Transnasal Humidified Rapid Insufflation Ventilatory Exchange in Endoscopic Esophageal Surgery.

OBJECTIVES: Transnasal humidified rapid insufflation ventilatory exchange (THRIVE) describes apneic oxygenation using humidified high flow nasal-cannula oxygen. Although it has been described as a sole mode of oxygenation in endoscopic laryngotracheal surgery, its use in endoscopic esophageal surgery under general anesthesia with neuromuscular paralysis has not previously been described. The objective of this study is to assess the safety and efficacy of THRIVE in esophagology. METHODS: We conducted a retrospective review of adult patients undergoing esophageal procedures under general anesthesia who were oxygenated using THRIVE at two academic institutions. Demographic, clinical, and anesthesiologic data were collected and analyzed. RESULTS: 14 cases performed from March 2021 to March 2022 met inclusion criteria. 13/14 (92.9%) of patients were able to maintain oxygenation throughout the entirety of their procedure. The mean apneic time was 17.9 minutes with a maximum of 32 minutes. One patient required "rescue" intubation due to failure to maintain oxygenation. Excluding the sole THRIVE failure, the median SpO2 at the conclusion of surgery was 99% (range 94-100%). A linear regression model yielded an increase in EtCO2 of 0.95 mmHg/min or 0.127 kPa/min. SpO2 was negatively associated with both tobacco pack-year smoking history (R2 = 0.343, P = .014) and BMI (R2 = 0.238, P = .038). CONCLUSION: THRIVE is a feasible, safe, and efficacious means of apneic oxygenation for patients undergoing esophageal endoscopic surgery under general anesthesia with neuromuscular paralysis, which may be particularly beneficial in patients with airway stenosis, as post-intubation changes can have severe clinical implications for this patient population. Obese patients and tobacco smokers may be at increased risk of oxygen desaturation when using THRIVE.

Diagnosing Laryngopharyngeal Reflux: A Comparison between 24-hour pH-Impedance Testing and Pharyngeal Probe (Restech) Testing, with Introduction of the Sataloff Score.

OBJECTIVE: The purpose of this study was to compare the diagnostic utility of pH monitoring using 24-hour esophageal pH-Impedance (HEMII-pH) testing versus pharyngeal pH (Restech) testing (Respiratory Technology Corporation, Houston, Texas) for diagnosing laryngopharyngeal reflux (LPR). METHODS: Retrospectively, patients were reviewed who had completed a Reflux Symptom Index (RSI) survey and stroboscopy within 60 days before or after undergoing simultaneous esophageal pH-Impedance monitoring and Restech testing. Reflux Finding Score (RFS) was determined by 4 blinded observers. 80.45% of patients were on anti-reflux medications at the time of study and had incomplete response to treatment for reflux. Improvement on reflux treatment was determined by evaluating presenting pre-pH monitoring RFS, post treatment RFS, and improvement of symptoms. Pearson correlation coefficients were calculated to assess relationships among RSI, RFS, and test results from HEMII-pH and Restech tests. RESULTS: Eighty-seven patients were included in the analysis. The inter-rater reliability of the RFS determination was 74.57%, and the intra-rater reliability was 67.00%. Subjects who had a positive RYAN Score had a significant correlation with RFS (r of 0.222 and p-value of 0.0492). There was no correlation between RFS and number or percent time of reflux events, longest event, total number of events, or percent of time at alkaline pH for either HEMII-PH or Restech test. RSI correlated better with HEMII-pH test than with Restech for percent time spent in both upright (r of 0.226 and p-value of 0.029) and supine position (r of 0.261 and a p-value of 0.032). Restech correlated better with total patient symptom Scores including cough, heartburn, burping, and throat clearing, with a r of 0.242 and a p-value of 0.048. Restech detected more percent time in reflux for total reflux, supine reflux, and upright reflux (p-value less than 0.0001). Restech also detected longer event times than Impedance (p-value of less than 0.0001). When diagnosis of LPR is based on the definition of CRC, the Sataloff Score test had 70.45% sensitivity and 80.95% specificity. The RYAN Score had a sensitivity of 72%, and a specificity of 56.45%, and the Wu Score had a sensitivity of 62.16%, and specificity of 54.05%. When the Sataloff and Wu Score were used together, the sensitivity was 71.45%, specificity 100%, positive predictive value of 100%, and a negative predictive value of 59.46%. CONCLUSION: The amount of time of reflux events correlates with symptoms better than the number of events. The HEMII-pH test was able to detect more events of pH<4 than Restech, possibly because there might have been more acid events below than above the upper esophageal sphincter, while Restech detected more total events. Restech recorded longer event times than HEMII-pH test. Since length of time correlates with RFS (probably reflecting laryngeal inflammation), and since laryngeal clearance of acid is more similar to pharyngeal than esophageal clearance, this finding might prove valuable clinically. The Sataloff Score has a sensitivity of 70.45%, and a specificity of 80.95% and appears useful clinically to detect mild to moderate that is missed by the RYAN Score. A combination of Sataloff Score and Wu Score may be clinically valuable to identify LPR with an increased sensitivity of 71.45% and increased specificity of 100%. The Wu Score is not yet available for the general clinical use, but the Sataloff Score is.

The Validity and Reliability of the Reflux Finding Score.

Laryngopharyngeal reflux (LPR) disease is common. The incidence of newly diagnosed cases has increased substantially due to awareness and development of new diagnostic measurements. The reflux finding score (RFS) and reflux symptom index (RSI) are believed to be useful in the assessment process, including after the initiation of therapy. However, many authors have suggested concerns about the reliability and validity of the RFS. OBJECTIVE: To evaluate the validity and reliability of the RFS. METHODS: Ninety-two patients diagnosed with LPR who had undergone 24-hour pH-Impedance tests were included. All patients underwent stroboscopy and 24-Hour pH-Impedance monitoring within thirty days. Fifty-nine patients filled out a RSI prior to stroboscopic exam. The RFS was determined by four blinded observers: one otolaryngology resident, two laryngology fellows, and one laryngologist. Stroboscopic images were reviewed again one year later to assess intrarater reliability. RFS and RSI were correlated with 24-hour pH Impedance testing. RESULTS: The Kappa value between reviewers was 0.479. The percent agreement of the four observers for total RFS was 74.04%.The percent agreement between reviewers for subglottic edema was 78.77%; for ventricular obliteration was 65.55%; for erythema/hyperemia was 69.62%, for vocal fold edema was 68.32%; for diffuse laryngeal edema was 66.86%, for posterior commissure hypertrophy was 73.54%; for granuloma/granulation was 96.80%; for thick endolaryngeal mucus was 72.81%. The intrarater reliability of the four observers for total RFS was 67.5% with an intrarater reliability range of 50%-90%. The intrarater reliability for subglottic edema was 85% with a range of 70%-100%; for ventricular obliteration was 77.50% with a range of 70%-90%; for erythema/hyperemia was 65.00% with a range of 50%-90%; for vocal fold edema was 52.50% with a range of 30%-70%; for diffuse laryngeal edema was 62.50% with a range of 20%-80%; for posterior commissure hypertrophy was 52.50% with a range of 10%-80%; for granuloma/granulation was 100%; for thick endolaryngeal mucus was 55.00% with a range of 10%-90%. There was no correlation between RFS and any parameter of the 24-Hr pH-Impedance Test. RSI had a significant correlation with number of upright events (r value of 0.271, R2 of 0.0733 and P-value of 0.037), total symptoms experienced (r value of 0.0.267, R2 of 0.0715 and P-value of 0.041), and symptom correlation score (r value of -0.297, R2 of 0.0884 and P-value of 0.022). CONCLUSION: Many authors have expressed concerns about the reliability and validity of the RFS. In our study we found a fair/substantial interrater reliability, and a modest intra-rater reliability. We found no correlation between the RFS and 24-Hr pH Impedance testing. This study suggests that the concerns about the validity and reliability of the RFS may be warranted. This widely used clinical score should be interpreted with caution and further research and refinement should be considered.

Immediate and Short-term Effects of Straw Phonation in Air or Water on Vocal Fold Vibration and Supraglottic Activity of Adult Patients with Voice Disorders Visualized with Strobovideolaryngoscopy: A Pilot Study.

Purpose The first purpose of this study was to investigate and compare the short-term effects after a semi-occluded vocal tract (SOVT) therapy session consisting of straw phonation (SP) in air or water on vocal fold vibration and supraglottic activity of adult patients with voice disorders, visualized with strobovideolaryngoscopy (SVL). The second purpose of this study was to investigate and compare immediate changes in the patients' vocal fold vibration and supraglottic activity during SP in air or water, visualized with SVL. Methods Twelve adult patients with voice disorders (eight women and four men, mean age 52 years) were assigned randomly to one of two study groups: SP in air or SP in water. Immediately before and after a therapy session of 15 min, participants underwent a rigid SVL to determine the short-term effects of the SP session. At the posttherapy examination, flexible SVL while performing SP was added to determine the effects occurring during SP. The visual-perceptual ratings were performed blindly and in random order by three laryngologists, using the Voice-Vibratory Assessment with Laryngeal Imaging rating form for stroboscopy. ResultsShort-term effects after SP: After the SP-in-air session, the supraglottic mediolateral compression decreased significantly. The SP-in-water session led to significantly increased left vibrational amplitude. Immediate effects during SP: During SP in air, a significantly increased left amplitude and mucosal wave, and significantly decreased mediolateral supraglottic activity, were found. SP in water tended to decrease the vibrational amplitude during performance of the task. A trend toward higher anteroposterior supraglottic compression was observed during both SP in air and water, being more prominent in the latter. Conclusion SP in air led to less false vocal fold adduction and consequently less hyperfunction. The small increment in anteroposterior supraglottic activity during SP in air and water might be related to epilarynx narrowing, an economic phenomenon associated with SOVT exercises. The effects on vibrational amplitude were rather ambiguous. The small reduction in amplitude during SP in water is expected to diminish vocal fold impact stress and therefore creates an ideal basis for voice therapy. The increment in amplitude and mucosal wave during SP in air might indicate insufficient supraglottic pressure to obtain the favorable effects of semi-occlusion. Whether or not the rise in amplitude after the SP-in-water session is due to voice efficiency or voice fatigue remains unknown. Future larger-scale investigation in subgroups of voice patients is needed to explore these hypotheses.

Takedown of Painful Ankle Arthrodesis to Total Ankle Arthroplasty: A Case Series of 77 Patients.

Treatment of painful or malaligned ankle arthrodesis can present as a challenging issue. Several published studies have demonstrated that takedown of a painful ankle arthrodesis to total ankle arthroplasty can assist in restoring some sagittal plane motion and improving functional scores. The goal of this study was to contribute to the limited body of literature with the largest cohort and longest follow-up to date. A retrospective analysis was performed on patient and surgical characteristics of those who underwent a conversion of a painful ankle arthrodesis to a total ankle arthroplasty by 1 of 3 experienced total ankle arthroplasty surgeons from February 2003 to December 2016 with ≥2 years of follow up. Seventy-seven subjects were included for evaluation, with an implant retention rate of 88% (68 of 77) and mean follow-up of 8.3 years (range 2.6 to 15.8). Of the 11 (14%) failures (defined as retrieval or exchange of metallic components), 8 (10%) were revised to a total ankle replacement, 2 (2%) underwent revision arthrodesis, and 1 (1%) elected for below-the-knee amputation. The mean time since the primary arthrodesis was 8.6 years (range 1 to 44), and the longer time interval between primary arthrodesis to takedown total ankle arthroplasty did not correlate with poorer outcome scores or increased risk of failure. The mean American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot, Buechel-Pappas, and visual analog pain scale scores improved from preoperative values, with less satisfaction noted in those who needed revision surgery. The conversion of a painful ankle arthrodesis to a total ankle implant is a viable option to obtain range of motion and improved patient satisfaction scores similar to primary total ankle replacement.

Laboratory Evaluation of Spasmodic Dysphonia.

OBJECTIVES: To evaluate the utility of comprehensive laboratory evaluation in patients with spasmodic dysphonia (SD). STUDY DESIGN: Retrospective chart review. METHODS: A review of the medical records of 40 patients diagnosed with spasmodic dysphonia from 2009-2018 was preformed to evaluate abnormal test results that were significant when compared with abnormal results of the general population and for any other clinically relevant pathology. RESULTS: Erythrocyte sedimentation rate, ceruloplasmin levels, and anti-AChR were found to be elevated at levels considered statistically significant (p <0.05). Furthermore, we found levels of cholesterol, thyroid-stimulating hormone (TSH), T3, fasting blood glucose, creatine kinase, immunoglobulin, antinuclear antibody (ANA), and alpha-fetoprotein (AFP) levels to be abnormal at a greater rate in our population, but these were not statistically significant. Workup revealed several underlying conditions including thyroid neoplasms, hypothyroidism, and laryngopharyngeal reflux. Additionally, brain MRI revealed age-related ischemic pathology in an elevated number of patients, but with no obvious clinical sequalae. CONCLUSION: There is an association between serological values and spasmodic dysphonia that can aid in diagnosing pathology, as well as establishing a directed workup. Additionally, our study shows the utility of comprehensive evaluation in identifying undetected disease.

Utility of Audiometry in the Evaluation of Patients Presenting with Dysphonia.

OBJECTIVES: Hearing loss has been implicated in dysphonia secondary to voice misuse, although the data supporting this claim are scant. Determining the prevalence of hearing loss in patients with dysphonia and correlating it with self-perception of vocal handicap may help clarify the value of audiometry in evaluation of patients with dysphonia. METHODS: This is a retrospective chart review of all new voice patients (n = 405) presenting with dysphonia to the primary investigator between 2015 and 2018. Each new patient routinely undergoes audiometric and voice objective analyses. Main outcomes measured include prevalence, severity of hearing loss, and voice handicap index-10 (VHI-10). RESULTS: Of the 405 subjects reviewed, mean age was 49.0 years (SD = 17.4). 60.7% of subjects were female and 39.3% male. Patients with hearing loss defined as >25 dB in worse ear with pure tone average (PTA) thresholds at 0.5, 1, 2, and 3 kHz (PTA-S) accounted for 18% of the total cohort. The prevalence of previously undiagnosed hearing loss in this cohort was 13.1% (53 of 405 subjects). Of these subjects, 62.3% (33 subjects) reported no perception of hearing loss while 37.7% (20 subjects) suspected they had some hearing loss, yet never sought evaluation. Only increased PTA-S, speech discrimination, Reflux Symptom Index, and female gender demonstrated a significant relationship with VHI-10 when analyzed with multivariate linear regression analysis. CONCLUSIONS: The prevalence of hearing loss in patients presenting with dysphonia in this cohort is similar to normative population data. This study has also demonstrated that the majority of these patients did not perceive any hearing loss. The reasons behind this may be a result of or associated with the patients' dysphonia. Furthermore, clinicians should consider performing audiometric evaluation in patients with abnormal VHI-10 scores in the appropriate clinical context.

The Development of an Emergency Medicine Resident Research Program in the United States Military.

INTRODUCTION: The Accreditation Council for Graduate Medical Education stipulates that residents should participate in scholarly activity. As of 2019 that verbiage will be changed to, "Residents must participate in scholarship." However, scholarly activity is not clearly defined. We set out to define our graduation research requirement in a measurable way and structure a research curriculum that better prepared residents to conduct scholarly activity. MATERIALS AND METHODS: This study compares resident scholarly output in several categories before and after the initiation of a revised research curriculum and graduation requirement. Scholarly activity was measured by comparing the production of Pubmed Indexed (PMID) publications, online publications, and conference presentations of two Emergency Medicine Residency classes. The intervention class was represented by the class of 2018 which exposed 16 residents to the new curriculum and graduation requirement for the full three years of their residency. The comparison class was represented by the class of 2015 which exposed 16 residents to the old curriculum and old graduation requirement. The old graduation requirement and curriculum were undefined. The new requirement involved two options, participate in original research starting from the process of question formulation and carried through manuscript drafting or publishing at first author PMID of any kind. The new curriculum involved monthly journal clubs, two annual deep dives, and an 8-day Intern Research Course modeled after the Emergency Medicine Basic Research Skills workshop sponsored by the American College of Emergency Physicians. In addition to the new curriculum, several new leadership positions were created at both the staff and resident level that solely focused on the promotion of scholarly activity. In addition to creating a culture within the department that encouraged scholarship, these overlapping leadership positions also helped create continuity in a program that could easily be hampered by frequent staff turnover due to new military assignments and military deployments. RESULTS: Resident scholarly activity in the form of PMIDs increased from 4 to 22. The production of online publications was 0 and 12, respectively. There were 2 and 11 conference presentations, respectively. CONCLUSION: Resident scholarly activity increased following the institution of a new research curriculum and graduation requirement.

Thyroglossal duct cyst surgery: A ten-year single institution experience.

OBJECTIVE: 1. Review our institution's experience with thyroglossal duct cyst (TGDC) excision. 2. Determine if TGDC recurrence rate is influenced by surgical technique, preoperative infection, or surgeon specialty. METHODS: We conducted a retrospective analysis of patients at a tertiary care children's hospital who underwent excision of neck mass with proven TGDC histopathology from 2005 to 2015. Patient demographics and presentation, preoperative workup, surgeon specialty, procedural details, lesion characteristics, and complication rates including recurrence were evaluated. RESULTS: 108 patients (51% male, 49% female), aged 6 months to 20 years (mean 6 years) met inclusion criteria. 121 procedures were performed: 96 (79.3%) for primary disease and 25 (20.7%) for recurrence. 32 patients (27.8%) were infected preoperatively, resulting in a significant increase in complications and recurrence (46.9% and 28.1%, respectively). Lack of preoperative imaging was correlated with increased recurrence (p = 0.0002). Recurrence rate after treatment for primary (11.3%) versus secondary disease (24%) was not significant (p = 0.23). Total recurrence rate differed between ENT (9.1%) and Pediatric Surgery (PS) (27.3%) (p = 0.0172). Difference was not significant for recurrence in primary disease (10% ENT, 14.8% PS, p = 0.49), but was for secondary disease (5.6% ENT, 71.4% PS, p = 0.002). Modifications of Sistrunk's procedure did not result in increased rates of recurrence (p = 0.1273). CONCLUSION: Preoperative TGDC infection and lack of imaging led to a significant increase in postoperative complications, including recurrence. Surgical specialty significantly affected recurrence rates following excision overall and in secondary disease. Recurrence was not affected by surgical technique, age, sex, cyst size, or mucin rich histology.

Medical Student Volunteerism Addresses Patients' Social Needs: A Novel Approach to Patient-Centered Care.

BACKGROUND: Healthcare providers must be equipped to recognize and address patients' psychosocial needs to improve overall health outcomes. To give future healthcare providers the tools and training necessary to identify and address psychosocial issues, Lankenau Medical Center in partnership with the Philadelphia College of Osteopathic Medicine designed the Medical Student Advocate (MSA) program. METHODS: The MSA program places volunteer second-year osteopathic medical students in care coordination teams at Lankenau Medical Associates, a primary care practice serving a diverse patient population in the Philadelphia, PA, region. As active members of the team, MSAs are referred high-risk patients who have resource needs such as food, employment, child care, and transportation. MSAs work collaboratively with patients and the multidisciplinary team to address patients' nonmedical needs. RESULTS: From August 2013 to August 2015, 31 osteopathic medical students volunteered for the MSA program and served 369 patients with 720 identified needs. Faculty and participating medical students report that the MSA program provided an enhanced understanding of the holistic nature of patient care and a comprehensive view of patient needs. CONCLUSION: The MSA program provides students with a unique educational opportunity that encompasses early exposure to patient interaction, social determinants of health, population health, and interdisciplinary collaboration. Students develop skills to help them build patient relationships, understand the psychosocial factors shaping health outcomes, and engage with other healthcare professionals. This work in the preclinical years provides students with the knowledge to help them perform more effectively in the changing healthcare environment.

Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.

BACKGROUND: The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2, a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS: Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS: We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of dynamin 2 and stimulates its GTPase activity under physiologic conditions (37°C and 100mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by dynamin 2. Arc also increases the GTPase activity of dynamin 3, an isoform of implicated in dendrite remodeling, but does not affect the activity of dynamin 1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity.

Pulmonary pleomorphic carcinoma metastasis to the midfoot.

Metastases to the bones in the foot are extremely uncommon, occurring in approximately 0.01% of all metastatic bone disease. We describe a case of an 82-year-old female with a metastatic pulmonary sarcomatoid carcinoma lesion to the midfoot. This rare and aggressive pulmonary malignancy has a poor prognosis. The purpose of the present case report was to highlight the key roles that medical history and biopsy, combined with a multispecialty approach, play in accurately diagnosing and appropriately treating a patient with metastatic bone disease.

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells.

BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state. METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells. RESULTS: Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1µM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. CONCLUSIONS AND GENERAL SIGNIFICANCE: These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.

Modified Blair tibiotalar arthrodesis for post-traumatic avascular necrosis of the talus: a case report.

Surgical treatment of post-traumatic avascular necrosis of the talus coupled with collapse often results in limited treatment options. Of those options, the Blair tibiotalar arthrodesis has been beneficial in preserving limb length and subtalar motion. The complications associated with Blair tibiotalar arthrodesis have led to modifications to improve stability and functional outcomes with rigid internal fixation. We present the case of a 29-year-old female with a history of an open fracture dislocation of the talus 10 years previously, with subsequent development of avascular necrosis of the talus. The purpose of the present case report was to describe the surgical approach and use of an anterior compression plate to augment the modified Blair tibiotalar arthrodesis.

Fluorescence techniques in analysis of protein-ligand interactions.

Fluorescence spectroscopy may serve as a universal tool for the study of protein-ligand interactions. Applications of fluorometry have made use of various aspects of fluorescence such as intensity, emission and excitation spectra, lifetime, quantum yield, polarization state, and anisotropy, as well as energy transfer and other electronic phenomena. An experimentalist has to consider each of these characteristics carefully, frequently in combination with each other, for the analysis of protein-ligand complexes and for the determination of binding constants. Most of the available techniques are of a rather general nature and a wealth of possibilities exists for their utilization. In this chapter we will provide a short survey of selected techniques that can be used for measuring binding constants and probing protein-ligand interactions. Basic principles and phenomena are discussed followed by experimental considerations and examples of binding constant determination. Emphasis is placed on steady-state techniques that employ the use of intrinsic protein fluorescence, labeled ligands, as well as anisotropy and resonance energy transfer.

Studies on the dissociation and urea-induced unfolding of FtsZ support the dimer nucleus polymerization mechanism.

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (K(d) = 9 µM) indicates a significant fraction (~10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay.

Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134-206 of SNAP-25 (synaptosome-associated protein of 25kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

Oligomerization state of dynamin 2 in cell membranes using TIRF and number and brightness analysis.

Dynamin 2 is an ubiquitously expressed ∼100 kDa GTPase involved in receptor-mediated endocytosis, Golgi budding, and cytoskeletal reorganization. Dynamin molecules assemble around the necks of budding vesicles and constrict membranes in a GTP-dependent process, resulting in vesicle release. The oligomerization state of dynamin 2 in the membrane is still controversial. We investigated dynamin 2 within the plasma membrane of live cells using total internal reflection microscopy coupled with number and brightness analysis. Our results demonstrate that dynamin 2 is primarily tetrameric throughout the entire cell membrane, aside from punctate structures that may correspond to regions of membrane vesiculation.