perioperative dissociation, for postoperative PTSD symptoms with the PCL-S is practical and could promote early referral, evaluation, and treatment.

Prevention of intraoperative awareness in a high-risk surgical population.

BACKGROUND: Unintended intraoperative awareness, which occurs when general anesthesia is not achieved or maintained, affects up to 1% of patients at high risk for this complication. We tested the hypothesis that a protocol incorporating the electroencephalogram-derived bispectral index (BIS) is superior to a protocol incorporating standard monitoring of end-tidal anesthetic-agent concentration (ETAC) for the prevention of awareness. METHODS: We conducted a prospective, randomized, evaluator-blinded trial at three medical centers. We randomly assigned 6041 patients at high risk for awareness to BIS-guided anesthesia (with an audible alert if the BIS value was <40 or >60, on a scale of 0 to 100, with 0 indicating the suppression of detectable brain electrical activity and 100 indicating the awake state) or ETAC-guided anesthesia (with an audible alert if the ETAC was <0.7 or >1.3 minimum alveolar concentration). In addition to audible alerts, the protocols included structured education and checklists. Superiority of the BIS protocol was assessed with the use of a one-sided Fisher's exact test. RESULTS: A total of 7 of 2861 patients (0.24%) in the BIS group, as compared with 2 of 2852 (0.07%) in the ETAC group, who were interviewed postoperatively had definite intraoperative awareness (a difference of 0.17 percentage points; 95% confidence interval [CI], -0.03 to 0.38; P=0.98). Thus, the superiority of the BIS protocol was not demonstrated. A total of 19 cases of definite or possible intraoperative awareness (0.66%) occurred in the BIS group, as compared with 8 (0.28%) in the ETAC group (a difference of 0.38 percentage points; 95% CI, 0.03 to 0.74; P=0.99), with the superiority of the BIS protocol again not demonstrated. There was no difference between the groups with respect to the amount of anesthesia administered or the rate of major postoperative adverse outcomes. CONCLUSIONS: The superiority of the BIS protocol was not established; contrary to expectations, fewer patients in the ETAC group than in the BIS group experienced awareness. (Funded by the Foundation for Anesthesia Education and Research and others; BAG-RECALL ClinicalTrials.gov number, NCT00682825.).

Postoperative recovery with bispectral index versus anesthetic concentration-guided protocols.

BACKGROUND: Use of the bispectral index (BIS) monitor has been suggested to decrease excessive anesthetic drug administration, leading to improved recovery from general anesthesia. The purpose of this substudy of the B-Unaware and BAG-RECALL trials was to assess whether a BIS-based anesthetic protocol was superior to an end-tidal anesthetic concentration-based protocol in decreasing recovery time and postoperative complications. METHODS: Patients at high risk for awareness were randomized to either BIS-guided or end-tidal anesthetic concentration-guided general anesthesia in the original trials. Outcomes included time to postanesthesia care unit discharge readiness, time to achieve a postoperative Aldrete score of 9-10, intensive care unit length of stay, postoperative nausea and vomiting, and severe postoperative pain. Univariate Cox regression and chi-square tests were used for statistical analyses. RESULTS: The BIS cohort was not superior in time to postanesthesia care unit discharge readiness (hazard ratio, 1.0; 95% CI, 1.0-1.1; n = 2,949), time to achieve an Aldrete score of 9-10 (hazard ratio, 1.2; 95% CI, 1.0-1.4; n = 706), intensive care unit length of stay (hazard ratio, 1.0; 95% CI, 0.9-1.1; n = 2,074), incidence of postoperative nausea and vomiting (absolute risk reduction, -0.5%; 95% CI, -5.8 to 4.8%; n = 789), or incidence of severe postoperative pain (absolute risk reduction, 4.4%; 95% CI, -2.3 to 11.1%; n = 759). CONCLUSIONS: In patients at high risk for awareness, the BIS-guided protocol is not superior to an anesthetic concentration-guided protocol in time needed for postoperative recovery or in the incidences of common postoperative complications.

Mouse class III myosins: kinase activity and phosphorylation sites.

As class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation.

Anesthesia awareness and the bispectral index.

BACKGROUND: Awareness during anesthesia is a serious complication with potential long-term psychological consequences. Use of the bispectral index (BIS), developed from a processed electroencephalogram, has been reported to decrease the incidence of anesthesia awareness when the BIS value is maintained below 60. In this trial, we sought to determine whether a BIS-based protocol is better than a protocol based on a measurement of end-tidal anesthetic gas (ETAG) for decreasing anesthesia awareness in patients at high risk for this complication. METHODS: We randomly assigned 2000 patients to BIS-guided anesthesia (target BIS range, 40 to 60) or ETAG-guided anesthesia (target ETAG range, 0.7 to 1.3 minimum alveolar concentration [MAC]). Postoperatively, patients were assessed for anesthesia awareness at three intervals (0 to 24 hours, 24 to 72 hours, and 30 days after extubation). RESULTS: We assessed 967 and 974 patients from the BIS and ETAG groups, respectively. Two cases of definite anesthesia awareness occurred in each group (absolute difference, 0%; 95% confidence interval [CI], -0.56 to 0.57%). The BIS value was greater than 60 in one case of definite anesthesia awareness, and the ETAG concentrations were less than 0.7 MAC in three cases. For all patients, the mean (+/-SD) time-averaged ETAG concentration was 0.81+/-0.25 MAC in the BIS group and 0.82+/-0.23 MAC in the ETAG group (P=0.10; 95% CI for the difference between the BIS and ETAG groups, -0.04 to 0.01 MAC). CONCLUSIONS: We did not reproduce the results of previous studies that reported a lower incidence of anesthesia awareness with BIS monitoring, and the use of the BIS protocol was not associated with reduced administration of volatile anesthetic gases. Anesthesia awareness occurred even when BIS values and ETAG concentrations were within the target ranges. Our findings do not support routine BIS monitoring as part of standard practice. (ClinicalTrials.gov number, NCT00281489 [ClinicalTrials.gov].).

Bispectral index monitoring, duration of bispectral index below 45, patient risk factors, and intermediate-term mortality after noncardiac surgery in the B-Unaware Trial.

BACKGROUND: Postoperative mortality has been associated with cumulative anesthetic duration below an arbitrary processed electroencephalographic threshold (bispectral index [BIS] <45). This substudy of the B-Unaware Trial tested whether cumulative duration of BIS values lower than 45, cumulative anesthetic dose, comorbidities, or intraoperative events were independently associated with postoperative mortality. METHODS: The authors studied 1,473 patients (mean ± SD age, 57.9 ± 14.4 yr; 749 men) who underwent noncardiac surgery at Barnes-Jewish Hospital in St. Louis, Missouri. Multivariable Cox regression analysis was used to determine whether perioperative factors were independently associated with all-cause mortality. RESULTS: A total of 358 patients (24.3%) died during a follow-up of 3.2 ± 1.1 yr. There were statistically significant associations among various perioperative risk factors, including malignancy and intermediate-term mortality. BIS-monitored patients did not have lower mortality than unmonitored patients (24.9 vs. 23.7%; difference = 1.2%, 95% CI, -3.3 to 5.6%). Cumulative duration of BIS values less than 45 was not associated with mortality (multivariable hazard ratio, 1.03; 95% CI, 0.93-1.14). Increasing mean and cumulative end-tidal anesthetic concentrations were not associated with mortality. The multivariable Cox regression model showed a good discriminative ability (c-index = 0.795). CONCLUSIONS: This study found no evidence that either cumulative BIS values below a threshold of 40 or 45 or cumulative inhalational anesthetic dose is injurious to patients. These results do not support the hypothesis that limiting depth of anesthesia either by titration to a specific BIS threshold or by limiting end-tidal volatile agent concentrations will decrease postoperative mortality.

Relationship between bispectral index values and volatile anesthetic concentrations during the maintenance phase of anesthesia in the B-Unaware trial.

BACKGROUND: Hypnotic depth during anesthesia affects electroencephalography waveforms and electroencephalogram-derived indices, such as the bispectral index (BIS). Titrating anesthetic administration against the BIS assumes reliable relationships between BIS values, electroencephalogram waveforms, and effect site concentration, beyond loss of responsiveness. Associations among BIS, end-tidal anesthetic concentrations (ETAC), and patient characteristics were examined during anesthetic maintenance, using B-Unaware trial data. METHODS: Pharmacokinetically stable ETAC epochs during intraoperative anesthetic maintenance were analyzed. A generalized estimating equation determined independent relationships among BIS, ETAC (in age-adjusted minimum alveolar concentration equivalents), patient characteristics, and 1-yr mortality. Further individual and population characteristics were explored graphically. RESULTS: A total of 3,347,523 data points from 1,100 patients were analyzed over an ETAC range from 0.42 to 1.51 age-adjusted minimum alveolar concentration. A generalized estimating equation yielded a best predictive equation: BIS = 62.9-1.6 (if age younger than 60 yr) -1.6 (if female) -2.5 (if American Society of Anesthesiologists physical status more than 3) -2.6 (if deceased at 1 yr) -2.5 (if N2O was not used) -1.4 (if midazolam dose more than 2 mg) -1.3 (if opioid dose more than 50 morphine equivalents) -15.4 × age-adjusted minimum alveolar concentration. Although a population relationship between ETAC and BIS was apparent, interindividual variability in the strength and reliability of this relationship was large. Decreases in BIS with increasing ETAC were not reliably observed. Individual-patient linear regression yielded a median slope of -8 BIS/1 age-adjusted minimum alveolar concentration (interquartile range -30, 0) and a median correlation coefficient of -0.16 (interquartile range -0.031, -0.50). CONCLUSIONS: Independent of pharmacokinetic confounding, BIS frequently correlates poorly with ETAC, is often insensitive to clinically significant changes in ETAC, and is vulnerable to interindividual variability. BIS is therefore incapable of finely guiding volatile anesthetic titration during anesthetic maintenance.

Association of perioperative risk factors and cumulative duration of low bispectral index with intermediate-term mortality after cardiac surgery in the B-Unaware Trial.

BACKGROUND: Current data suggest that mortality after noncardiac surgery may be associated with persistent hypotension and the cumulative duration of low processed electroencephalogram-based bispectral index (BIS). This study assessed the relationships among cumulative duration of low BIS (BIS < 45), intermediate-term mortality, and anesthetic dose after cardiac surgery. METHODS: The authors studied 460 patients (mean age, 63.0 +/- 13.1 yr; 287 men) who underwent cardiac surgery between September 2005 and October 2006 at Washington University Medical Center, St Louis, Missouri. By using multivariable Cox regression analysis, perioperative factors were evaluated for their potential association with intermediate-term all-cause mortality. RESULTS: A total of 82 patients (17.8%) died during a median follow-up of 3 yr (interquartile range, 2.7-3.3 yr). Comparing patients who died with those who survived, there was no statistically significant difference in the relationship between end-tidal anesthetic gas concentrations during the anesthetic maintenance phase and the BIS. Cumulative duration of low BIS was independently associated with intermediate-term mortality. The 1.29 adjusted hazard ratio (95% CI, 1.12-1.49) for intermediate-term mortality with cumulative duration of low BIS translated into a 29% increased risk of death for every cumulative hour spent with a BIS less than 45. The final multivariable Cox regression model showed a good discriminative ability (c-index of 0.78). CONCLUSIONS: This study found an association between cumulative duration of low BIS and mortality in the setting of cardiac surgery. Notably, this association was independent of both volatile anesthetic concentration and duration of anesthesia, suggesting that intermediate-term mortality after cardiac surgery was not causally related to excessive anesthetic dose.

Cloning and distribution of myosin 3B in the mouse retina: differential distribution in cone outer segments.

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.

A randomized study in diabetic patients undergoing cardiac surgery comparing computer-guided glucose management with a standard sliding scale protocol.

OBJECTIVE: The aim of this study was to compare a standard insulin protocol with a computer-guided glucose management system to determine which method achieves tighter glucose control. DESIGN: A prospective, randomized trial. SETTING: A cardiothoracic intensive care unit (ICU) in a large academic medical center. PARTICIPANTS: Forty patients with diabetes mellitus who were scheduled for cardiac surgery. INTERVENTIONS: After induction of anesthesia and for the first 9 hours in the ICU, each subject received a standardized infusion of a 10% glucose solution at a rate of 1.0 mL/kg/h (ideal body weight). The subjects were then randomized to have their glucose controlled by either a paper-based insulin protocol or by a computer-guided glucose management system (CG). The desired range for blood glucose was set between 90 and 150 mg/dL. MEASUREMENTS AND MAIN RESULTS: There were no differences between groups in baseline characteristics. Patients in the CG group spent more time in the desired range during both the intraoperative phase (49% v 27%, p = 0.001) and the ICU phase (84% v 60%, p < 0.0001). There were no statistical differences between groups in the number of hypoglycemia episodes. CONCLUSIONS: The computer-guided glucose management system achieved tighter blood glucose control than a standard paper-based protocol in diabetic patients undergoing cardiac surgery. However, the low proportion of blood glucose recordings within the desired range in both groups during the intraoperative period reflects the challenges associated with achieving normoglycemia during cardiac surgery.

A new compartment at stereocilia tips defined by spatial and temporal patterns of myosin IIIa expression.

Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)-myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.

Retina-specific protein fascin 2 is an actin cross-linker associated with actin bundles in photoreceptor inner segments and calycal processes.

PURPOSE: Fascin 2 is a retinal-specific member of the fascin family of actin filament-bundling proteins. Fascin 2 mutation in humans results in autosomal dominant retinitis pigmentosa or macular degeneration. To investigate the role of fascin 2 in photoreceptor survival, the authors examined its localization in photoreceptors and characterized its interactions with actin filaments in vitro. METHODS: Fascin 2 localization was determined by immunohistochemistry and transgenic expression of green fluorescent protein (GFP)-tagged fascin 2 in Xenopus laevis rods. Fascin 2 actin-binding and actin-bundling activity were examined in sedimentation assays using bacterially expressed fusion proteins and polymerized actin. To assess the role of phosphorylation of a conserved serine (amino acid 39) in fascin 2 on subcellular localization and actin-binding, effects of serine mutants were also examined in transgenic Xenopus and in in vitro assays. RESULTS: Fascin 2 is localized to actin filament bundles of the photoreceptor inner segment and calycal processes. Like fascin 1, fascin 2 binds and cross-links actin filaments. Mutation of serine 39 to an aspartic acid reduced fascin 2 binding of actin filaments and abolished fascin 2 bundling of actin filaments in vitro but produced no detectable effect on GFP-tagged fascin 2 localization in transgenic Xenopus. CONCLUSIONS: These observations suggest that fascin 2 plays a role in the assembly or stabilization of inner segment and calycal process actin filament bundles in photoreceptors and that serine 39 phosphorylation reduces actin-binding and cross-linking activity and, thus, is likely to regulate the inner segment actin cytoskeleton.

Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain.

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.

Myo3A, one of two class III myosin genes expressed in vertebrate retina, is localized to the calycal processes of rod and cone photoreceptors and is expressed in the sacculus.

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

Characterization of peripherin/rds and rom-1 transport in rod photoreceptors of transgenic and knockout animals.

PURPOSE: Peripherin/rds and rom-1 have structural roles in morphogenesis and stabilization of the outer segment, but little is known about their transport and sorting to the rod outer segment. Peripherin/rds and rom-1 trafficking were studied in several knockout and transgenic animal models. METHODS: Rod outer segment formation and distribution of peripherin/rds and rom-1 were examined by immunohistochemistry, electron microscopy, and molecular biological methods in wild-type, rhodopsin-knockout, and peripherin/rds-knockout mice. C-terminally truncated peripherin/rds (Xper38)-GFP chimeric protein sorting was followed by immunofluorescence microscopy in transgenic Xenopus. RESULTS: In developing wild-type photoreceptors, peripherin/rds was detected exclusively in the distal tip of the connecting cilium in advance of outer segment formation. Rhodopsin-knockout mice failed to create normal rod outer segments and instead, elaborated membranous protrusions at the distal cilium tip. Peripherin/rds and rom-1 localized to this ciliary membrane in rhodopsinless photoreceptors. In transgenic Xenopus, a C-terminally truncated peripherin/rds-GFP fusion predominantly localized to its normal location within disc rims. In developing rds mice, rom-1 accumulated primarily in distal ciliary membranes. CONCLUSIONS: Peripherin/rds transport and localization are polarized to the site of outer segment morphogenesis before disc formation in developing photoreceptors. Peripherin/rds and rom-1 trafficking is maintained in rhodopsin-knockouts, suggesting that rim proteins and rhodopsin have separate transport pathways. The presence of truncated peripherin/rds-GFP in the outer segment supports previous evidence that peripherin/rds mice form homotetramers for outer segment targeting. The finding that rom-1 transports to the outer segment domain in rds mice suggests that rom-1 may possess its own sorting and transport signals.

Disruption of kinesin II function using a dominant negative-acting transgene in Xenopus laevis rods results in photoreceptor degeneration.

PURPOSE: Kinesin II is a motor protein that moves on microtubules and whose importance in ciliary and flagellar transport has been well documented. In the current study, the role of kinesin II in rod photoreceptors was examined by expressing a dominant negative-acting transgene that disrupts kinesin II function in Xenopus laevis rods of transgenic tadpoles. METHODS: A previously characterized dominant negative-acting kinesin II transgene tagged with enhanced green fluorescent protein (EGFP) driven by the Xenopus rod opsin promoter was used to make Xenopus transgenic tadpoles to disrupt kinesin II function specifically in rod photoreceptors. Transgenic tadpole retinas were examined to ascertain transgene expression pattern and morphologic phenotype. Rod-to-cone ratios were determined in experimental and control retinas. RESULTS: Visualized by its EGFP tag, the kinesin II transgene was expressed in rods in a mosaic pattern in the retina. Subcellular localization of transgenic kinesin II was similar to that of endogenous kinesin II subunit photoreceptor expression-that is, it was localized to the connecting cilium, inner segment, and synapse. However, in kinesin II transgene-expressing animals, fluorescence was transient. Ocular fluorescence was lost 6 days after its first detection. The disappearance of fluorescence was due to degeneration of rods expressing the transgene. Retinas of 7- to 9-day old kinesin II transgenic tadpoles had significantly fewer rods than did control retinas. CONCLUSIONS: The observation that rod degeneration is produced by expression of a dominant negative-acting kinesin II transgene in Xenopus rods is consistent with previous studies in mice, suggesting that kinesin II function is required for photoreceptor survival.

Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.

The occurrence of antibodies to heparin-platelet factor 4 in cardiac and thoracic surgical patients receiving desirudin or heparin for postoperative venous thrombosis prophylaxis.

INTRODUCTION: This randomized, exploratory study compared the incidence of heparin-dependent antibodies associated with subcutaneous (SC) desirudin or heparin given for deep-vein thrombosis prophylaxis following cardiac and thoracic surgery. MATERIALS AND METHODS: Adult patients scheduled for elective cardiac or thoracic surgery received desirudin 15 mg SC twice daily or unfractionated heparin 5000 units SC thrice daily. Duration of thrombosis prophylaxis was determined by the treating physician. Primary outcome measure was the incidence of new antibody formation directed against platelet factor 4 (PF4)/heparin complex. Secondary outcomes included bleeding and thrombotic complications. Blood was tested for anti-PF4/heparin antibodies at baseline, after surgery prior to study drug administration, postdrug day (PDD) 2, PDD 7, and at 1 month. Doppler studies were done before discharge. RESULTS: Of 120 patients, 61 received desirudin, 59 received heparin. New PF4/heparin antibodies occurred in 10.2% and 13.6% of desirudin- and heparin-treated patients, respectively. Among desirudin patients with no heparin exposure, none (0/36) developed PF4/heparin antibodies versus 17.1% with heparin exposure. Incidence of deep venous thrombosis was 4.9% and 3.4% in the desirudin and heparin groups, respectively. Two heparin-group patients developed pulmonary embolism. Two patients per group had bleeding events; no patients required re-exploration for bleeding complications. Median chest tube output was similar with desirudin (900 mL) and heparin (692 mL) as was blood transfusion requirements of more than 2 units (5/61, desirudin; 2/59 heparin). CONCLUSIONS: The incidence of thrombotic events was low in both groups. There were no safety concerns, and desirudin was not associated with anti-PF4/heparin antibodies.