The fatigue paradox: Team perceptions of physician fatigue.

OBJECTIVES: Ongoing calls to implement fatigue risk management in residency education assume a shared understanding of physician fatigue as a workplace hazard, yet we lack empirical evidence that all health care team members maintain this assumption. Thus, this study seeks to explore how health care team members understand the role of physician fatigue in an effort to inform the implementation of fatigue risk management in residency training and medical practice. METHODS: This study uses constructivist grounded theory to explore perceptions of workplace fatigue and its impact on clinical practice. We conducted individual semi-structured interviews with physicians, nurses and senior residents across four hospitals in 8 different specialties for a total of 40 participants. Constant comparative analysis guided data analysis and led to the final grounded theory. RESULTS: While participants outlined multiple problematic manifestations of physician fatigue on clinical performance, they were reluctant to acknowledge any negative impact of fatigue on patient care. We refer to these contradictions as the fatigue paradox. Four key themes sustain the fatigue paradox: the indefatigable physician, blind spots, faith in safety nets and the minimisation of fatigue-related events. CONCLUSIONS: This study suggests that health care team members do not universally feel that physician fatigue is problematic for patient care, despite providing multiple examples to the contrary. This paradoxical understanding of fatigue likely exists because the system relies on fatigued physicians, particularly trainees, and provides few mechanisms to critically examine fatigue. Successful implementation of fatigue risk management in residency training may prove elusive if clinical supervisors are skeptical of the potentially negative impact of workplace fatigue.

Engagement: what is it good for? The role of learner engagement in healthcare simulation contexts.

Learner engagement matters, particularly in simulation-based education. Indeed, it has been argued that instructional design only matters in the service of engaging learners in a simulation encounter. Yet despite its purported importance, our understanding of what engagement is, how to define it, how to measure it, and how to assess it is limited. The current study presents the results of a critical narrative review of literature outside of health sciences education, with the aim of summarizing existing knowledge in these areas and providing a research agenda to guide future scholarship on learner engagement in healthcare simulation. Building on this existing knowledge base, we provide a working definition for engagement and provide an outline for future research programs that will help us better understand how health professions' learners experience engagement in the simulated setting. With this in hand, additional research questions can be addressed including: how do simulation instructional design features (fidelity, range of task difficulty, feedback, etc.) affect engagement? What is the relationship between engagement and simulation learning outcomes? And how is engagement related to or distinct from related variables like cognitive load, motivation, and self-regulated learning?

Association between known or strongly suspected malignant hyperthermia susceptibility and postoperative outcomes: an observational population-based study.

PURPOSE: Whether current standards of care management for malignant hyperthermia (MH)-susceptible patients result in acceptable postoperative clinical outcomes at a population level is not known. Our objective was to determine if patients with susceptibility to MH experienced similar outcomes as patients without MH susceptibility after surgery under general anesthesia. METHODS: This was a retrospective, population-based cohort study from 1 April 2009 until 31 March 2016 in the Canadian province of Ontario. Participants were adults who underwent common in- or outpatient surgeries under general anesthesia. The exposure studied was either known or strongly suspected MH susceptibility as determined by usage of a specific physician billing code. The primary outcome was a composite of all-cause death, hospital readmission, or major postoperative complications, all within 30 postoperative days. Separate analyses were employed, based on whether a patient had in- or outpatient surgery. Inverse probability of exposure weighting based on the propensity score was used to estimate adjusted exposure effects. RESULTS: The cohort included 957,876 patients (583,254 in- and 374,622 outpatients). There were 2,900 (0.3%) patients with a known or strong suspicion of MH susceptibility. For inpatients, the primary outcome occurred in 146,192 (25.1%) of the non-MH-susceptible group and in 337 (20.1%) of the MH-susceptible group (unadjusted risk difference [RD], -5.0%; 95% confidence interval [CI], -6.9 to -3.1%; P < 0.001). In outpatients, the primary outcome occurred in 9,146 (2.4%) of the non-MH-susceptible group and in 32 (2.6%) of the MH-susceptible group (RD, 0.2%; 95% CI, -0.7 to 1.1%; P = 0.72). After adjustment, MH susceptibility was not associated with the primary outcome in either the inpatients (adjusted risk difference [aRD], 1.2%; 95% CI, -1.3 to 3.6%; P = 0.35) or outpatients (aRD, -0.1%; 95% CI -1.0 to 0.9%; P = 0.90). CONCLUSIONS: Among adults in Ontario who underwent common surgeries under general anesthesia from 2009 to 2016, known or strongly suspected MH was not associated with a higher risk of adverse postoperative outcomes. These findings support the current standard of care management for MH-susceptible patients.

RéSUMé: OBJECTIF: Nous ignorons si les normes actuelles de gestion des soins de patients susceptibles d'hyperthermie maligne (HM) aboutissent à des résultats cliniques postopératoires acceptables à l'échelle d'une population. Notre objectif a été de déterminer si des patients présentant une susceptibilité à l'HM présentaient une évolution comparable à celle des patients non connus susceptibles après chirurgie sous anesthésie générale. MéTHODES: Il s'agissait d'une étude de cohorte rétrospective, basée sur une population de la province canadienne de l'Ontario allant du 1er avril 2009 au 31 mars 2016. Les participants étaient des adultes, hospitalisés ou ambulatoires, ayant subi des interventions sous anesthésie générale. L'exposition étudiée était une susceptibilité à l'HM connue ou fortement suspectée, déterminée par l'utilisation d'un code de facturation spécifique des médecins. Le critère d'évaluation principal était un critère composite incluant les décès toutes causes confondues, les réadmissions hospitalières ou les complications postopératoires majeures qui étaient survenus dans un délai de 30 jours postopératoires. Des analyses séparées ont été utilisées, selon que les patients avaient été hospitalisés ou opérés en chirurgie d'un jour. La probabilité inverse de la pondération de l'exposition basée sur le score pour la propension a servi à estimer les effets ajustés de l'exposition. RéSULTATS: La cohorte a inclus 957 876 patients (583 254 patients hospitalisés et 374 622 patients ambulatoires). Parmi eux, 2 900 patients (0,3 %) avaient une susceptibilité à l'HM connue ou fortement suspectée. Pour les patients hospitalisés, le critère d'évaluation principal est survenu chez 146 192 (25,1 %) des patients du groupe non susceptible d'HM et chez 337 (20,1 %) patients du groupe susceptible d'HM (différence de risques [DR] non ajustée : −5,0 %; intervalle de confiance [IC] à 95 % : −6,9 % à −3,1 %; P < 0,001). Pour les patients ambulatoires, le critère d'évaluation principal est survenu chez 9 146 (2,4 %) des patients du groupe non susceptible d'HM et chez 32 (2,6 %) patients du groupe susceptible d'HM (différence de risques [DR] non ajustée : 0,2 %; IC à 95 % : −0,7 % à 1,1 %; P = 0,72). Après ajustement, la susceptibilité à l'HM ne s'est pas avérée associée au critère d'évaluation principal dans le groupe de patients hospitalisés (différence de risques ajustée [DRa], 1,2 %; IC à 95 % : −1,3 % à 3,6 %; P = 0,35) ou dans le groupe de patients ambulatoires (DRa : −0,1 %; IC à 95 % : −1,0 % à 0,9 %; P = 0,90). CONCLUSIONS: Parmi les adultes de la province de l'Ontario ayant subi des interventions chirurgicales usuelles sous anesthésie générale entre 2009 et 2016, l'HM connue ou fortement suspectée n'a pas été associée à un plus grand risque d'évolution postopératoire défavorable. Ces constatations sont en faveur du maintien des normes des soins actuels pour la gestion des patients susceptibles d'HM.

Association Between Handover of Anesthesia Care and Adverse Postoperative Outcomes Among Patients Undergoing Major Surgery.

Importance: Handing over the care of a patient from one anesthesiologist to another occurs during some surgeries and might increase the risk of adverse outcomes. Objective: To assess whether complete handover of intraoperative anesthesia care is associated with higher likelihood of mortality or major complications compared with no handover of care. Design, Setting, and Participants: A retrospective population-based cohort study (April 1, 2009-March 31, 2015 set in the Canadian province of Ontario) of adult patients aged 18 years and older undergoing major surgeries expected to last at least 2 hours and requiring a hospital stay of at least 1 night. Exposure: Complete intraoperative handover of anesthesia care from one physician anesthesiologist to another compared with no handover of anesthesia care. Main Outcomes and Measures: The primary outcome was a composite of all-cause death, hospital readmission, or major postoperative complications, all within 30 postoperative days. Secondary outcomes were the individual components of the primary outcome. Inverse probability of exposure weighting based on the propensity score was used to estimate adjusted exposure effects. Results: Of the 313 066 patients in the cohort, 56% were women; the mean (SD) age was 60 (16) years; 49% of surgeries were performed in academic centers; 72% of surgeries were elective; and the median duration of surgery was 182 minutes (interquartile [IQR] range, 124-255). A total of 5941 (1.9%) patients underwent surgery with complete handover of anesthesia care. The percentage of patients undergoing surgery with a handover of anesthesiology care progressively increased each year of the study, reaching 2.9% in 2015. In the unweighted sample, the primary outcome occurred in 44% of the complete handover group compared with 29% of the no handover group. After adjustment, complete handovers were statistically significantly associated with an increased risk of the primary outcome (adjusted risk difference [aRD], 6.8% [95% CI, 4.5% to 9.1%]; P < .001), all-cause death (aRD, 1.2% [95% CI, 0.5% to 2%]; P = .002), and major complications (aRD, 5.8% [95% CI, 3.6% to 7.9%]; P < .001), but not with hospital readmission within 30 days of surgery (aRD, 1.2% [95% CI, -0.3% to 2.7%]; P = .11). Conclusions and Relevance: Among adults undergoing major surgery, complete handover of intraoperative anesthesia care compared with no handover was associated with a higher risk of adverse postoperative outcomes. These findings may support limiting complete anesthesia handovers.

Interaction of HLA-DR and CD74 at the cell surface of antigen-presenting cells by single particle image analysis.

Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.

Patient barrier acceptance during airway management among anesthesiologists: a simulation pilot study.

BACKGROUND: Protection of healthcare providers (HCP) has been a serious challenge in the management of patients during the coronavirus 2019 (COVID-19) pandemic. Additional physical barriers have been created to enhance personal protective equipment (PPE). In this study, user acceptability of two novel barriers was evaluated and the performance of airway management using PPE alone versus PPE plus the additional barrier were compared. METHODS: An open-label, double-armed simulation pilot study was conducted. Each participant performed bag-mask ventilation and endotracheal intubation using a GlideScope in two scenarios: 1) PPE donned, followed by 2) PPE donned plus the addition of either the isolation chamber (IC) or aerosol box (AB). Endotracheal intubation using videolaryngoscopy was timed. Participants completed pre- and post-simulation questionnaires. RESULTS: Twenty-nine participants from the Department of Anesthesia were included in the study. Pre- and post-simulation questionnaire responses supported the acceptance of additional barriers. There was no significant difference in intubating times across all groups (PPE vs. IC 95% CI, 26.3, 35.1; PPE vs. AB 95% CI, 25.9, 35.5; IC vs. AB 95% CI, 23.6, 39.1). Comparison of post-simulation questionnaire responses between IC and AB showed no significant difference. Participants did not find the additional barriers negatively affected communication, visualization, or maneuverability. CONCLUSIONS: Overall, the IC and AB were comparable, and there was no negative impact on performance under testing conditions. Our study suggests the positive acceptance of additional patient protection barriers by anesthesia providers during airway management.

Cell bio-imaging reveals co-expression of HLA-G and HLA-E in human preimplantation embryos.

The non-classical major histocompatibility complex (MHC) class Ib antigens, termed HLA-G and HLA-E, have been associated with fetal maternal tolerance. The role of HLA-G in the preimplantation embryo remains unclear although immunoprotection, adhesion and cell signalling mechanisms have been suggested. Unlike HLA-G, HLA-E protein expression has not been previously studied in preimplantation embryos. Embryos and model trophoblast cell lines JEG-3 and BeWo were labelled with the HLA-G- and HLA-E-specific monoclonal antibodies MEMG9 and MEME07. Flow cytometry, confocal microscopy and single particle fluorescence imaging techniques were employed to investigate the spatial and temporal expression of these receptors. Lipid raft analysis and adhesion assays were performed to investigate the role of these receptors in cell membrane domains and in promoting adhesion by cell-to-cell contact. HLA-E and HLA-G were co-localized in the trophectoderm of day 6 blastocysts. Analysis on trophoblast cell lines revealed that 37% of HLA-G and 41% of HLA-E receptors were co-localized as tetramers or higher order homodimer clusters. HLA-G receptors did not appear to play a role in either cell adhesion or immunoreceptor signalling via lipid raft platforms on the cell membrane. A possible role of HLA-G and HLA-E in implantation via immunoregulation or modulation of uterine maternal leukocytes is discussed.

A comparison of glidescope videolaryngoscopy to direct laryngoscopy for nasotracheal intubation.

BACKGROUND: In this study, we compared the effectiveness of direct laryngoscopy (DL) and the GlideScope videolaryngoscope (GVL) for nasotracheal intubation, as judged by the time to intubation (TTI-the primary outcome) and the ease of intubation. METHODS: Seventy patients requiring nasotracheal intubation for elective surgery were randomly allocated to intubation with the GVL or DL. TTI was assessed by a blinded observer. Operators were blinded until the start of laryngoscopy. A Visual Analog Scale assessed the ease of intubation. The number of intubation attempts, number of failures, glottic grades, amount of bleeding, usage of Magill forceps, and the severity of postoperative sore throat were recorded. RESULTS: The median TTI was 23.2 s faster with the GVL (43.5 s, interquartile range [IQR]: 39.8-67.3) than with DL (66.7 s, IQR: 53.8-89.9), P = 0.0023. Nasotracheal intubation was easier with the GVL than with DL (Visual Analog Scale 10 mm, IQR: 5.5-18, vs 20 mm, IQR: 10-32, P = 0.0041). The incidence of postoperative moderate or severe sore throat was significantly reduced in the GVL group (9% vs 34%, P = 0.018). Glottic exposure was significantly better with the GVL. Magill forceps were not used in the GVL group, but were used 49% of the time in the DL group, P < 0.0001. The incidence and severity of bleeding were similar between groups. CONCLUSIONS: Compared with DL, the GVL has superior performance characteristics when used for nasotracheal intubation and demonstrates an important reduction of postoperative sore throat. The GVL has a clear role in routine nasotracheal intubation.

Stochastic modeling of protein motions within cell membranes.

A simple model in which immobilizing events are imposed onto otherwise free Brownian diffusion [R. Metzler and J. Klafter, Phys. Rep. 339, 1 (2000) and a recent adaptation due to S. Khan and A. M. Reynolds, Physica A 350, 183 (2005)] is shown to encapsulate the peculiar transport characteristics of individual cell receptors within plasma membranes observed in single-particle tracking (SPT) experiments. These characteristics include the occurrence of normal diffusion; non-Gaussian subdiffusion; confined diffusion; a superdiffusive mode of transport that is not due to flow of the membrane or molecular motor attachment; and the occurrence of transitions between these transport modes. Model predictions are shown to be in close agreement with a reanalysis of existing SPT data.

Interaction of HLA-DR with actin microfilaments.

Capping of HLA-DR on the surface of a human lymphoblastoid cell line (RAJI) and a transfectant human fibroblast cell line (M1DR1) was studied by confocal microscopy. Capping was induced at 22 degrees C after treating cells with an HLA-DR specific monoclonal antibody, L243, followed by a secondary antibody conjugated with FITC. Cytoskeletal actin filaments (F-actin) accumulated under the caps were detected by rhodamine-phalloidin fluorescence. Two processes appear to take place: in the round lymphoblastoid cells, actin, initially distributed uniformly at the cell periphery, redistributes and becomes concentrated underneath HLA-DR patches or caps. In the non-round, substrate-attached fibroblasts, actin was organized in tightly packed filaments along the plasma membrane. It was observed that crosslinked HLA-DR receptors were associated with these filaments and were dragged toward the perinuclear area of the cells, where they coalesce to form a cap. The cytoskeleton-disrupting drugs that inhibit actin polymerisation were used to investigate the mechanism of capping of HLA-DR molecules. Sodium nitroprusside, a nitric oxide releasing agent, cytochalasin D both inhibited the percentage of capping in a dose-dependent manner. These data suggest that on antigen presenting cells, such as B cells and fibroblasts, actin microfilaments acts as a regulator of the movement and capping of HLA-DR receptors.

Digital fluorescence analysis of trafficking of single endosomes containing low-density lipoprotein in adrenocortical cells: facilitation of centripetal motion by adrenocorticotropic hormone.

Imaging of trafficking of endosomes containing low-density lipoprotein (LDL) is useful to analyze cholesterol transport in adrenocortical cells. At 60 min after the application of fluorescently labeled LDL to adrenocortical cells, individual endosomes containing LDL were demonstrated to undergo frequent switching between forward and reverse movement and immobility. The population of moving endosomes (>or=0.065 microm/s) was approximately 75% in control cells. The remaining endosomes were either slowly moving or temporarily immobile. At 3h after the LDL addition, endosomes were concentrated around the circumference of the cell nuclei. The endosome movement was inhibited by nocodazole, implying that endosomes undergo movement along microtubule networks. Anti-dynein antibodies inhibited the motion of endosomes towards the nucleus, and anti-kinesin antibodies inhibited peripherally directed motion. These results imply that both dynein-like and kinesin-like motor proteins bind to the same endosome, resulting in saltatory movements with centripetal or peripherally directed direction, depending on which motor binds to microtubules. Though the dynein and kinesin motors drive the endosomes very rapidly (microm/s), frequent saltatory motions of single endosomes may induce the very slow net centripetal motion (microm/h).The application of adrenocorticotropic hormone (ACTH) resulted in a facilitation of the centripetal motion of endosomes, resulting in the establishment of the concentration of endosomes around cell nuclei within 1 h.

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression.

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.

Effect of stylet angulation and endotracheal tube camber on time to intubation with the GlideScope.

PURPOSE: The GlideScope videolaryngoscope usually provides excellent glottic visualization, but directing an endotracheal tube (ETT) through the vocal cords is sometimes difficult. The goal of the study was to determine which of two ETT angles (60 degrees vs 90 degrees ) and cambers (forward vs reverse) was better, as determined by time to intubation (TTI). METHODS: Two hundred patients requiring orotracheal intubation for elective surgery were randomly allocated to one of four groups: A) 90 degrees angle, forward camber; B) 90 degrees angle, reverse camber; C) 60 degrees angle, forward camber; D) 60 degrees angle, reverse camber. Time to intubation was assessed by a blinded observer. Operators were blinded until the point of intubation. A visual analogue scale (VAS) assessed the ease of intubation. The number of intubation attempts, number of failures, glottic grades, and use of external laryngeal manipulation were recorded. RESULTS: The angle of the ETT had an impact on TTI but camber did not. The 90 degrees angle demonstrated a 13% faster TTI than the 60 degrees angle (47.1 +/- 21.2 sec vs 54.4 +/- 28.2 sec, P=0.042), and it resulted in easier intubation (VAS 16.4 +/- 14.2 mm vs 27.3 +/- 23.5 mm, P=0.0001). The overall incidence of a grade 1 or 2 Cormack-Lehane glottic view was 99%. CONCLUSIONS: In a heterogeneous group of operators and patients intubated with the GlideScope, a 90 degrees ETT angle provided the best result and should be the initial configuration. The camber of the ETT does not affect the time to intubation.

The GlideScope-specific rigid stylet and standard malleable stylet are equally effective for GlideScope use.

PURPOSE: The GlideScope videolaryngoscope usually provides excellent glottic visualization, but directing an endotracheal tube (ETT) through the vocal cords can be challenging. The goal of the study was to compare the dedicated GlideScope-specific rigid stylet to the standard malleable stylet, assessed by time to intubation (TTI). METHODS: Eighty patients requiring orotracheal intubation for elective surgery were randomly allocated to either the GlideScope rigid stylet (GRS) or a standard malleable stylet to facilitate intubation using the GlideScope. Time to intubation was recorded by blinded assessors; operators were blinded until after laryngoscopy. The operator assessed the ease of intubation using a visual analogue scale (VAS). The number of intubation attempts, number of failures, glottic grades, and use of external laryngeal manipulation were documented. RESULTS: The median TTI was 42.7 sec (inter-quartile range (IQR) 38.9-56.7) for the GRS group compared to 39.9 sec (IQR 34.1-48.2) for the control group (P=0.07). The median VAS score for ease of intubation was 20 (IQR 12.0-33.0) for the GRS group compared to 18 (IQR 9.5-29.5) for the control group (P=0.21). There was no significant difference in TTI or VAS between stylets. The overall incidence of a Cormack-Lehane grade I or II glottic view was 98%. CONCLUSIONS: In a group of experienced operators using the GlideScope, the dedicated GRS and the standard malleable ETT stylet are equally effective in facilitating endotracheal intubation.

Brachial plexus anesthesia compared to general anesthesia when a block room is available.

PURPOSE: Regional anesthesia is often felt to be beneficial to patient care but detrimental to operating room (OR) efficiency. In this report we compare how a block room (BR) affects OR time (ORT) utilization for brachial plexus anesthesia (BPA) in a busy upper limb practice. We also compare how anesthetic technique, BPA or general anesthesia (GA), impacts on the time to recovery and discharge in patients having outpatient upper limb surgery. METHODS: With the Ethics Committee's approval, a prospective study using hospital databases was undertaken. All patients presenting for surgery on the upper limb between November 1999 and April 2000 were eligible for analysis. A comparison was made of the various time intervals that comprise a patient's hospital stay for either GA or BPA. Demographic data (ASA, age, outpatient status), and location of BPA were analyzed. RESULTS: Use of the BR for BPA significantly reduced the pre-procedure anesthesia ORT when compared to BPA done in the OR (11.4 vs 32.9 min, P < 0.05; GA pre-procedure time was 17.8 min). In the ambulatory patient, BPA alone reduced post procedure anesthesia ORT, postanesthetic care unit, surgical day care unit, and total hospital times when compared to those receiving GA. On average those receiving a BPA spent 1.5 hr less in hospital (P < 0.01). Additionally, fewer admissions (2.4 vs 5.4%) occurred in the BPA group. CONCLUSION: The use of a BR reduces the anesthesia ORT associated with BPA. Secondly, BPA improves the recovery time phase of outpatients undergoing surgery on the upper limb.

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging.

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.

Id proteins negatively regulate basic helix-loop-helix transcription factor function by disrupting subnuclear compartmentalization.

Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect.

Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A.

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.

Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe.

The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.