An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

A molecular roadmap of reprogramming somatic cells into iPS cells.

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.

Modeling Rett syndrome with stem cells.

The discovery that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) raised the exciting possibility of modeling diseases with patient-specific cells. Marchetto et al. (2010) now use iPSC technology to generate, characterize, and treat an in vitro model for the autism spectrum disorder Rett syndrome.

Randomized controlled trial of urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE): Rationale and design.

Diuresis to achieve decongestion is a central aim of therapy in patients hospitalized for acute decompensated heart failure (ADHF). While multiple clinical trials have investigated initial diuretic strategies for a designated period of time, there is a paucity of evidence to guide diuretic titration strategies continued until decongestion is achieved. The use of urine chemistries (urine sodium and creatinine) in a natriuretic response prediction equation accurately estimates natriuresis in response to diuretic dosing, but a randomized clinical trial is needed to compare a urine chemistry-guided diuresis strategy with a strategy of usual care. The urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE) trial is designed to test the hypothesis that protocolized diuretic therapy guided by spot urine chemistry through completion of intravenous diuresis will be superior to usual care and improve outcomes over the 14 days following randomization. ESCALATE will randomize and obtain complete data on 450 patients with acute heart failure to a diuretic strategy guided by urine chemistry or a usual care strategy. Key inclusion criteria include an objective measure of hypervolemia with at least 10 pounds of estimated excess volume, and key exclusion criteria include significant valvular stenosis, hypotension, and a chronic need for dialysis. Our primary outcome is days of benefit over the 14 days after randomization. Days of benefit combines patient symptoms captured by global clinical status with clinical state quantifying the need for hospitalization and intravenous diuresis. CLINICAL TRIAL REGISTRATION: NCT04481919.

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

The prevalence, characteristics, and risk factors of external cervical resorption: a retrospective practice-based study.

OBJECTIVES: External cervical root resorption (ECR) is a poorly understood and aggressive form of resorption. The purpose of this study was to examine the prevalence, characteristics, and risk factors associated with the occurrence of ECR in patients seeking endodontic care from private practice settings. MATERIALS AND METHODS: Records of 343 patients with 390 teeth diagnosed with ECR were identified from 3 private endodontic practices from 2008 to 2022. The patients' demographic information, systemic conditions, and dental history were recorded. The characteristics of the cases including Heithersay classification, pulpal and periapical status, and their management were documented. The association between case severity and potential predisposing factors was examined using chi-square analysis. RESULTS: The overall prevalence of ECR among patients seeking endodontic care was low (< 1%). However, there was a greater than twofold increase in the pooled prevalence from 2016 to 2021 (0.99%) compared to the data from 2010 to 2015 (0.46%). The most commonly affected teeth were anterior teeth (48.7%). Class II (30.0%) and class III (45.4%) defects were the most often identified. Patients with a history of trauma or orthodontic treatment were significantly more likely to be diagnosed with severe cervical resorption (class III or IV) (p < 0.05). CONCLUSIONS: There has been an increase in the prevalence of ECR in patients seeking endodontic care. A history of orthodontic treatment and traumatic dental mechanical injuries may predict the severity of resorption. CLINICAL RELEVANCE: The upward trend in the occurrence of ECR warrants close monitoring of the patients at high risk of developing the condition to facilitate early detection and management.

Using Implementation Science to Guide the Process of Adapting a Patient Engagement Intervention for Inpatient Spinal Cord Injury/Disorder Rehabilitation.

OBJECTIVES: This study aimed to describe the process of adapting an evidence-based patient engagement intervention, enhanced medical rehabilitation (E-MR), for inpatient spinal cord injury/disease (SCI/D) rehabilitation using an implementation science framework. DESIGN: We applied the collaborative intervention planning framework and included a community advisory board (CAB) in an intervention mapping process. SETTING: A rehabilitation hospital. PARTICIPANTS: Stakeholders from inpatient SCI/D rehabilitation (N=7) serving as a CAB and working with the research team (N=7) to co-adapt E-MR. INTERVENTIONS: E-MR. MAIN OUTCOME MEASURES: Logic model and matrices of change used in CAB meetings to identify areas of intervention adaptation. RESULTS: The CAB and research team implemented adaptations to E-MR, including (1) identifying factors influencing patient engagement in SCI/D rehabilitation (eg, therapist training); (2) revising intervention materials to meet SCI/D rehabilitation needs (eg, modified personal goals interview and therapy trackers to match SCI needs); (3) incorporating E-MR into the rehabilitation hospital's operations (eg, research team coordinated with CAB to store therapy trackers in the hospital system); and (4) retaining fidelity to the original intervention while best meeting the needs of SCI/D rehabilitation (eg, maintained core E-MR principles while adapting). CONCLUSIONS: This study demonstrated that structured processes guided by an implementation science framework can help researchers and clinicians identify adaptation targets and modify the E-MR program for inpatient SCI/D rehabilitation.

Scleroderma and multiple teeth with invasive cervical resorption: is there a connection?

Scleroderma is a chronic connective tissue disease generally classified as an autoimmune rheumatic disease. Symptoms may include thickening of the skin, calcifications, Raynaud syndrome, and esophageal problems. Invasive cervical resorption is an aggressive form of external resorption localized in the cervical part of the tooth. Its etiology remains uncertain. This case report describes invasive cervical resorption affecting 4 teeth in a 44-year-old woman with scleroderma and speculates on the possible relationships between these disease entities.

High-sensitivity small-molecule detection of microcystin-LR cyano-toxin using a terahertz-aptamer biosensor.

We demonstrate the rapid and highly sensitive detection of a small molecule, microcystin-LR (MC-LR) toxin using an aptasensor based on a terahertz (THz) emission technique named the terahertz chemical microscope (TCM). The main component of the TCM is the sensing plate, which consists of a thin silicon layer deposited on a sapphire substrate, with a natural SiO2 layer formed on the top of the Si layer. The DNA aptamer is linked to the oxidized top surface of the silicon layer by a one-step reaction (click chemistry) between the DBCO-labeled aptamer and an azido group that binds to the surface. Using density functional theory (DFT) calculations, the number of active sites on the surface has been estimated to be 3.8 × 1013 cm-2. Aptamer immobilization and MC-LR binding have been optimized by adjusting the aptamer concentration and the binding buffer composition. When MC-LR binds with the DNA aptamer, it causes a change in the chemical potential at the surface of the sensing plate, which leads to a change in the amplitude of the THz signal. Compared with other bio-sensing methods such as surface plasmon resonance (SPR), TCM is a rapid assay that can be completed in 15 min (10 min incubation and 5 min data acquisition). Moreover, our results show that the aptamer-based TCM can detect MC-LR with an excellent detection limit of 50 ng L-1, which is 20 times more sensitive compared with SPR measurements of MC-LR.

The expanding reaction toolkit for DNA-encoded libraries.

Over the past decade, DNA-encoded libraries (DELs) have emerged as a leading platform for small molecule drug discovery among pharmaceutical companies, biotech companies and academic drug hunters alike. This revolutionary technology has tremendous potential that is yet to be fully realized, as the exploration of therapeutically relevant chemical space is fueled by the ever-expanding repertoire of DNA-compatible reactions used to construct the libraries. Advances in direct coupling reactions, like photo-catalytic cross couplings, unique cyclizations such as the formation of 1,2,4-oxadiazoles, and new functional group transformations are valuable contributions to the DEL reaction toolkit, and indicate where future reaction development efforts should focus in order to maximize the productivity of DELs.

Central airway collapse is related to obesity independent of asthma phenotype.

BACKGROUND AND OBJECTIVE: Late-onset non-allergic asthma in obesity is characterized by an abnormally compliant, collapsible lung periphery; it is not known whether this abnormality exists in proximal airways. We sought to compare collapsibility of central airways between lean and obese individuals with and without asthma. METHODS: A cross-sectional study comparing luminal area and shape (circularity) of the trachea, left mainstem bronchus, right bronchus intermedius and right inferior lobar bronchus at RV and TLC by CT was conducted. RESULTS: In 11 lean controls (BMI: 22.4 (21.5, 23.8) kg/m2 ), 10 lean individuals with asthma (23.6 (22.0, 24.8) kg/m2 ), 10 obese controls (45.5 (40.3, 48.5) kg/m2 ) and 21 obese individuals with asthma (39.2 (35.8, 42.9) kg/m2 ), lumen area and circularity increased significantly with an increase in lung volume from RV to TLC for all four airways (P < 0.05 for all). Changes in area and circularity with lung volume were similar in obese individuals with and without asthma, and both obese groups had severe airway collapse at RV. In multivariate analysis, change in lumen area was related to BMI and change in circularity to waist circumference, but neither was related to asthma diagnosis. CONCLUSION: Excessive collapse of the central airways is related to obesity, and occurs in both obese controls and obese asthma. Increased airway collapse could contribute to ventilation abnormalities in obese individuals particularly at lower lung volumes, and complicate asthma in obese individuals.

Neuro-Ophthalmic Complications in Patients Treated With CTLA-4 and PD-1/PD-L1 Checkpoint Blockade.

BACKGROUND: In recent years, CTLA-4 and PD-1/PD-L1 checkpoint inhibitors have proven to be effective and have become increasingly popular treatment options for metastatic melanoma and other cancers. These agents work by enhancing autologous antitumor immune responses. Immune-related ophthalmologic complications have been reported in association with checkpoint inhibitor use but remain incompletely characterized. This study seeks to investigate and further characterize the neuro-ophthalmic and ocular complications of immune checkpoint blockade treatment. METHODS: A survey was distributed through the secure electronic data collection tool REDCap to neuro-ophthalmology specialists in the North American Neuro-Ophthalmology Society listserv. The study received human subjects approval through the University of California at Los Angeles Institutional Review Board. The survey identified patients sent for neuro-ophthalmic consultation while receiving one or more of a PD-1 inhibitor (pembrolizumab, nivolumab, or cemiplimab); PD-L1 inhibitor (atezolizumab, avelumab, or durvalumab); or the CTLA-4 inhibitor ipilimumab. Thirty-one patients from 14 institutions were identified. Patient demographics, neuro-ophthalmic diagnosis, diagnostic testing, severity, treatment, clinical response, checkpoint inhibitor drug used, and cancer diagnosis was obtained. RESULTS: The checkpoint inhibitors used in these patients included pembrolizumab (12/31), nivolumab (6/31), combined ipilimumab with nivolumab (7/31, one of whom also received pembrolizumab during their course of treatment), durvalumab (3/31), ipilimumab (2/31), and cemiplimab (1/31). Malignant melanoma (16/31) or nonsmall cell lung carcinoma (6/31) were the most common malignancies. The median time between first drug administration and the time of ophthalmological symptom onset was 14.5 weeks. Eleven patients had involvement of the optic nerve, 7 patients had inflammatory orbital or extraocular muscle involvement, 6 patients had ocular involvement from neuromuscular junction dysfunction, 4 patients had cranial nerve palsy, and 4 patients had non neuro-ophthalmic complications. Use of systemic corticosteroids with or without stopping the checkpoint inhibitor resulted in improvement of most patients with optic neuropathy, and variable improvement for the other ophthalmic conditions. CONCLUSION: This study describes the variable neuro-ophthalmic adverse events associated with use of immune checkpoint inhibitors and contributes a more thorough understanding of their clinical presentations and treatment outcomes. We expect this will increase awareness of these drug complications and guide specialists in the care of these patients.

Case Report: Phenazopyridine-Induced Sulfhemoglobinemia in an 83-Year-Old Presenting with Dyspnea.

BACKGROUND: Sulfhemoglobinemia is a rare dyshemoglobinemia that presents similarly to methemoglobinemia. CASE REPORT: An 83-year-old woman with stage IV ovarian cancer presented to the Emergency Department after a near syncopal spell and was found to be cyanotic with a pulse oximetry reading of 71%. Pulse oximetry improved to only the mid-80s range with administration of high-flow oxygen. Her arterial blood gas on supplemental high-flow oxygen demonstrated a PaO2 of 413 mm Hg and methemoglobin of 1.2%, but also noted the interference of the co-oximetry with sulfhemoglobinemia. Further history revealed that the patient had recently been started on phenazopyridine. The phenazopyridine was stopped, an exchange transfusion was offered but declined, and the patient was discharged to home hospice. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: The diagnosis of sulfhemoglobinemia can be challenging given that routine co-oximetry does not identify it. The clue to the diagnosis is that the cyanotic-appearing patient has a normal or elevated PaO2 and seems to be less ill than expected, given the degree of cyanosis. Sulfhemoglobinemia does not reverse with the administration of methylene blue.

Pilot Study of Hyperfractionated Dosing of Lutetium-177-Labeled Antiprostate-Specific Membrane Antigen Monoclonal Antibody J591 (177 Lu-J591) for Metastatic Castration-Resistant Prostate Cancer.

LESSONS LEARNED: Hyperfractionation of lutetium-177 (177 Lu)-J591 for patients with metastatic castration-resistant prostate cancer did not appear to have any additional advantage over the single dose 177 Lu-J591 or fractionated two-dose 177 Lu-J591 therapy. Definite conclusions were challenging because of the small sample size of this study, and so further studies are needed to evaluate the viability of the hypothesis. On the basis of available data, a registration study of 177 Lu-J591 (also known as TLX591) is planned and will use the two-dose fractionation schedule (Telix Pharma Q3 2019 update https://telixpharma.com/news-media/). BACKGROUND: Phase I and II single-dose studies of lutetium-177 (177 Lu)-J591, a radio-labeled antibody binding prostate-specific membrane antigen (PSMA), demonstrated safety and efficacy with dose response. Modest dose fractionation of 177 Lu-J591 (2 doses) has less myelosuppression per similar cumulative dose, allowing higher doses to be administered safely. We hypothesized that additional dose fractionation would allow a higher cumulative dose, potentially with less toxicity and more efficacy. METHODS: Men with progressive metastatic castration-resistant prostate cancer and adequate organ function were enrolled. 177 Lu-J591 was administered at 25 mCi/m2 every 2 weeks until the emergence of related grade 2 toxicity. 177 Lu-J591 imaging was performed and circulating tumor cell (CTC) counts were measured before and after treatment along with standard monitoring. RESULTS: Six subjects in a single cohort, with a median age of 68.6 years, were enrolled. Patients received three to six doses (cumulative 75-150 mCi/m2 ). Two (33%) patients had >30% prostate-specific antigen (PSA) decline and three (50%) had CTC count decline. Two (33%) experienced grade (Gr) 4 neutropenia (without fever), three (50%) had Gr 4 thrombocytopenia (without hemorrhage), and two (33%) required platelet transfusions. Following hematological improvement, two patients developed worsening cytopenia during prostate cancer progression; bone marrow biopsies revealed infiltrative tumor replacing normal marrow elements without myelodysplasia. Targeting of known disease sites was seen on planar imaging in all. CONCLUSION: Hyperfractionation of 177 Lu-J591 is feasible but does not seem to have significant advantages over the two-dose fractionation regimen.

Preliminary Assessment of Intravoxel Incoherent Motion Diffusion-Weighted MRI (IVIM-DWI) Metrics in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects aging populations. Current MRI techniques are often limited in their sensitivity to underlying neuropathological changes. PURPOSE: To characterize differences in voxel-based morphometry (VBM), apparent diffusion coefficient (ADC), and intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) metrics in aging populations. Additionally, to investigate the connection between cognitive assessments and neuroimaging metrics. STUDY TYPE: Prospective/cross-sectional. POPULATION: In all, 49 subjects, including 13 with AD dementia, 12 with mild cognitive impairment (MCI), and 24 healthy controls (HC). FIELD STRENGTH/SEQUENCE: 3T/magnetization-prepared rapid acquisition gradient echo (MP-RAGE) and IVIM-DWI ASSESSMENT: All participants completed a cognitive screening battery prior to MRI. IVIM-DWI maps (pure diffusion coefficient [D], pseudodiffusion coefficient [D*], and perfusion fraction [f]) were generated from a biexponential fit of diffusion MRI data. VBM was performed on the standard T1 -weighted MP-RAGE structural images. Group-wise templates were used to compare across groups. STATISTICAL TESTS: Analysis of covariance (ANCOVA) with gender and age as covariates (familywise error [FWE] corrected, post-hoc comparisons using Bonferroni correction) for group comparisons. Partial-η2 and Hedges' g were used for effect-size analysis. Spearman's correlations (false discovery rate [FDR]-corrected) for the relationship between cognitive scores and imaging. RESULTS: Clusters of significant group-wise differences were found mainly in the temporal lobe, hippocampus, and amygdala using all VBM and IVIM methods (P < 0.05 FWE). While VBM showed significant changes between MCI and AD groups and between HC and AD groups, no significant clusters were observed between HC and MCI using VBM. ADC and IVIM-D demonstrated significant changes, at P < 0.05 FWE, between HC and MCI, notably in the amygdala and hippocampus. Several voxel-based correlations were observed between neuroimaging metrics and cognitive tests within the cognitively impaired groups (P < 0.05 FDR). DATA CONCLUSION: These findings suggest that IVIM-DWI metrics may be earlier biomarkers for AD-related changes than VBM. The use of these techniques may provide novel insight into subvoxel neurodegenerative processes. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2 J. MAGN. RESON. IMAGING 2020;52:1811-1826.

The effect of current and former tobacco use on outcomes after primary reverse total shoulder arthroplasty.

BACKGROUND: The purpose of this study was to determine the influence of current and former tobacco use on minimum 2-year clinical and radiographic outcomes after reverse total shoulder arthroplasty (RTSA). METHODS: Review of primary RTSA patient data identified 186 patients with at least 2 years of follow-up. Patients were classified as nonsmokers (76 patients), former smokers (89 patients), or current smokers (21 patients). Assessment included preoperative and postoperative visual analog scale pain scores, American Shoulder and Elbow Surgeons scores, strength, range of motion, complications, revisions, and narcotic use. Radiographs were analyzed for signs of loosening or mechanical failure. RESULTS: Overall mean age of the patients was 70 (48-87) years, and mean follow-up was 2.6 (2.0-5.7) years. Smokers (62.1 years) were significantly younger than nonsmokers (70.7 years) and former smokers (70.8 years; P = .00002). All patients had significant improvements in pain, American Shoulder and Elbow Surgeons score, strength, and forward flexion range of motion; however, smokers had higher visual analog scale pain scores (mean, 2.5) than nonsmokers (mean, 1.8) or former smokers (mean, 1.0; P = .014). Otherwise, no differences were found regarding any of the postoperative parameters (P > .05). CONCLUSIONS: Aside from increased patient-reported pain, current tobacco use does not appear to negatively affect outcomes after primary RTSA. The RTSA design obviates the need for a functioning rotator cuff, possibly mitigating tobacco's negative effects previously demonstrated in rotator cuff repair and anatomic total shoulder arthroplasty. Former users obtained outcomes similar to those of nonusers, suggesting that tobacco use is a modifiable risk factor to achieve optimal pain relief after RTSA.

Swallow-Induced Eyelid Myokymia: A Novel Synkinesis Syndrome.

Herein, we describe a novel manifestation of facial nerve synkinesis, swallow-induced eyelid myokymia, and hypothesise that this phenomenon is due to synkinetic facial nerve innervations of the stylohyoid-posterior digastric complex of suprahyoid muscles and orbicularis oculi muscle. In our patient's case, onabotulinum toxin A treatment provided good therapeutic response. Swallow-induced eyelid myokymia is a unique and previously unreported variety of facial nerve synkinesis.

Label-free cocaine aptasensor based on a long-period fiber grating.

In this Letter, we combined a promising bioreceptor, a cocaine aptamer MN6, with an ultrasensitive optical platform long-period fiber grating (LPFG) to create a new cocaine biosensor. The cocaine induces a conformational rearrangement of the aptamer which changes the refractive index around the LPFG producing a measurable shift of the transmission spectrum. We were able to track subtle interaction between the receptor and cocaine molecules over a concentration range of 25 to 100 µM. The presented biosensor does not require labeling or signal enhancement, resulting in a simple user-friendly device.

A reanalysis of protein tyrosine phosphatases inhibitory studies using the unnatural substrate analogue p-nitrophenyl phosphate.

The determination of inhibition mode is extremely important in the understanding of drug interactions and biological mechanisms. The data presented by Hjortness et al. in their recent papers [1,2] on the inhibition of various Protein Tyrosine Phosphatases addresses this issue in an exemplary manner, determining the mode of inhibition based on global fitting of the data to multiple models of inhibition. However, Protein Tyrosine Phosphatases are known to undergo substrate induced conformational changes, so inhibition models which are based on enzyme that adhere to Michaelis-Menten single substrate kinetics may not be appropriate for examining these interactions. To examine the appropriateness of these models, the reported raw data was examined using a recently developed template for global data fitting in Excel. Based on the sum of squared residuals this analysis demonstrates that the excel template was able to match or improve on the reported fittings and demonstrates that a better fit can be achieved with a model that takes into account p-nitrophenyl phosphate-based substrate activation. Whether the substrate activation observed with this model substrate has physiological relevance is debatable, however, it does correspond to the known conformational rearrangement these enzymes undergo when working on their larger peptide substrates.