Universal Urine Drug Screening with Rapid Confirmation upon Admission to Labor and Delivery.

OBJECTIVE: This study aimed to describe our experience with universal urine drug screening (UDS) with rapid confirmation (RC) via liquid chromatography mass spectrometry (LC-MS) before infant's discharge, in efforts to increase detection of neonates at risk of neonatal opioid withdrawal syndrome (NOWS) while reducing patient burden related to false positive results. STUDY DESIGN: Two-phase retrospective study of all pregnant women admitted to our labor and delivery (L&D) unit before (phase 1, April 2018-March 2019) and after (phase 2, October 2019-September 2020) RC of UDS was initiated. Urine samples were obtained on admission and screened for drugs using an enzyme immunoassay with positive results reflexed to confirmation via LC-MS. The turnaround time for LC-MS was 1 week in phase 1 and 24 hours in phase 2. For mothers with positive LC-MS confirmation, the infant's meconium was sent for drug screening. Positive results were determined to be true or false positive based on urinary LC-MS results. The primary outcome was the rate of opioid-positive mothers who were unanticipated. The secondary outcome was the difference in rate of neonates who were observed for NOWS, before and after implementation of RC with LC-MS. RESULTS: In phase 2, a total of 2,395 deliveries occurred of which 2,122 (88.6%) had available UDS results. Fifty-two (2.5%) women had a positive UDS for at least one drug with LC-MS confirmation. Of those, 25 were true positive and 27 were false positive. Twenty-one (84%) true positive mothers were taking opioids and 8 (37%) of them were unanticipated positives. Among mothers with positive UDS for opioids, the neonatal observation rate for development of NOWS was 100% (22/22) and 48% (21/44) before and after implementation of LC-MS RC, respectively. CONCLUSION: Universal UDS and LC-MS RC in L&D may improve detection of unanticipated positive mothers whose infants are at risk of NOWS. RC of positive results allows intervention only for confirmed cases. KEY POINTS: · Universal UDS can detect more infants at risk of NOWS.. · Rapid confirmation of positive UDS reduces burden.. · Only confirmed infants should be observed in the neonatal intensive care unit.. · Child Protective Services should only be notified of confirmed opioid-positive results..

Disseminating child abuse clinical decision support among commercial electronic health records: Effects on clinical practice.

Objectives: The use of electronic health record (EHR)-embedded child abuse clinical decision support (CA-CDS) may help decrease morbidity from child maltreatment. We previously reported on the development of CA-CDS in Epic and Allscripts. The objective of this study was to implement CA-CDS into Epic and Allscripts and determine its effects on identification, evaluation, and reporting of suspected child maltreatment. Materials and Methods: After a preimplementation period, CA-CDS was implemented at University of Wisconsin (Epic) and Northwell Health (Allscripts). Providers were surveyed before the go-live and 4 months later. Outcomes included the proportion of children who triggered the CA-CDS system, had a positive Child Abuse Screen (CAS) and/or were reported to Child Protective Services (CPS). Results: At University of Wisconsin (UW), 3.5% of children in the implementation period triggered the system. The CAS was positive in 1.8% of children. The proportion of children reported to CPS increased from 0.6% to 0.9%. There was rapid uptake of the abuse order set.At Northwell Health (NW), 1.9% of children in the implementation period triggered the system. The CAS was positive in 1% of children. The child abuse order set was rarely used. Preimplementation, providers at both sites were similar in desire to have CA-CDS system and perception of CDS in general. After implementation, UW providers had a positive perception of the CA-CDS system, while NW providers had a negative perception. Discussion: CA-CDS was able to be implemented in 2 different EHRs with differing effects on clinical care and provider feedback. At UW, the site with higher uptake of the CA-CDS system, the proportion of children who triggered the system and the rate of positive CAS was similar to previous studies and there was an increase in the proportion of cases of suspected abuse identified as measured by reports to CPS. Our data demonstrate how local environment, end-users' opinions, and limitations in the EHR platform can impact the success of implementation. Conclusions: When disseminating CA-CDS into different hospital systems and different EHRs, it is critical to recognize how limitations in the functionality of the EHR can impact the success of implementation. The importance of collecting, interpreting, and responding to provider feedback is of critical importance particularly with CDS related to child maltreatment.

Exploitation, Labor and Sex Trafficking of Children and Adolescents: Health Care Needs of Patients.

Exploitation and labor and sex trafficking of children and adolescents is a major public health problem in the United States and throughout the world. Significant numbers of US and non-US-born children and adolescents (including unaccompanied immigrant minors) are affected by this growing concern and may experience a range of serious physical and mental health problems associated with human trafficking and exploitation (T/E). Despite these considerations, there is limited information available for health care providers regarding the nature and scope of T/E and how providers may help recognize and protect children and adolescents. Knowledge of risk factors, recruitment practices, possible indicators of T/E, and common medical, mental, and emotional health problems experienced by affected individuals will assist health care providers in recognizing vulnerable children and adolescents and responding appropriately. A trauma-informed, rights-based, culturally sensitive approach helps providers identify and treat patients who have experienced or are at risk for T/E. As health care providers, educators, and leaders in child advocacy and development, pediatricians play an important role in addressing the public health issues faced by children and adolescents who experience exploitation and trafficking. Working across disciplines with professionals in the community, health care providers can offer evidence-based medical screening, treatment, and holistic services to individuals who have experienced T/E and assist vulnerable patients and families in recognizing signs of T/E.

Dissemination of child abuse clinical decision support: Moving beyond a single electronic health record.

BACKGROUND: Child maltreatment is a leading cause of pediatric morbidity and mortality. We previously reported on development and implementation of a child abuse clinical decision support system (CA-CDSS) in the Cerner electronic health record (EHR). Our objective was to develop a CA-CDSS in two different EHRs. METHODS: Using the CA-CDSS in Cerner as a template, CA-CDSSs were developed for use in four hospitals in the Northwell Health system who use Allscripts and two hospitals in the University of Wisconsin health system who use Epic. Each system had a combination of triggers, alerts and child abuse-specific order sets. Usability evaluation was done prior to launch of the CA-CDSS. RESULTS: Over an 18-month period, a CA-CDSS was embedded into Epic and Allscripts at two hospital systems. The CA-CDSSs vary significantly from each other in terms of the type of triggers which were able to be used, the type of alert, the ability of the alert to link directly to child abuse-specific order sets and the order sets themselves. CONCLUSIONS: Dissemination of CA-CDSS from one EHR into the EHR in other health care systems is possible but time-consuming and needs to be adapted to the strengths and limitations of the specific EHR. Site-specific usability evaluation, buy-in of multiple stakeholder groups and significant information technology support are needed. These barriers limit scalability and widespread dissemination of CA-CDSS.

A Comprehensive Pediatric Acute Sexual Assault Protocol: From Emergency Department to Outpatient Follow-up.

OBJECTIVE: Close medical follow-up after pediatric acute sexual assault is recommended and may mitigate adverse consequences and decrease long-term comorbidities. The objectives are to (1) examine adherence to a comprehensive outpatient medical follow-up protocol after evaluation in the emergency department in a pediatric population and (2) identify characteristics associated with patient adherence to inform the utilization of a medical follow-up protocol after pediatric acute sexual assault. METHODS: A retrospective medical record review was conducted of patients younger than 18 years presenting to the emergency department from January 1, 2010, to December 31, 2013, with a discharge diagnosis suggestive of sexual assault/abuse. We examined differences in demographics, assault characteristics, and medical/legal needs of patients who were evaluated in follow-up versus patients who were not. RESULTS: Of 182 patients, 60.4% completed follow-up appointments with the child protection center. Younger patients had follow-up rates higher than older patients (70.2% vs 50%; odds ratio [OR], 0.42). For patients where child protective services or law enforcement were called, follow-up rates were 74.2% and 64.7%, respectively (OR, 2.5; OR, 3.1). All patients with anogenital injuries on initial examination were seen in follow-up. The majority of patients who followed-up were accompanied by a caregiver/relative (95%). CONCLUSIONS: (1) Caregivers should be integrated into the evaluation to facilitate compliance with follow-up; (2) child abuse specialists may be consulted to facilitate specific interventions and recommendations; (3) professionals should work as a multidisciplinary team; and (4) the patient's psychological status should be evaluated, and mental health interventions recommended.

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks.

BACKGROUND: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study's objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. METHODS: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. RESULTS: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. CONCLUSION: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks.

Domestic Minor Sex Trafficking: Medical Follow-up for Victimized and High-Risk Youth.

Domestic minor sex trafficking (DMST) has become an increasingly recognized issue associated with both immediate and long-term physical and mental health consequences. Guidelines have focused on potential risk factors, recruitment practices, and health consequences for these youth assisting in identification and intervention efforts. However, recommendations have not been established for continuous medical intervention and follow-up for this vulnerable patient population that includes both patients involved in and at high risk for DMST. Our goal is to highlight preliminary recommendations for and the importance of medical visits for these youth. A comprehensive physical examination, STI testing and treatment, and pregnancy prevention options are important to address the patients' concerns for their body and identify acute and chronic injuries. Further, collaborating with other medical and non-medical providers can provide essential resources for the multifaceted needs of DMST patients.

A Multisymptomatic Child With Bipolar Disorder: How to Track and Sequence Treatment.

Treatment sequences for the multisymptomatic child with bipolar disorder are not adequately described or based on a systematic clinical trial database, and systems for longitudinal tracking of symptoms are rarely utilized. We present a patient whose symptoms of depression, anxiety, attention-deficit/hyperactivity disorder, oppositional behavior, and mania are rated by a parent and plotted on a weekly basis in the Child Network under a Johns Hopkins Institutional Review Board-approved protocol. This 9-year-old girl remained inadequately responsive to lithium or risperidone. We describe a range of other treatment options and a possible sequence for their introduction. We encourage the use of systematic longitudinal ratings to help better visualize course of symptom fluctuations and response of the child to treatment. Given the highly fluctuating course of many symptoms in very young children as illustrated here, prospective monitoring appears essential. The current case also highlights the great unmet need for comparative effectiveness data in children less than 10 years of age to better guide clinical therapeutics.

The Child Network for Parents to Track Their Child's Mood and Behavior.

INTRODUCTION: A wide range of psychiatric disorders are common in young children, especially in those at high risk because of a parent with a unipolar or bipolar mood disorder in the United States. Yet in part because most children are seen in primary care, these illnesses are often not recognized or treated in a timely fashion. To begin to address this problem, we started the Child Network. METHODS: The Child Network is for parents of children age 2-12 with mood or behavioral symptoms or at high risk for them. The parents rate the severity of symptoms of depression, anxiety, attention-deficit/hyperactivity disorder (ADHD), oppositional behavior, and mania on a once a week basis on a secure website under a Johns Hopkins Intramural Review Board (IRB)-approved protocol. These ratings can then be printed out along with any treatments given to aide in visualization of symptom course. A demographics form, which includes diagnoses given to the child in the community, and a symptom checklist are filled out upon Network entry. We report on the retrospective diagnoses and prospective treatment of the first 65 parents to join the Network. RESULTS: The most common diagnoses were anxiety disorders and ADHD followed by disruptive behavioral disorders and bipolar spectrum disorders. Prospective ratings of two or more consecutive weeks of moderate to severe rating in any of the five symptom domains paralleled these diagnoses given in the community. Atypical antipsychotics, anticonvulsant mood stabilizers, and medications for ADHD were among the most widely used drugs. An illustrated example of symptom course is presented. DISCUSSION: Many children continued to show substantial symptom severity despite treatment with an average of 2.2 medications. The Child Network provides a useful longitudinal approach to visualize the course of symptoms, which should help lead to earlier and more effective treatment.

Sex Trafficking of Minors.

Sex trafficking is an increasingly recognized global health crisis affecting every country and region in the world. Domestic minor sex trafficking is a subset of commercial sexual exploitation of children, defined as engagement of minors (<18 years of age) in sexual acts for items of value (eg, food, shelter, drugs, money) involving children victimized within US borders. These involved youth are at risk for serious immediate and long-term physical and mental health consequences. Continued efforts are needed to improve preventive efforts, identification, screening, appropriate interventions, and subsequent resource provision for victimized and high-risk youth.

Domestic Minor Sex Trafficking Patients: A Retrospective Analysis of Medical Presentation.

STUDY OBJECTIVE: To describe the clinical characteristics of patients referred for domestic minor sex trafficking (DMST) to improve identification and intervention. DESIGN: Retrospective cohort study. SETTING: The Lawrence A. Aubin, Sr Child Protection Center at Hasbro Children's Hospital where patients are evaluated by child abuse pediatricians in outpatient, emergency department, and inpatient settings. PARTICIPANTS: A total of 41 patients younger than the age of 18 years referred for the evaluation of DMST involvement between August 1, 2013 and March 30, 2015. INTERVENTIONS AND MAIN OUTCOME MEASURES: We collected demographic, social-environmental, medical, and psychiatric variables from the medical records of patients referred for evaluation who have self-disclosed, been reported with evidence, and/or have histories that place them at high risk for DMST involvement. RESULTS: Children had frequent contact with medical providers, with 81% seen in the year before referral for DMST. Childhood maltreatment and family dysfunction were identified (sexual abuse, 21/37 or 57%; parental substance abuse, 22/37 or 60%) in the 41 patients. Children had medical problems (eg, sexually transmitted infection, 13/41 or 32%), psychiatric needs (eg, acute suicidality, 8/41 or 20%; at least 1 previous psychiatric admission, 19/41 or 46%), and substance use (36/41 or 88%). Although 26/41 (63%) had runaway and 17/41 (42%) lived in a group home placement, 28/41 (68%) currently lived at home and 29/41 (71%) presented with a parent/guardian or relative. CONCLUSION: Children referred for DMST present frequently to physicians and have complex medical and psychiatric needs. Medical providers' increased awareness of this health issue would inform victim identification and intervention.

Xenobiotic phenols in early pregnancy amniotic fluid.

We found detectable levels of three phytoestrogens (enterolactone, daidzein and genistein) and bisphenol A (BPA) in 21 residual amniotic fluid specimens that were collected before 20 weeks gestation. Samples were obtained by amniocentesis from women who were referred to the Mount Sinai Medical center because of advanced maternal age. Phytoestrogens were present in higher concentrations than BPA. Enterolactone was detected at the highest concentration (median 95.9 microg/L), followed by daidzein and genistein (9.5 and 1.4 microg/L, respectively). BPA was present at very low concentrations (10%>LOD of 0.5 microg/L). The relative concentration of the chemicals measured in amniotic fluid were identical to those in urine reported by other studies, i.e. enterolactone>daidzein>genistein>>BPA. Amniotic fluid is a source of fetal exposure to polar xenobiotics that come from the mother.

Functional requirements for the optimal catalytic configuration of the AChE active center.

Functional analysis of the HuAChE active center architecture revealed that accommodation of structurally diverse substrates and other ligands is achieved through interactions with specific subsites such as the acyl pocket, cation binding site, hydrophobic site or the oxyanion hole. Recent studies have begun to unravel the role of this active center architecture in maintaining the optimal catalytic facility of the enzyme through inducing proper alignment of the catalytic triad. The exact positioning of the catalytic glutamate (Glu334) seems to be determined by a hydrogen bond network including several polar residues and water molecules. Disruption of this network by replacement of Ser229 by alanine is thought to remove the Glu334 carboxylate from the vicinity of His447 abolishing catalytic activity. The proper orientation of the catalytic histidine side chain is maintained by these polar interactions as well as through "aromatic trapping" by residues lining the HuAChE active center gorge. Thus, replacement of aromatic residues in the vicinity of His447, as in the F295A/F338A or in the Y72N/Y124Q/W286A/F295L/F297V/Y337A (hexamutant which mimicks the aromatic lining of HuBChE) enzymes, resulted in a dramatic decrease in catalytic activity, which was proposed to originate from catalytically nonproductive mobility of His447. Yet, HuBChE is catalytically efficient indicating that "aromatic trapping" is not the only way to conformationally stabilize the His447 side chain. A possible restriction of this mobility in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Both simulations and reactivities of the actual F295X/F338A enzymes, carrying various aliphatic residues at position 295, indicate that of the bulky amino acids, like leucine or isoleucine, only methionine was capable of maintaining the catalytically viable conformation of His447. The F295M/F338A HuAChE was only two-fold less reactive than the F338A enzyme toward acetylthiocholine, and exhibited wild type-like reactivity toward covalent modifiers of the catalytic Ser203. The findings are consistent with the notion that different combinations of steric interference and specific polar interactions serve to maintain the position of His447 and thereby the high efficiency of the catalytic machinery. The two seemingly conflicting demands on the architecture of the active center-flexible accommodation of substrate and optimal juxtaposition of residues of the catalytic triad, demonstrate the truly amazing molecular design of the AChE active center.

The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers.

The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.

Lessons from functional analysis of AChE covalent and noncovalent inhibitors for design of AD therapeutic agents.

Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).

Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE.

The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.

Is aromaticity essential for trapping the catalytic histidine 447 in human acetylcholinesterase?

Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.