Calcium Bistriflimide-Mediated Sulfur(VI)-Fluoride Exchange (SuFEx): Mechanistic Insights toward Instigating Catalysis.

We report a mechanistic investigation of calcium bistriflimide-mediated sulfur(VI)-fluoride exchange (SuFEx) between sulfonyl fluorides and amines. We determine the likely pre-activation resting state─a calcium bistriflimide complex with ligated amines─thus allowing for corroborated calculation of the SuFEx activation barrier at ∼21 kcal/mol, compared to 21.5 ± 0.14 kcal/mol derived via kinetics experiments. Transition state analysis revealed: (1) a two-point calcium-substrate contact that activates the sulfur(VI) center and stabilizes the leaving fluoride and (2) a 1,4-diazabicyclo[2.2.2]octane additive that provides Brønsted-base activation of the nucleophilic amine. Stable Ca-F complexes upon sulfonamide formation are likely contributors to inhibited catalytic turnover, and a proof-of-principle redesign provided evidence that sulfonamide formation is feasible with 10 mol % calcium bistriflimide.

Early Functional Status After Surgery for Congenital Heart Disease: A Single-Center Retrospective Study.

OBJECTIVES: The objective of this study is to investigate the change in functional status in infants, children, and adolescents undergoing congenital heart surgery using the Functional Status Scale. DESIGN: A single-center retrospective study. SETTING: A 26-bed cardiac ICU in a free-standing university-affiliated tertiary children's hospital. PATIENTS: All patients 0-18 years who underwent congenital heart surgery from January 1, 2014, to December 31, 2017. INTERVENTIONS: None. MEASUREMENTS AND MIN RESULTS: The primary outcome variable was change in Functional Status Scale scores from admission to discharge. Additionally, two binary outcomes were derived from the primary outcome: new morbidity (change in Functional Status Scale ≥ 3) and unfavorable functional outcome (change in Functional Status Scale ≥ 5); their association with risk factors was assessed using modified Poisson regression. Out of 1,398 eligible surgical encounters, 65 (4.6%) and 15 (1.0%) had evidence of new morbidity and unfavorable functional outcomes, respectively. Higher Surgeons Society of Thoracic and the European Association for Cardio-Thoracic Surgery score, single-ventricle physiology, and longer cardiopulmonary bypass time were associated with new morbidity. Longer hospital length of stay was associated with both new morbidity and unfavorable outcome. CONCLUSIONS: This study demonstrates the novel application of the Functional Status Scale on patients undergoing congenital heart surgery. New morbidity was noted in 4.6%, whereas unfavorable outcome in 1%. There was a small change in the total Functional Status Scale score that was largely attributed to changes in the feeding domain. Higher Society of Thoracic and the European Association for Cardio-Thoracic Surgery score, single-ventricle physiology, and longer cardiopulmonary bypass times were associated with new morbidity, whereas longer hospital length of stay was associated with both new morbidity and unfavorable outcome. Further studies with larger sample size will need to be done to confirm our findings and to better ascertain the utility of Functional Status Scale on this patient population.

Isolated Acetabuloplasty and Labral Repair for Combined-Type Femoroacetabular Impingement: Are We Doing Too Much?

PURPOSE: To evaluate patient outcomes after isolated arthroscopic volumetric acetabular osteoplasty and labral repair for the treatment of patients with combined femoroacetabular impingement (FAI) lesions. METHODS: A review of a prospectively collected registry identified 86 patients (106 hips) with an average age of 38.1 years (range, 17-59 years) with combined-type FAI that underwent isolated acetabular osteoplasty and labral repair. Preoperative α-angle, degree of radiographic degenerative changes, and presence of a crossover sign were recorded. Clinical outcomes were assessed with the modified Harris Hip Score (mHHS), International Hip Outcome Tool-12 (iHOT-12), Hip Outcome Score Sport-Specific Subscale (HOS-SSS), and patient satisfaction score (out of 10) at a minimum 2-year follow-up. RESULTS: Clinical follow-up was obtained at a mean follow-up of 37.2 months (range, 27.9-79.2 months). Patients with Tönnis grade 0 and I findings had significantly higher mHHS (83.5 vs 71.5, P = .01), HOS-SSS (81.3 vs 59.9, P = .02), and iHOT-12 scores (71.1 vs 58.8, P = .04) compared to patients with Tonnis grade II changes. However, patient satisfaction scores (8.0 vs 7.2, P = .45) were no different. No significant difference was noted between unilateral and bilateral hip patient outcome scores. Patient age and preoperative α-angles did not correlate with any outcome scores (all R2 <0.05). There were no cases of revision surgery or progression to arthroplasty. CONCLUSIONS: Isolated acetabular decompression may adequately address the underlying impingement in combined-type FAI while avoiding the risks associated with femoral-sided decompression. Good to excellent patient-reported outcomes and satisfaction scores were noted with significantly higher scores in patients with minimal arthritic change. Patient age and preoperative α-angle had less effect on postoperative outcomes. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Incidence, risk factors, and outcomes of acute kidney injury in adults undergoing surgery for congenital heart disease.

BACKGROUND: Acute kidney injury after cardiac surgery is a frequent and serious complication among children with congenital heart disease (CHD) and adults with acquired heart disease; however, the significance of kidney injury in adults after congenital heart surgery is unknown. The primary objective of this study was to determine the incidence of acute kidney injury after surgery for adult CHD. Secondary objectives included determination of risk factors and associations with clinical outcomes. METHODS: This single-centre, retrospective cohort study was performed in a quaternary cardiovascular ICU in a paediatric hospital including all consecutive patients ⩾18 years between 2010 and 2013. RESULTS: Data from 118 patients with a median age of 29 years undergoing cardiac surgery were analysed. Using Kidney Disease: Improving Global Outcome creatinine criteria, 36% of patients developed kidney injury, with 5% being moderate to severe (stage 2/3). Among higher-complexity surgeries, incidence was 59%. Age ⩾35 years, preoperative left ventricular dysfunction, preoperative arrhythmia, longer bypass time, higher Risk Adjustment for Congenital Heart Surgery-1 category, and perioperative vancomycin use were significant risk factors for kidney injury development. In multivariable analysis, age ⩾35 years and vancomycin use were significant predictors. Those with kidney injury were more likely to have prolonged duration of mechanical ventilation and cardiovascular ICU stay in the univariable regression analysis. CONCLUSIONS: We demonstrated that acute kidney injury is a frequent complication in adults after surgery for CHD and is associated with poor outcomes. Risk factors for development were identified but largely not modifiable. Further investigation within this cohort is necessary to better understand the problem of kidney injury.

Radiology Case of the Month: An Unusual Etiology of Increased Abdominal Girth in a 42-Year-Old Man.

A 42-year-old man with diabetes and hypertension presented to the emergency room after experiencing a several month history of gradually increasing abdominal girth with the sudden onset of abdominal pain.

Criteria for Early Pacemaker Implantation in Patients With Postoperative Heart Block After Congenital Heart Surgery.

BACKGROUND: Guidelines recommend observation for atrioventricular node recovery until postoperative days (POD) 7 to 10 before permanent pacemaker placement (PPM) in patients with heart block after congenital cardiac surgery. To aid in surgical decision-making for early PPM, we established criteria to identify patients at high risk of requiring PPM. METHODS: We reviewed all cases of second degree and complete heart block (CHB) on POD 0 from August 2009 through December 2018. A decision tree model was trained to predict the need for PPM amongst patients with persistent CHB and prospectively validated from January 2019 through March 2021. Separate models were developed for all patients on POD 0 and those without recovery by POD 4. RESULTS: Of the 139 patients with postoperative heart block, 68 required PPM. PPM was associated with older age (3.2 versus 1.0 years; P=0.018) and persistent CHB on POD 0 (versus intermittent CHB or second degree heart block; 87% versus 58%; P=0.001). Median days [IQR] to atrioventricular node recovery was 2 [0-5] and PPM was 9 [6-11]. Of the 100 cases of persistent CHB (21 in the validation cohort), 59 (59%) required PPM. A decision tree model identified 4 risk factors for PPM in patients with persistent CHB: (1) aortic valve replacement, subaortic stenosis repair, or Konno procedure; (2) ventricular L-looping; (3) atrioventricular valve replacement; (4) and absence of preoperative antiarrhythmic agent (in POD 0 model only). The POD 4 model specificity was 0.89 [0.67-0.99] and positive predictive value was 0.94 [95% CI 0.81-0.98], which was stable in prospective validation (positive predictive value 1.0). CONCLUSIONS: A data-driven analysis led to actionable criteria to identify patients requiring PPM. Patients with left ventricular outflow tract surgery, atrioventricular valve replacement, or ventricular L-Looping could be considered for PPM on POD 4 to reduce risks of temporary pacing and improve care efficiency.

Quantifying Electronic Health Record Data: A Potential Risk for Cognitive Overload.

OBJECTIVES: To quantify and describe patient-generated health data. METHODS: This is a retrospective, single-center study of patients hospitalized in the pediatric cardiovascular ICU between February 1, 2020, and February 15, 2020. The number of data points generated over a 24-hour period per patient was collected from the electronic health record. Data were analyzed by type, and frontline provider exposure to data was extrapolated on the basis of patient-to-provider ratios. RESULTS: Thirty patients were eligible for inclusion. Nineteen were hospitalized after cardiac surgery, whereas 11 were medical patients. Patients generated an average of 1460 (SD 509) new data points daily, resulting in frontline providers being presented with an average of 4380 data points during a day shift (7:00 am to 7:00 pm). Overnight, because of a higher patient-to-provider ratio, frontline providers were exposed to an average of 16 060 data points. There was no difference in data generation between medical and surgical patients. Structured data accounted for >80% of the new data generated. CONCLUSIONS: Health care providers face significant generation of new data daily through the contemporary electronic health record, likely contributing to cognitive burden and putting them at risk for cognitive overload. This study represents the first attempt to quantify this volume in the pediatric setting. Most data generated are structured and amenable to data-optimization systems to mitigate the potential for cognitive overload and its deleterious effects on patient safety and health care provider well-being.

Role of Texting as a Source of Cognitive Burden in a Pediatric Cardiovascular ICU.

OBJECTIVES: To characterize frontline provider perception of clinical text messaging and quantify clinical texting data in a pediatric cardiovascular ICU (CICU). METHODS: This is a mixed-methods, retrospective single center study. A survey of frontline CICU providers (pediatric fellows, nurse practitioners, and physician assistants) was conducted to assess attitudes characterizing text messaging on cognitive burden. Text messaging data were abstracted and quantified between January 29, 2020, and April 18, 2020, and the patterns of text messages were analyzed per shift and by provider. RESULTS: The survey was completed by 33 of 39 providers (85%). Out of responders, 78% indicated that clinical text messaging frequently or very frequently disrupts critical thinking and workflow. They also felt that the burden of messages was worse during the night shift. Through abstraction, 31 926 text messages were identified. A median of 15 (interquartile range: 12-19) messages per hour were received. A median of 5 messages were received per hour per provider during the day shift and 6 during the night shift. From the entire study period, there were total 2 hours of high-frequency texting (≥15 texts per hour) during the day shift and 68 hours during the night shift. CONCLUSION: In our study, providers in the CICU received a large number of texts with a disproportionate burden during the night shift. Text messages are a potential source of cognitive overload for providers. Optimization of text messaging may be needed to mitigate cognitive burden for frontline providers.

Rapid induction of autoantibodies during ARDS and septic shock.

BACKGROUND: Little is known about the induction of humoral responses directed against human autoantigens during acute inflammation. We utilized a highly sensitive antibody profiling technology to study autoantibodies in patients with acute respiratory distress syndrome (ARDS) and severe sepsis, conditions characterized by intensive immune activation leading to multiple organ dysfunction. METHODS: Using Luciferase Immunoprecipitation Systems (LIPS), a cohort of control, ARDS and sepsis patients were tested for antibodies to a panel of autoantigens. Autoantibody titers greater than the mean plus 3 SD of the 24 control samples were used to identify seropositive samples. Available longitudinal samples from different seropositive ARDS and sepsis patient samples, starting from within the first two days after admission to the intensive care, were then analyzed for changes in autoantibody over time. RESULTS: From screening patient plasma, 57% of ARDS and 46% of septic patients without ARDS demonstrated at least one statistically significant elevated autoantibody compared to the controls. Frequent high titer antibodies were detected against a spectrum of autoantigens including potassium channel regulator, gastric ATPase, glutamic decarboxylase-65 and several cytokines. Analysis of serial samples revealed that several seropositive patients had low autoantibodies at early time points that often rose precipitously and peaked between days 7-14. Further, the use of therapeutic doses of corticosteroids did not diminish the rise in autoantibody titers. In some cases, the patient autoantibody titers remained elevated through the last serum sample collected. CONCLUSION: The rapid induction of autoantibodies in ARDS and severe sepsis suggests that ongoing systemic inflammation and associated tissue destruction mediate the break in tolerance against these self proteins.

Benefits of Multifaceted Chemopreventives in the Suppression of the Oral Squamous Cell Carcinoma (OSCC) Tumorigenic Phenotype.

Over one third of patients who have undergone oral squamous cell carcinoma (OSCC) surgical resections develop life-threatening and often untreatable recurrences. A variety of drugs, intended for management of recurrent or disseminated cancers, were designed to exploit cancer cells' reliance upon overexpressed receptors and gratuitous signaling. Despite their conceptual promise, clinical trials showed these agents lacked efficacy and were often toxic. These findings are consistent with evasion of pathway-targeted treatments via extensive signaling redundancies and compensatory mechanisms common to cancers. Optimal secondary OSCC chemoprevention requires long-term efficacy with multifaceted, nontoxic agents. Accordingly, this study evaluated the abilities of three complementary chemopreventives, that is, the vitamin A derivative fenretinide (4-HPR, induces apoptosis and differentiation, inhibits signaling proteins, and invasion), the estrogen metabolite 2-methoxyestradiol (2-ME, apoptosis-inducing, antiangiogenic), and the humanized mAb to the IL6R receptor tocilizumab (TOC, reduces IL6 signaling) to suppress OSCC gratuitous signaling and tumorigenesis. Modeling studies demonstrated 4-HPR's high-affinity binding at STAT3's dimerization site and c-Abl and c-Src ATP-binding kinase sites. Although individual agents suppressed cancer-promoting pathways including STAT3 phosphorylation, STAT3-DNA binding, and production of the trans-signaling enabling sIL6R, maximal chemopreventive effects were observed with agent combinations. OSCC tumor xenograft studies showed that locally delivered TOC, TOC+4-HPR, and TOC+4-HPR+2-ME treatments all prevented significant tumor growth. Notably, the TOC+4-HPR+2-ME treatment resulted in the smallest overall increase in tumor volume. The selected agents use diverse mechanisms to disrupt tumorigenesis at multiple venues, that is, intracellular, tumor cell-ECM, and tumor microenvironment; beneficial qualities for secondary chemopreventives. Cancer Prev Res; 10(1); 76-88. ©2016 AACR.

Programmatic Approach to Management of Tetralogy of Fallot With Major Aortopulmonary Collateral Arteries: A 15-Year Experience With 458 Patients.

BACKGROUND: Tetralogy of Fallot with major aortopulmonary collateral arteries is a complex and heterogeneous condition. Our institutional approach to this lesion emphasizes early complete repair with the incorporation of all lung segments and extensive lobar and segmental pulmonary artery reconstruction. METHODS AND RESULTS: We reviewed all patients who underwent surgical intervention for tetralogy of Fallot and major aortopulmonary collateral arteries at Lucile Packard Children's Hospital Stanford (LPCHS) since November 2001. A total of 458 patients underwent surgery, 291 (64%) of whom underwent their initial procedure at LPCHS. Patients were followed for a median of 2.7 years (mean 4.3 years) after the first LPCHS surgery, with an estimated survival of 85% at 5 years after first surgical intervention. Factors associated with worse survival included first LPCHS surgery type other than complete repair and Alagille syndrome. Of the overall cohort, 402 patients achieved complete unifocalization and repair, either as a single-stage procedure (n=186), after initial palliation at our center (n=74), or after surgery elsewhere followed by repair/revision at LPCHS (n=142). The median right ventricle:aortic pressure ratio after repair was 0.35. Estimated survival after repair was 92.5% at 10 years and was shorter in patients with chromosomal anomalies, older age, a greater number of collaterals unifocalized, and higher postrepair right ventricle pressure. CONCLUSIONS: Using an approach that emphasizes early complete unifocalization and repair with incorporation of all pulmonary vascular supply, we have achieved excellent results in patients with both native and previously operated tetralogy of Fallot and major aortopulmonary collateral arteries.

Time and Cost of Diagnosis for Symptomatic Femoroacetabular Impingement.

BACKGROUND: Femoroacetabular impingement (FAI) and labral tears are common causes of hip pain that are often not promptly or properly diagnosed. To our knowledge, no reports have defined the time and cost of diagnosis of labral tears associated with FAI. HYPOTHESIS: Patients with labral tears associated with FAI undergo extraneous diagnostic testing and pain and incur a significant amount of health care costs before they receive appropriate surgical management for their pathology. STUDY DESIGN: Economic and decision analysis; Level of evidence, 4. METHODS: A total of 78 patients diagnosed with symptomatic FAI were surveyed. A standardized questionnaire asked patients about time to diagnosis, symptoms, health care providers visited, imaging tests, and treatments prior to diagnosis. Costs were calculated based on 2012 national Medicare data. RESULTS: Patients in the cohort saw an average of 4.0 health care providers, had an average of 3.4 diagnostic imaging tests, and tried an average of 3.1 treatments prior to diagnosis. The average total amount spent per patient prior to diagnosis was US$2456.97. The calculated minimum cost of diagnosis, including a visit to an orthopaedic surgeon as well as an anteroposterior pelvis and lateral hip radiograph and 1 magnetic resonance arthrogram, was US$690.62. The average duration between onset of symptoms and diagnosis of labral tear was 32.0 months. CONCLUSION: The average amount of health care dollars spent per patient prior to receiving a diagnosis of acetabular labral tear was US$1766.35 higher than the calculated minimum cost. This figure is based on Medicare payment amounts, which may significantly underestimate the actual charges at many hospitals, thereby increasing the total cost of diagnosis. CLINICAL RELEVANCE: The costs and pain associated with this time, along with the potential long-term degradation of the hip joint, make it important for all health care professionals to recognize and appropriately manage or refer the patient.

Topical application of a mucoadhesive freeze-dried black raspberry gel induces clinical and histologic regression and reduces loss of heterozygosity events in premalignant oral intraepithelial lesions: results from a multicentered, placebo-controlled clinical trial.

PURPOSE: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions. EXPERIMENTAL DESIGN: This placebo-controlled multicenter study investigated the effects of topical application of bioadhesive gels that contained either 10% w/w freeze-dried black raspberries (BRB) or an identical formulation devoid of BRB placebo to biopsy-confirmed OIN lesions (0.5 g × q.i.d., 12 weeks). Baseline evaluative parameters (size, histologic grade, LOH events) were comparable in the randomly assigned BRB (n = 22) and placebo (n = 18) gel cohorts. Evaluative parameters were: histologic grade, clinical size, and LOH. RESULTS: Topical application of the BRB gel to OIN lesions resulted in statistically significant reductions in lesional sizes, histologic grades, and LOH events. In contrast, placebo gel lesions demonstrated a significant increase in lesional size and no significant effects on histologic grade or LOH events. Collectively, these data strongly support BRB's chemopreventive impact. A cohort of very BRB-responsive patients, as demonstrated by high therapeutic efficacy, was identified. Corresponding protein profiling studies, which demonstrated higher pretreatment levels of BRB metabolic and keratinocyte differentiation enzymes in BRB-responsive lesions, reinforce the importance of local metabolism and differentiation competency. CONCLUSIONS: Results from this trial substantiate the LOH reductions identified in the pilot BRB gel study and extend therapeutic effects to significant improvements in histologic grade and lesional size.

All-arthroscopic iliotibial band autograft harvesting and labral reconstruction technique.

The labrum is essential for stability, movement, and prevention of arthritis in the hip. In cases of labral damage where repair of a labral tear is not possible, reconstruction can be a useful alternative. Several different autografts have been used, including the iliotibial band (ITB), the ligamentum teres capitis, and the gracilis tendon. Authors have reported both open and arthroscopic techniques for reconstruction with good preliminary results. However, an all-arthroscopic labral reconstruction technique including the graft harvest and reconstruction portions of a labral reconstruction procedure using an ITB autograft has not been previously described. We describe a technique for an all-arthroscopic labral reconstruction performed using a novel method for arthroscopic harvest of the ITB. The decreased invasiveness of our described technique for labral reconstruction may potentially minimize scarring, bodily disfigurement, infection, and postoperative pain associated with the graft harvesting incision.

Synthetic biology for translational research.

Synthetic biology involves the engineering of proteins, signaling pathways and even whole organisms using modular designs and formats. A major tool of synthetic biology is artificial gene synthesis, which provides a direct means from a conceptualized DNA sequence to the corresponding physical DNA for the construction of a variety of biological components. To date, synthetic biology has often been used to answer fundamental questions in basic research, but now is poised to greatly enhance translational research. In this review, we discuss several translational applications of synthetic biology including the construction of novel diagnostics and vaccines, development of new synthetic pathways for drug screening and biosynthesis, and the creation of engineered viruses and microbes to fight human disease. Together these and other novel translational applications of artificial gene synthesis and synthetic biology have the opportunity to make major advances for improving human health.

Antibody-profiling technologies for studying humoral responses to infectious agents.

Analyses of humoral responses against different infectious agents are critical for infectious disease diagnostics, understanding pathogenic mechanisms, and the development and monitoring of vaccines. While ELISAs are often used to measure antibody responses to one or several targets, new antibody-profiling technologies, such as protein microarrays, can now evaluate antibody responses to hundreds, or even thousands, of recombinant antigens at one time. These large-scale studies have uncovered new antigenic targets, provided new insights into vaccine research and yielded an overview of immunoreactivity against almost the entire proteome of certain pathogens. However, solid-phase antigen arrays also have drawbacks that limit the type of information obtained, including suboptimal detection of conformational epitopes, high backgrounds due to impure antigens and a narrow dynamic range of detection. We have developed a solution-phase antibody-profiling technology, luciferase immunoprecipitation systems (LIPS), which harnesses light-emitting recombinant antigen fusion proteins to quantitatively measure patient antibody titers. Owing to the highly linear light output of the luciferase reporter, some antibodies can be detected without serum dilution in a dynamic range of detection often spanning seven orders of magnitude. When LIPS is applied iteratively with multiple target antigens, a high-definition antibody profile is obtained. Here, we discuss the application of these different antibody-profiling technologies and their associated limitations with particular emphasis on protein microarrays. We also describe LIPS in detail and discuss several clinically relevant uses of the technology. Together, these new technologies offer new tools for understanding humoral responses to known and emerging infectious agents.

Extraordinary antigenicity of the human Ro52 autoantigen.

Autoantibody levels to the SSA complex, composed of Ro52 and Ro60 proteins, are commonly measured in the diagnoses of Sjögren's Syndrome (SjS), as well as other rheumatological diseases. One of these proteins, Ro52, is an interferon-inducible member of the tripartite motif family bearing a RING motif functioning as an E3 ligase that ubiquitinates interferon regulatory factor 8 and other proteins. Using Luciferase Immunoprecipitation Systems (LIPS) we explored the antigenicity of Ro52 in detail. Analysis of antibody responses against Ro52 and 20 other established antigens revealed that Ro52 had the highest antibody titers and most likely represents one of the most immunogenic human proteins. While the antibody titers in many of the SjS patients were significantly and substantially higher than the controls, all healthy individuals had anti-Ro52 autoantibodies. N- and C-terminal fragments of Ro52 showed immunoreactivity in these serum samples, but the sums of these antibody titers were significantly lower than the antibody titers directed against the full-length Ro52. Antibody profiling of controls and SjS patients with three different N-terminal fragments of Ro52 revealed that the coiled-coil region was the most useful diagnostic (66% sensitivity), followed by the B-box (31% sensitivity), and then the RING-finger (24% sensitivity). The C-terminal region of Ro52, containing the B30.2 domain, showed higher antibody titers in SjS patients compared to controls and this region was responsible for the high level of Ro52 immunoreactivity in healthy individuals. Analysis of immunoreactivity to TRIM5, a Ro52-related protein, and the B30.2 domain from BTN1 and pyrin, failed to show significant antibody titers with the control or SjS patient serum. These results highlight the unusually high level of Ro52 antigenicity and demonstrate that autoantibodies are directed at both linear and conformational epitopes spanning the entire molecule.