Grand Multiparity and Obstetric Outcomes in a Contemporary Cohort: The Role of Increasing Parity.

OBJECTIVE: Evidence is inconsistent regarding grand multiparity and its association with adverse obstetric outcomes. Few large American cohorts of grand multiparas have been studied. We assessed if increasing parity among grand multiparas is associated with increased odds of adverse perinatal outcomes. STUDY DESIGN: Multicenter retrospective cohort of patients with parity ≥ 5 who delivered a singleton gestation in New York City from 2011 to 2019. Outcomes included postpartum hemorrhage, preterm delivery, hypertensive disorders of pregnancy, shoulder dystocia, birth weight > 4,000 and <2,500 g, and neonatal intensive care unit (NICU) admission. Parity was analyzed continuously, and multivariate analysis determined if increasing parity and other obstetric variables were associated with each adverse outcome. RESULTS: There were 2,496 patients who met inclusion criteria. Increasing parity among grand multiparas was not associated with any of the prespecified adverse outcomes. Odds of postpartum hemorrhage increased with history (adjusted odds ratio [aOR]: 2.65, 95% confidence interval [1.83, 3.84]) and current cesarean delivery (aOR: 4.59 [3.40, 6.18]). Preterm delivery was associated with history (aOR: 12.36 [8.70-17.58]) and non-White race (aOR: 1.90 [1.27, 2.84]). Odds of shoulder dystocia increased with history (aOR: 5.89 [3.22, 10.79]) and birth weight > 4,000 g (aOR: 9.94 [6.32, 15.65]). Birth weight > 4,000 g was associated with maternal obesity (aOR: 2.92 [2.22, 3.84]). Birth weight < 2,500 g was associated with advanced maternal age (aOR: 1.69 [1.15, 2.48]), chronic hypertension (aOR: 2.45 [1.32, 4.53]), and non-White race (aOR: 2.47 [1.66, 3.68]). Odds of hypertensive disorders of pregnancy increased with advanced maternal age (aOR: 1.79 [1.25, 2.56]), history (aOR: 10.09 [6.77-15.04]), and non-White race (aOR: 2.79 [1.95, 4.00]). NICU admission was associated with advanced maternal age (aOR: 1.47 [1.06, 2.02]) and non-White race (aOR: 2.57 [1.84, 3.58]). CONCLUSION: Among grand multiparous patients, the risk factor for adverse maternal, obstetric, and neonatal outcomes appears to be occurrence of those adverse events in a prior pregnancy and not increasing parity itself. KEY POINTS: · Increasing parity is not associated with adverse obstetric outcomes among grand multiparas.. · Prior adverse pregnancy outcome is a risk factor for the outcome among grand multiparas.. · Advanced maternal age is associated with adverse obstetric outcomes among grand multiparas..

Cesarean Birth Morbidity among Women with SARS-CoV-2.

OBJECTIVE: The objective of this study was to compare maternal outcomes of women with and without severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections who underwent cesarean births. STUDY DESIGN: This was a matched cohort study of pregnant women who had a cesarean birth between March 15, 2020, and May 20, 2020. Cases included women who tested positive for SARS-CoV-2. For every case, two patients who tested negative for SARS-CoV-2 were matched by maternal age, gestational age, body mass index, primary or repeat cesarean birth, and whether the procedure was scheduled or unscheduled. We compared rates of adverse postcesarean complications (intraoperative bladder or bowel injury, estimated blood loss more than or equal to 1,000 mL, hemoglobin drop more than 3 g/dL, hematocrit drop more than 10%, need for blood transfusion, need for hysterectomy, maternal intensive care unit admission, postoperative fever, and development of surgical site infection), with the primary outcome being a composite of those outcomes. We also assessed duration of postoperative stay. Fisher's exact tests were performed to compare the primary outcome between both groups. RESULTS: Between March and May 2020, 202 women who subsequently underwent cesarean birth were tested for SARS-CoV-2. Of those 202, 43 (21.3%) patients were positive. They were matched to 86 patients who tested negative. There was no significant difference in the rate of composite adverse surgical outcomes between the groups (SARS-CoV-2 infected 27.9%, SARS-CoV-2 uninfected 25.6%; p = 0.833). There was a higher rate of postoperative fevers (20.9 vs. 5.8%; p = 0.015), but that did not result in a longer length of stay (p = 0.302). CONCLUSION: Pregnant women with SARS-CoV-2 who underwent a cesarean birth did not have an increased risk of adverse surgical outcomes, other than fever, compared with pregnant women without SARS-CoV-2. KEY POINTS: · Women with SARS-CoV-2 had more postoperative fevers.. · Length of stay did not differ based on SARS-CoV-2 status.. · Composite postoperative outcome did not differ based on SARS-CoV-2 status..

Temporal Trend in SARS-CoV-2 Symptoms in Pregnant Women.

OBJECTIVE: The objective of this study was to examine temporal trends in the clinical presentation of patients diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in pregnancy. STUDY DESIGN: This is a retrospective cohort study of pregnant women who were universally screened for SARS-CoV-2 and tested positive. This multi-center study of admissions to labor and delivery units in New York City and Long Island included all SARS-CoV-2-infected pregnant women admitted to labor and delivery units between April 10th and June 4th 2020. Six Northwell Health hospitals and Maimonides Medical Center were included in the study. The main measures of the study included patient reports of COVID-19 symptoms: fever, cough, chest pain, shortness of breath, nausea, vomiting, and intensive care unit (ICU) admissions. The main outcome measure was the percentage of all infected women who reported any of the above symptoms. RESULTS: In total, 427 infected pregnant women were included in the study. There was a statistically significant decline in the percentage of patients presenting with any symptoms over the course of the study. In addition, disease severity, symptoms of fever, cough, and chest pain/shortness of breath also significantly declined over time, and no ICU admissions were noted after the third week of April. CONCLUSION: There was a temporal shift away from symptomatic presentation in pregnant women diagnosed with SARS-CoV-2 over the course of the first months of the epidemic in New York. Further studies are necessary to elucidate the cause of this change in presentation among pregnant women, to determine whether this trend is also observed in other patient populations. KEY POINTS: · Retrospective cohort review of 427 SARS-CoV-2-infected pregnant women admitted to labor and delivery units.. · A significant decline in the percentage of patients presenting with symptoms over time was noted.. · Further studies are necessary to elucidate the cause of this change in presentation.. · Theories for the noted trend: viral evolution, decreased viral inoculums, and prolonged polymerase chain reaction positivity..

Adverse outcomes in early term versus full-term deliveries among higher-order cesarean births.

OBJECTIVE: In an attempt to avoid emergency deliveries of women with multiple prior scars, providers may choose to schedule those repeat cesarean births prior to 39 weeks. Our primary goal was to compare rates of assisted ventilation use between neonates with early term (37°-386 weeks) and full-term (39°-396 weeks) deliveries among women with three or more prior cesarean births. METHODS: A retrospective cohort study of women with three or more previous cesarean births. The study group consisted of women who delivered at early term (37°-386 weeks). The control group consisted of women who delivered at full term (39°-396 weeks gestation). Women with a history of pre-gestational diabetes, gestational hypertension and chronic hypertension were excluded. Data were extracted from the 2017 United States Natality database. Characteristics were compared between groups for potential confounders. Primary outcome, neonatal assisted ventilation use greater than 6 h, and other secondary outcomes (including immediate assisted ventilation in the neonate and uterine rupture) were compared between groups. Multivariable logistic regression analyses were performed to adjust for potential confounding factors between groups. RESULTS: A total of 28,584 women with three or more prior cesarean births were included. There were 12,391 women who delivered at early term, and 16,193 who delivered at full term. Neonates born from women who delivered at early term had an increased risk of assisted ventilation use greater than 6 h compared with neonates who delivered at full term (assisted ventilation greater than 6 h, adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) [1.59-2.73]). Neonates delivered at early term were also more likely to need immediate ventilation use than were neonates delivered at full term (aOR 1.52, 95% CI [1.33-1.73]). Women who delivered at early term had a higher rate of uterine rupture compared with women who delivered at full term (OR 5.67, 95% CI [2.33-13.79]). CONCLUSION: Higher order cesarean births performed early term had an increased risk of neonatal assisted ventilation use greater than 6 h compared with full-term births. These results argue against delivering women with multiple prior uterine scars before term in an attempt to avoid emergency sections.

The use of bilateral transversus abdominis plane blocks with liposomal bupivacaine on postoperative cesarean delivery patients during COVID-19 pandemic is associated with reduced narcotics use and reduced length of stay.

BACKGROUND: The use of transversus abdominis plane blocks has been previously shown in both large-scale studies and our own institution to significantly reduce postoperative pain and opioid use. In addition, the use of bilateral transversus abdominis plane blocks using liposomal bupivacaine in combination with neuraxial morphine significantly reduced post-cesarean-delivery pain and opioid use. During the COVID-19 crisis, our anesthesia department in a collaborative effort with our obstetric colleagues thought that the use of bilateral transversus abdominis plane blocks with liposomal bupivacaine could reduce the use of opioids to treat postoperative pain and might result in decreased length of stay. METHODS: After institutional review board approval, a retrospective study of 288 patients who underwent cesarean delivery under spinal or epidural (neuraxial) anesthesia at Maimonides Medical Center in Brooklyn, NY was conducted. Historical controls were from 142 consecutive patients from 1 January 2012 through 12 May 2012. An additional set of controls consisted of 30 consecutive patients from 10 March 2020 through 13 April 2020. The primary outcome data analyzed were the use of opioids and length of stay. RESULTS: Post cesarean delivery, patients who received both bilateral transversus abdominis plane blocks with liposomal bupivacaine and neuraxial morphine was associated with a significant decrease in the number of patients using post operative opioids, 54%-60% decreased to 18% (p < 0.001), and a decreased length of stay; 3.1 days was reduced to 2.39 (p < 0.001). CONCLUSION: Neuraxial opioids combined with liposomal bupivacaine transversus abdominis plane blocks provided significant pain relief for patients post cesarean delivery, required less post operative opioids, and facilitated earlier discharge that may aid in reducing patient exposure and hospital burden secondary to COVID-19.

Histologic and Immunohistochemical Evaluation of 65 Placentas From Women With Polymerase Chain Reaction-Proven Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

CONTEXT.­: Coronavirus disease 2019 (COVID-19) has been shown to have effects outside of the respiratory system. Placental pathology in the setting of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains a topic of great interest because earlier studies have shown mixed results. OBJECTIVE.­: To ascertain whether maternal SARS-CoV-2 infection is associated with any specific placental histopathology, and to evaluate the virus's propensity for direct placental involvement. DESIGN.­: Placentas from 65 women with polymerase chain reaction-proven SARS-CoV-2 infection underwent histologic evaluation using Amsterdam consensus group criteria and terminology. Another 85 placentas from women without SARS-CoV-2 constituted the negative control group. A total of 64 of the placentas from the SARS-CoV-2-positive group underwent immunohistochemical staining for SARS-CoV-2 nucleocapsid protein. RESULTS.­: Pathologic findings were divided into maternal vascular malperfusion, fetal vascular malperfusion, chronic inflammatory lesions, amniotic fluid infection sequence, increased perivillous fibrin, intervillous thrombi, increased subchorionic fibrin, meconium-laden macrophages (M-LMs) within fetal membranes, and chorangiosis. There was no statistically significant difference in prevalence of any specific placental histopathology between the SARS-CoV-2-positive and SARS-CoV-2-negative groups. There was no immunohistochemical evidence of SARS-CoV-2 virus in any of the 64 placentas that underwent staining for viral nucleocapsid protein. CONCLUSIONS.­: Our study results and a literature review suggest that there is no characteristic histopathology in most placentas from women with SARS-CoV-2 infection. Likewise, direct placental involvement by SARS-CoV-2 is a rare event.

The Relationship between Status at Presentation and Outcomes among Pregnant Women with COVID-19.

OBJECTIVE: This study was aimed to compare maternal and pregnancy outcomes of symptomatic and asymptomatic pregnant women with novel coronavirus disease 2019 (COVID-19). STUDY DESIGN: This is a retrospective cohort study of pregnant women with COVID-19. Pregnant women were divided into two groups based on status at admission, symptomatic or asymptomatic. All testing was done by nasopharyngeal swab using polymerase chain reaction (PCR) for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Initially, nasopharyngeal testing was performed only on women with a positive screen (symptoms or exposure) but subsequently, testing was universally performed on all women admitted to labor and delivery. Chi-square and Wilcoxon's rank-sum tests were used to compare outcomes between groups. RESULTS: Eighty-one patients were tested because of a positive screen (symptoms [n = 60] or exposure only [n = 21]) and 75 patients were universally tested (all asymptomatic). In total, there were 46 symptomatic women and 22 asymptomatic women (tested based on exposure only [n = 12] or as part of universal screening [n = 10]) with confirmed COVID-19. Of symptomatic women (n = 46), 27.3% had preterm delivery and 26.1% needed respiratory support while none of the asymptomatic women (n = 22) had preterm delivery or need of respiratory support (p = 0.007 and 0.01, respectively). CONCLUSION: Pregnant women who presented with COVID19-related symptoms and subsequently tested positive for COVID-19 have a higher rate of preterm delivery and need for respiratory support than asymptomatic pregnant women. It is important to be particularly rigorous in caring for COVID-19 infected pregnant women who present with symptoms. KEY POINTS: · Respiratory support is often needed for women who present with symptoms.. · Low rate of severe disease in women who present without symptoms.. · There were no neonatal infections on day 0 of life..

Caring for Pregnant Patients with COVID-19: Practical Tips Getting from Policy to Practice.

Novel coronavirus disease 2019 (COVID-19) is a pandemic with most American cases in New York. As an institution residing in a high-prevalence zip code, with over 8,000 births annually, we have cared for over 80 COVID-19-infected pregnant women, and have encountered many challenges in applying new national standards for care. In this article, we review how to change outpatient and inpatient practices, develop, and disseminate new hospital protocols, and we highlight the psychosocial challenges for pregnant patients and their providers. KEY POINTS: · Novel coronavirus disease 2019 (COVID-19) information rapidly changes.. · Multidisciplinary communication is key.. · This study addresses psychosocial challenges..

Hyperemesis Gravidarum: A Review of Recent Literature.

BACKGROUND: In the United States, hyperemesis gravidarum is the most common cause of hospitalization during the first half of pregnancy and is second only to preterm labor for hospitalizations in pregnancy overall. In approximately 0.3-3% of pregnancies, hyperemesis gravidarum is prevalent and this percentage varies on account of different diagnostic criteria and ethnic variation in study populations. Despite extensive research in this field, the mechanism of the disease is largely unknown. Although cases of mortality are rare, hyperemesis gravidarum has been associated with both maternal and fetal morbidity. The current mainstay of treatment relies heavily on supportive measures until improvement of symptoms as part of the natural course of hyperemesis gravidarum, which occurs with progression of gestational age. However, studies have reported that severe, refractory disease manifestations have led to serious adverse outcomes and to termination of pregnancies. SUMMARY: Despite extensive research in the field, the pathogenesis of hyperemesis gravidarum remains unknown. Recent literature points to a genetic predisposition in addition to previously studied factors such as infectious, psychiatric, and hormonal contributions. Maternal morbidity is common and includes psychological effects, financial burden, clinical complications from nutritional deficiencies, gastrointestinal trauma, and in rare cases, neurological damage. The effect of hyperemesis gravidarum on neonatal health is still debated in literature with conflicting results regarding outcomes of birth weight and prematurity. Available therapy options remain largely unchanged in the past several decades and focus on parenteral antiemetic medications, electrolyte repletion, and nutritional support. Most studies of therapeutic options do not consist of randomized control studies and cross-study analysis is difficult due to considerable variation of diagnostic criteria. Key Messages: Hyperemesis gravidarum carries a significant burden on maternal health and US health care. Most published research on pathogenesis is observational and suggests multifactorial associations with hyperemesis gravidarum. Precise, strictly defined criteria for clinical diagnosis are likely to benefit meta-analyses of further research studies regarding pathogenesis as well as therapeutic options.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Diagnostic inconsistency of faecal immunochemical tests for haemoglobin in population screening of colorectal cancer.

BACKGROUND: There is currently very little data available on the consistency of quantitative and qualitative faecal immunochemical test (FIT) for colorectal cancer screening. METHODS: A representative random population (n=1889, 40-74 year olds) in Jiashan, China was invited for FIT screening in 2012. Faecal samples were collected by a single specimen collection device and simultaneously tested by a quantitative FIT (OC-SENSOR, OC) and two qualitative FITs (FIT A and FIT B with intrinsic positive haemoglobin cut-off concentrations of 20 µg Hb/g faeces and 40 µg Hb/g faeces, respectively). The observational criteria for a positive result of the qualitative FIT were set according to the density of the colour appearing in the test strip. The results produced by the quantitative and qualitative FIT for each sample were compared. κ coefficient was used to measure consistency. RESULTS: A total of 1368 (72.4%) individuals returned faecal samples. Both FIT A and FIT B precisely identified all faecal samples with haemoglobin concentration above 100 µg Hb/g faeces, but the overall consistency was poor for OC & FIT A (κ=0.32, 95% CI 0.20-0.44) and was moderate for OC & FIT B (κ=0.74, 95% CI 0.64-0.85). A more favourable consistency (κ=0.64, 95% CI 0.57-0.72) was achieved when a different positive criterion was employed for FIT A. CONCLUSIONS: The diagnostic inconsistency between quantitative and qualitative FITs mainly exists in the faecal samples with low haemoglobin concentrations. Refining the criterion for a positive result may be a feasible way to improve the accuracy of qualitative FIT.

Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Assembling DNA fragments by USER fusion.

Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct.

Targeted manipulation of mammalian genomes using designed zinc finger nucleases.

Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.

Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant.

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.