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Innovation in medical education is not only inevitable but a requirement. Manikin-based simulation is currently the gold standard for supplemental clinical training; however, this modality requires significant equipment and personnel to operate. Virtual reality (VR) is emerging as a new method of delivering medical simulation sessions that requires less infrastructure but also allows for greater accessibility and flexibility. VR has slowly been integrated into the medical curriculum in some hospitals; however, more widespread adoption would transform the delivery of medical education for future clinicians. This tutorial introduces educators to the BUILD REALITY (begin, use, identify, leverage, define, recreate, educate, adapt, look, identify, test, amplify) framework, a series of practical tips for designing and implementing a VR-based medical simulation environment in their curriculum. The suggestions are based on the relevant literature and the authors' personal experience in creating and implementing VR environments for medical trainees. Altogether, this paper provides guidance on conducting a needs assessment, setting objectives, designing a VR environment, and incorporating the session into the broader medical curriculum. [ABSTRACT FROM AUTHOR]

One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage. DNA binding | protein-protein interaction | structural analysis ssDNA mimicry

The mode of inhibition of mechanism-based inhibitor (2,Ki = 0.4 (mu)M) of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which is designed to mimic the combined key features of its natural substrates arabinose-5-phosphate and phoshoenolpyruvate into a single molecule, is investigated. A novel frequency-selective rotational-echo double-resonance (REDOR) experiment is developed and applied.

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2-/- mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor. [ABSTRACT FROM AUTHOR]

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity. [ABSTRACT FROM AUTHOR]

Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits. [ABSTRACT FROM AUTHOR]

Proton decoupled 31P and 13C solution NMR experiments were applied to mixtures of 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase, with each of its natural substrates, phosphoenolpyruvate and arabinose-5-phosphate (ASP), and product KDO8P to identify the formation of the enzyme–substrate and enzyme–product complexes. Effects arising from ligand interactions with the enzyme are reported via chemical shifts and line broadening with respect to those of the free ligands in solution, depending on the strength and dynamics of binding under thermodynamic equilibrium conditions. The characterization was done both at low and high field spectrometers, 200 and 500 MHz (1H frequencies), and in cases of 31P NMR measurements, it was demonstrated that only the low field spectrometer is capable of providing direct experimental evidence on the enzyme–ligand interactions. Since both the substrate A5P and the product KDO8P exhibit multiple anomeric forms in solution, evidence for the preference of recognition and binding of particular forms is sought. [Copyright &y& Elsevier]

In this report the mode of inhibition of mechanism-based inhibitor (2, K[sub i] = 0.4 µM) of 3-deoxyD-manno-2-octulosonate-8-phosphate synthase (KDO8PS), which was designed to mimic the combined key features of its natural substrates arabinose-5-phosphate (A5P) and phoshoenolpyruvate (PEP) into a single molecule, was investigated. Our earlier solid-state NMR observations identified the inhibitor to bind in a way that partly mimics ASP, while the phosphonate moiety of its PEP-mimicking part exhibits no interactions with enzyme residues. This result was apparently in disagreement with the competitive inhibition of 2 against PEP and with the later solved crystal structure of KDO8PS-2 binary complex identifying the interactions of its PEP-mimicking part with the enzyme residues that were not detected by solid-state NMR. To solve this discrepancy, further solid-state REDOR NMR and [sup 31]P solution NMR experiments were applied to a variety of enzyme complexes with the substrates and inhibitor. In particular, a novel frequency-selective REDOR experiment was developed and applied. Integration of the solution and solid-state NMR data clearly demonstrates that under conditions of stoichiometric enzyme-ligand ratio at thermodynamic equilibrium (a) PEP binding is unperturbed by the presence of 2 and (b) both PEP and 2 can bind simultaneously to the synthase, i.e., form a ternary complex with PEP occupying its own subsite and 2 occupying A5P's subsite. The latter observation suggests that under the conditions used in our NMR measurements, the inhibition pattern of 2 against PEP should have a mixed type character. Furthermore, the NMR data directly demonstrate the distinction between the relative binding strength of the two moieties of 2: enzyme interactions with PEP-mimicking moiety are much weaker than those with the A5P moiety. This observation is in agreement with KDO8PS-2 crystal structure showing only remote contacts of the phosphonate due to large structural... [ABSTRACT FROM AUTHOR]

Investigates the mechanism of action of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase. Use of solid-state REDOR nuclear magnetic resonance; Types of enzyme residues that bind the phosphate groups of each ligand; Structure of each basic enzyme residue.

Presents a study of spin transition in a manganese(III) porphyrin cation radical, its transformation to a dichloromanganese(IV) porphyrin, and chlorination of hydrocarbons by the latter. Effect of chemical oxidation of (TMP)MnIII(CI) (TMP=the tetramesitylporphyrinato dianion by Fe(CIO4); Detailed information on study.

Physiological changes associated with ageing could negatively impact the crisis resource management skills of acute care physicians. This study was designed to determine whether physician age impacts crisis resource management skills, and crisis resource management skills learning and retention using full-body manikin simulation training in acute care physicians. Acute care physicians at two Canadian universities participated in three 8-min simulated crisis (pulseless electrical activity) scenarios. An initial crisis scenario (pre-test) was followed by debriefing with a trained facilitator and a second crisis scenario (immediate post-test). Participants returned for a third crisis scenario 3–6 months later (retention post-test). For the 48 participants included in the final analysis, age negatively correlated with baseline Global Rating Scale (GRS; r=–0.30, P <0.05) and technical checklist scores (r =–0.44, P <0.01). However, only years in practice and prior simulation experience, but not age, were significant in a subsequent stepwise regression analysis. Learning from simulation-based education was shown with a mean difference in scores from pre-test to immediate post-test of 2.28 for GRS score (P <0.001) and 1.69 for technical checklist correct score (P <0.001); learning was retained for 3–6 months. Only prior simulation experience was significantly correlated with a decreased change in learning (r =–0.30, P <0.05). A reduced amount of prior simulation training and increased years in practice, but not age on its own, were significant predictors of low baseline crisis resource management performance. Simulation-based education leads to crisis resource management learning that is well retained for 3–6 months, regardless of age or years in practice. [ABSTRACT FROM AUTHOR]

UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain-plant homeodomain (TTD-PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD-PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide- binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a smallmolecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD-PHD conformations that are less efficient atH3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD-PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes. [ABSTRACT FROM AUTHOR]

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination. [ABSTRACT FROM AUTHOR]

Summary Polycomb repressive complexes PRC1 and PRC2 regulate expression of genes involved in proliferation and development. In mouse early embryos, however, canonical PRC1 localizes to paternal pericentric heterochromatin (pat-PCH), where it represses transcription of major satellite repeats. In contrast, maternal PCH (mat-PCH) is enriched for H3 lysine 9 tri-methylation (H3K9me3) and Hp1β. How PRC1 is targeted to pat-PCH, yet excluded from mat-PCH, has remained elusive. Here, we identify a PRC1 targeting mechanism that relies on Cbx2 and Hp1β. Cbx2 directs catalytically active PRC1 to PCH via its chromodomain (CD Cbx2 ) and neighboring AT-hook (AT Cbx2 ) binding to H3K27me3 and AT-rich major satellites, respectively. CD Cbx2 prevents AT Cbx2 from interacting with DNA at PCH marked by H3K9me3 and Hp1β. Loss-of-function studies show that Hp1β and not H3K9me3 prevents PRC1 targeting to mat-PCH. Our findings indicate that CD Cbx2 and AT Cbx2 separated by a short linker function together to integrate H3K9me3/HP1 and H3K27me3 states. [ABSTRACT FROM AUTHOR]

Summary: The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response. [Copyright &y& Elsevier]

Introduction: The Microsoft HoloLens is a head-mounted mixed reality device, which allows for overlaying hologram-like computer-generated elements onto the real world. This technology can be combined with preprocedural ultrasound during thoracic epidural placement to create a visual of the ideal needle angulation and trajectory in the users' field of view. This could result in a technically easier and potentially safer alternative to traditional blind landmark techniques., Methods: Patients were randomly assigned to one of two groups: (1) HoloLens-assisted thoracic epidural technique (intervention-group H) or (2) traditional thoracic epidural technique (control-group C). The primary outcome was needling time (defined as skin puncture to insertion of epidural catheter) during the procedure. The secondary outcomes were number of needle punctures, number of needle movements, number of bone contacts, and epidural failure. Procedural pain and recovery room pain levels were also evaluated., Results: Eighty-three patients were included in this study. The primary outcome of procedure time was reduced in the HoloLens group compared with control (4.5 min vs 7.3 min, p=0.02, 95% CI), as was the number of needle movements required (7.2 vs 14.4, p=0.01), respectively. There was no difference in intraprocedure or postprocedure pain, bone contacts, or total number of needle punctures. Three patients in the control group experienced epidural failure versus one patient in the HoloLens group., Conclusions: This study shows that thoracic epidural placement may be facilitated by using a guidance hologram and may be more technically efficient., Trial Registration Number: NCT04028284., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The REGAIN (Regional versus General Anesthesia for Promoting Independence after Hip Fracture) trial found similar ambulation and survival at 60 days with spinal versus general anesthesia for hip fracture surgery. Trial outcomes evaluating pain, prescription analgesic use, and patient satisfaction have not yet been reported., Objective: To compare pain, analgesic use, and satisfaction after hip fracture surgery with spinal versus general anesthesia., Design: Preplanned secondary analysis of a pragmatic randomized trial. (ClinicalTrials.gov: NCT02507505)., Setting: 46 U.S. and Canadian hospitals., Participants: Patients aged 50 years or older undergoing hip fracture surgery., Intervention: Spinal or general anesthesia., Measurements: Pain on postoperative days 1 through 3; 60-, 180-, and 365-day pain and prescription analgesic use; and satisfaction with care., Results: A total of 1600 patients were enrolled. The average age was 78 years, and 77% were women. A total of 73.5% (1050 of 1428) of patients reported severe pain during the first 24 hours after surgery. Worst pain over the first 24 hours after surgery was greater with spinal anesthesia (rated from 0 [no pain] to 10 [worst pain imaginable]; mean difference, 0.40 [95% CI, 0.12 to 0.68]). Pain did not differ across groups at other time points. Prescription analgesic use at 60 days occurred in 25% (141 of 563) and 18.8% (108 of 574) of patients assigned to spinal and general anesthesia, respectively (relative risk, 1.33 [CI, 1.06 to 1.65]). Satisfaction was similar across groups., Limitation: Missing outcome data and multiple outcomes assessed., Conclusion: Severe pain is common after hip fracture. Spinal anesthesia was associated with more pain in the first 24 hours after surgery and more prescription analgesic use at 60 days compared with general anesthesia., Primary Funding Source: Patient-Centered Outcomes Research Institute .

Introduction: Older patients undergoing cardiac surgery carry the highest risk for developing major postoperative neurocognitive disorder (postoperative NCD or P-NCD) with up to 25% incidence 3 months after surgery. P-NCD is associated with significant morbidity, mortality, loss of independence, premature retirement and increased healthcare costs. This multicentre randomised trial is investigating the efficacy of postoperative dexmedetomidine sedation in reducing the incidence of major P-NCD after cardiac surgery compared with standard protocols. CODEX will be the largest interventional trial with major P-NCD as the primary outcome., Methods and Analysis: CODEX is recruiting patients ≥60 years old, undergoing elective cardiac surgery and without pre-existing major cognitive dysfunction or dementia. Eligible participants are randomised to receive postoperative dexmedetomidine or standard institutional sedation protocols in the intensive care unit. Baseline preoperative cognitive function is assessed with the computer-based Cogstate Brief Battery. The primary outcome, major P-NCD, 3 months after surgery is defined as a decrease in cognitive function ≥1.96 SD below age-matched, non-operative controls. Secondary outcomes include delirium, major P-NCD at 6/12 months, depressive symptoms, mild P-NCD and quality of surgical recovery at 3/6/12 months. The specific diagnostic criteria used in this protocol are consistent with the recommendations for clinical assessment and management of NCD from the Nomenclature Consensus Working Group on perioperative cognitive changes. Intention-to-treat analysis will compare major P-NCD at 3 months between study groups., Ethics and Dissemination: CODEX was approved by Sunnybrook Health Sciences Centre Research Ethics Board (REB) (Project ID 1743). This will be the first multicentre, randomised controlled trial to assess the efficacy of a pharmacological intervention to reduce the incidence of major P-NCD after cardiac surgery in patients ≥60 years old. Dissemination of the study results will include briefings of key findings and interpretation, conference presentations and peer-reviewed publications., Trial Registration Number: NCT04289142., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: In traumatically injured patients, excessive blood loss necessitating the transfusion of red blood cell (RBC) units is common. Indicators of early RBC transfusion in the pre-hospital setting are needed. This study aims to evaluate the association between hypothermia (<36°C) and transfusion risk within the first 24 hours after arrival to hospital for a traumatic injury., Methods: We completed an audit of all traumatically injured patients who had emergent surgery at a single tertiary care center between 2010 and 2014. Using multivariable logistic regression analysis, we evaluated the association between pre-hospital hypothermia and transfusion of ≥1 unit of RBC within 24 hours of arrival to the trauma bay., Results: Of the 703 patients included to evaluate the association between hypothermia and RBC transfusion, 203 patients (29%) required a transfusion within 24 hours. After controlling for important confounding variables, including age, sex, coagulopathy (platelets and INR), hemoglobin, and vital signs (blood pressure and heart rate), hypothermia was associated with a 68% increased odds of transfusion in multivariable analysis (OR: 1.68; 95% CI: 1.11-2.56)., Conclusions: Hypothermia is strongly associated with RBC transfusion in a cohort of trauma patients requiring emergent surgery. This finding highlights the importance of early measures of temperature after traumatic injury and the need for intervention trials to determine if strategies to mitigate the risk of hypothermia will decrease the risk of transfusion and other morbidities.

RAD6 is a ubiquitin E2 protein with roles in a number of different biological processes. Here, using affinity purification coupled with mass spectrometry, we identify a number of new RAD6 binding partners, including the poorly characterized ubiquitin E3 ligases KCMF1 (potassium channel modulatory factor 1) and UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4), a protein that can bind N-end rule substrates, and which was recently linked to lysosome-mediated degradation and autophagy. NMR, combined with in vivo and in vitro interaction mapping, demonstrate that the KCMF1 C terminus binds directly to RAD6, whereas N-terminal domains interact with UBR4 and other intracellular vesicle- and mitochondria-associated proteins. KCMF1 and RAD6 colocalize at late endosomes and lysosomes, and cells disrupted for KCMF1 or RAD6 function display defects in late endosome vesicle dynamics. Notably, we also find that two different RAD6A point mutants (R7W and R11Q) found in X-linked intellectual disability (XLID) patients specifically lose the interaction with KCMF1 and UBR4, but not with other previously identified RAD6 interactors. We propose that RAD6-KCMF1-UBR4 represents a unique new E2-E3 complex that targets unknown N-end rule substrates for lysosome-mediated degradation, and that disruption of this complex via RAD6A mutations could negatively affect neuronal function in XLID patients., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53. CUL7 binds directly to p53, and small interfering RNA-mediated knockdown of CUL7 results in the elevation of p53 protein levels. This antiapoptotic role of CUL7 enables this novel oncogene to cooperate with Myc to drive transformation. Deregulated ectopic expression of c-Myc and CUL7 promotes Rat1a cell growth in soft agar, and knockdown of CUL7 significantly blocks human neuroblastoma SHEP cell growth in an anchorage-independent manner. Furthermore, using public microarray data sets, we show that CUL7 mRNA is significantly overexpressed in non-small cell lung carcinoma and is associated with poor patient prognosis. We provide experimental evidence to show CUL7 is a new oncogene that cooperates with Myc in transformation by blocking Myc-induced apoptosis in a p53-dependent manner.

Proton decoupled 31P and 13C solution NMR experiments were applied to mixtures of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, with each of its natural substrates, phosphoenolpyruvate and arabinose-5-phosphate (ASP), and product KDO8P to identify the formation of the enzyme-substrate and enzyme-product complexes. Effects arising from ligand interactions with the enzyme are reported via chemical shifts and line broadening with respect to those of the free ligands in solution, depending on the strength and dynamics of binding under thermodynamic equilibrium conditions. The characterization was done both at low and high field spectrometers, 200 and 500 MHz (1H frequencies), and in cases of 31P NMR measurements, it was demonstrated that only the low field spectrometer is capable of providing direct experimental evidence on the enzyme-ligand interactions. Since both the substrate A5P and the product KDO8P exhibit multiple anomeric forms in solution, evidence for the preference of recognition and binding of particular forms is sought.