Exploring the Professional Identity of Hospital Pharmacists in British Columbia.

Background: Pharmacists lack a cohesive professional identity, with only limited previous research on the formation of a professional identity for pharmacy. In particular, there is sparse information on the professional identity of pharmacists who practise in hospital settings. Objectives: To determine hospital pharmacists' professional identity and the characteristics of an ideal pharmacist and ideal practice setting. Methods: This qualitative study used key informant interviews with semistructured questions. A maximum variation sampling strategy was used to recruit a cross-section of pharmacists from different geographic areas of British Columbia who were practising in a variety of roles. The interviews were transcribed and then analyzed thematically. Results: Nineteen pharmacists participated in the study. Seven themes pertaining to hospital pharmacists' professional identity were generated, specifically medication expert, therapy optimizer, collaborator, educator, researcher, patient advocate, and unknown professional. Similarities were found with personas previously identified in a population of primarily community pharmacists. The ideal pharmacist was described as being a medication expert, a collaborator, and a leader. The ideal practice setting was characterized as being adequately funded and allowing pharmacists to practise to their full scope. Conclusions: Hospital pharmacists' professional identity is based on being a medication expert who is seen as an essential member of a collaborative team.

Contexte: Les pharmaciens manquent d'une identité professionnelle cohérente et les recherches antérieures portant sur la formation d'une identité professionnelle de la profession sont limitées. En particulier, les informations sur l'identité professionnelle des pharmaciens exerçant en milieu hospitalier sont rares. Objectif: Déterminer l'identité professionnelle des pharmaciens d'hôpitaux ainsi que les caractéristiques d'un pharmacien idéal et d'un milieu d'exercice idéal. Méthodes: Pour cette étude qualitative, des questions d'entretien semi-structurées ont été utilisées auprès d'informateurs clés. Une stratégie d'échantillonnage à variation maximale a été utilisée pour recruter un échantillon représentatif de pharmaciens de différentes régions géographiques de la Colombie-Britannique pratiquant divers rôles. Les entretiens ont ensuite été retranscrits puis analysés par thème. Résultats: Dix-neuf pharmaciens ont participé à l'étude. Sept thèmes relatifs à l'identité professionnelle des pharmaciens d'hôpitaux se sont dessinés: expert en médicaments, optimisateur thérapeutique, collaborateur, éducateur, chercheur, défenseur des patients et professionnel méconnu. Des similitudes se sont dégagées avec des identités précédemment cernées dans une population constituée principalement de pharmaciens communautaires. Le pharmacien idéal a été décrit comme étant un expert en médicaments, un collaborateur et un leader. Le milieu de pratique idéal a quant à lui été décrit comme un milieu adéquatement financé permettant aux pharmaciens d'exercer pleinement leurs compétences. Conclusions: L'identité professionnelle du pharmacien hospitalier repose sur le fait d'être un expert en médicaments. Cet expert est considéré comme membre essentiel d'une équipe collaborative.

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK.

Erythrocytosis and thrombotic events in kidney transplant recipients prescribed a sodium glucose cotransport-2 inhibitor.

INTRODUCTION: The safety and efficacy of sodium glucose cotransport-2 inhibitors (SGLT2i) in kidney transplant recipients remains uncertain. Transplant recipients may be at risk of thrombosis because of post-transplant erythrocytosis and SGLT2i are associated with an increase in hematocrit. METHODS: We determined SGLT2i use, the change in hematocrit and incidence of thrombotic events in kidney transplant recipients in 1700 prevalent patients in our center. RESULTS: Among the 42 patients treated with SGLT2i, the mean pre-transplant hematocrit was 31%, and none of the patients had a hematocrit ≥50%. The mean percent change in hematocrit measured at an average of 53 days after initiation of an SGLT2i was 11% and four patients (10%) had a hematocrit ≥ 50%. The mean hematocrit measured 3 months after treatment was 42% and two patients (5%) had a hematocrit ≥50%. One patient had a cerebellar stroke 14 months post-SGLT2i initiation when the hemoglobin was 173 grams/liter, and the hematocrit was 52%. CONCLUSIONS: All patients had a sustained increase in hematocrit 3 months after SGLT2i treatment. Hematocrit ≥50% occurred in 10%, and one patient had a thrombotic event that may or may not have been related to an increase in hematocrit. Clinicians may consider monitoring for erythrocytosis after starting and SGLT2i in kidney transplant recipients.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis.

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

Clinical Practice Guideline for Solid Organ Donation and Transplantation During the COVID-19 Pandemic.

The coronavirus 2019 (COVID-19) pandemic has disrupted health systems worldwide, including solid organ donation and transplantation programs. Guidance on how best to screen patients who are potential organ donors to minimize the risks of COVID-19 as well as how best to manage immunosuppression and reduce the risk of COVID-19 and manage infection in solid organ transplant recipients (SOTr) is needed. METHODS: Iterative literature searches were conducted, the last being January 2021, by a team of 3 information specialists. Stakeholders representing key groups undertook the systematic reviews and generation of recommendations using a rapid response approach that respected the Appraisal of Guidelines for Research and Evaluation II and Grading of Recommendations, Assessment, Development and Evaluations frameworks. RESULTS: The systematic reviews addressed multiple questions of interest. In this guidance document, we make 4 strong recommendations, 7 weak recommendations, 3 good practice statements, and 3 statements of "no recommendation." CONCLUSIONS: SOTr and patients on the waitlist are populations of interest in the COVID-19 pandemic. Currently, there is a paucity of high-quality evidence to guide decisions around deceased donation assessments and the management of SOTr and waitlist patients. Inclusion of these populations in clinical trials of therapeutic interventions, including vaccine candidates, is essential to guide best practices.

Structure of detergent-activated BAK dimers derived from the inert monomer.

A body of data supports the existence of core (α2-α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2-α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6-α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far.

Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations.

Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.

Virtual reality exergaming as adjunctive therapy in a sub-acute stroke rehabilitation setting: facilitators and barriers.

PURPOSE: To identify the facilitators and barriers perceived by clinicians to using an Exergaming Room as adjunct to conventional therapy. DESIGN: Phenomenological qualitative study using an interpretive description methodology. SUBJECTS: Ten clinicians (four physical therapists, six occupational therapists) from the Stroke Program at the Jewish Rehabilitation Hospital (nine female, one male, age range 25-50 years old) who referred clients to the Exergaming Room. METHODS: Ten to twenty minute semi-structured interviews were conducted with each clinician. Convenience sampling was used. A thematic analysis was performed on the data collected by grouping all the open codes into facilitators and barriers, and then categorized into levels, themes and subthemes. RESULTS: Facilitators and barriers were divided into three levels: organizational, individual and technological. Major facilitators at the organizational level were: institutional support; at the individual level: personal experience of referring clinician, presence of an expert clinician, and relevance of the Exergaming Room for stroke clients; and at the technological level: perceived ease of use of the exergames and possibility of providing additional therapy. Key barriers to successful implementation of the Exergaming Room at the organizational level were: scheduling difficulties and lack of staffing; at the individual level: client functional limitations; at the technological level: low precision in motion capture of the exergame systems. CONCLUSIONS: Multiple factors affect the implementation of new technology in rehabilitation settings. In order to successfully integrate exergame systems into practice, institutions are encouraged to take the identified factors (facilitators and barriers) into account. Implications for Rehabilitation Clinicians who have referred individuals with stroke to an "exergames" room over a 1-year period at a rehabilitation hospital have found the service to be highly relevant to their clients. The presence of an expert clinician, who evaluates the clients and builds an exergames activity program, was seen as an important facilitator by referring clinicians in the use of this service. An ideal Exergames Room should offer a wide variety of activities, including some that focus on motor, cognitive and/or communications abilities.

Ensemble Properties of Bax Determine Its Function.

BAX and BAK are essential mediators of intrinsic apoptosis that permeabilize the mitochondrial outer membrane. BAX activation requires its translocation from cytosol to mitochondria where conformational changes cause its oligomerization. To better understand the critical step of translocation, we examined its blockade by mutation near the C terminus (P168G) or by antibody binding near the N terminus. Similarities in the crystal structures of wild-type and BAX P168G but significant other differences suggest that cytosolic BAX exists as an ensemble of conformers, and that the distribution of conformers within the ensemble determines the different functions of wild-type and mutant proteins. We also describe the structure of BAX in complex with an antibody, 3C10, that inhibits cytosolic BAX by limiting exposure of the membrane-associating helix α9, as does the P168G mutation. Our data for both means of BAX inhibition argue for an allosteric model of BAX regulation that derives from properties of the ensemble of conformers.

Posttransplant Lymphoproliferative Disorder in Adults Receiving Kidney Transplantation in British Columbia: A Retrospective Cohort Analysis.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a major complication following kidney transplantation. OBJECTIVE: We undertook this study to characterize PTLD in kidney transplant patients in British Columbia with regard to incidence, patient and graft survival, histological subtypes, treatment modalities, and management of immunosuppression. DESIGN: Retrospective cohort analysis. SETTING: British Columbia. PATIENTS: All adult patients who underwent kidney transplantation in British Columbia between January 1, 1996, and December 31, 2012, were included. Patients less than 18 years of age at the time of first transplant and multiple organ transplant recipients were excluded from analysis. MEASUREMENTS: Patients with lymphoproliferative disorders that occurred subsequent to kidney transplantation were considered to have developed PTLD. METHODS: Cases of PTLD were identified by cross-referencing data abstracted from the provincial transplant agency's clinical database with the provincial cancer agency's lymphoma registry. Patients were followed up for the development of PTLD until December 31, 2012, and for outcomes of death and graft failure until December 31, 2014. Data collection was completed via an electronic chart review. RESULTS: Of 2217 kidney transplant recipients, 37 (1.7%) developed PTLD. Nine cases were early-onset PTLD, occurring within 1 year of transplant; of these cases, 6 were known/presumed Epstein-Barr virus mismatch, compared with only 2 of 28 late-onset cases. Patient survival for early-onset PTLD was 100% at 2 years post diagnosis. Late-onset PTLD had survival rates of 71.4% and 67.9% at 1 and 2 years, respectively. PTLD was associated with significantly decreased patient survival (P = .031) and graft survival (uncensored for death, P = .017), with median graft survival of PTLD and non-PTLD patients being 9.5 and 16 years, respectively. Immunosuppressant therapy was reduced in the majority of patients; additional therapies included rituximab monotherapy, CHOP-R, radiation, and surgery. LIMITATIONS: Limitations to this study include its retrospective nature and the unknown adherence of patients to prescribed immunosuppressant regimens. In addition, cumulative doses of immunosuppression received and the degree of immunosuppression reduction for PTLD management were not effectively captured. CONCLUSIONS: The incidence of PTLD in British Columbia following kidney transplantation was low and consistent with rates reported in the literature. The incidence of late-onset PTLD and its association with reduced patient and graft survival warrant further analysis of patients' long-term immunosuppression.

CONTEXTE: Le syndrome lymphoprolifératif post-greffe (SLPG) est une complication grave survenant à la suite d'une transplantation rénale. OBJECTIF DE L'ÉTUDE: Nous avons mené cette étude afin de caractériser le SLPG chez les receveurs d'une greffe rénale en Colombie-Britannique en ce qui a trait à son incidence, à la survie du patient et du greffon, aux sous-types histologiques, aux modalités de traitement et à la gestion de l'immunosuppression. CADRE ET TYPE D'ÉTUDE: Il s'agit d'une étude de cohorte rétrospective effectuée en Colombie-Britannique. SUJETS: Ont été inclus dans l'étude tous les patients adultes ayant subi une transplantation rénale entre le 1er janvier 1996 et le 31 décembre 2012 en Colombie-Britannique. Les patients âgés de moins de 18 ans au moment de l'intervention et les patients receveurs de greffe de multiples organes ont été exclus. MESURES: Tout cas de SL apparu après une greffe rénale étaient considérés comme un SLPG. MÉTHODOLOGIE: Les cas de SLPG ont été répertoriés en recoupant les données extraites de la base de données cliniques de l'agence provinciale de transplantation avec les données du registre des lymphomes tenu par l'agence provinciale de lutte contre le cancer. Les participants ont été suivis jusqu'au 31 décembre 2012 pour l'apparition du SLPG et jusqu'au 31 décembre 2014 pour les issues défavorables telles que la mort du patient ou le rejet du greffon. L'examen du dossier électronique des patients a complété la collecte des données. RÉSULTATS: Des 2 217 receveurs d'une greffe rénale répertoriés, seuls 37 (1,7 %) ont développé un SLPG. L'apparition du SLPG s'est faite de façon précoce, soit dans la première année post-greffe, pour neuf de ces patients, dont six représentaient un cas connu ou présumé de non-concordance pour le virus d'Epstein Barr (EBV). En comparaison, seuls deux des 28 patients ayant expérimenté un développement tardif du SLPG étaient présumés non-concordants pour l'EBV. Deux ans après le diagnostic, 100 % des patients ayant eu une apparition précoce du SLPG avaient survécu. Dans les cas de développement tardif de la maladie, le taux de survie passait à 71,4 % après un an et à 67,9 % après deux ans pour les patients. Le développement du SLPG a été associé avec une réduction significative de la chance de survie du patient (p = 0,031) et du greffon (p = 0,017, cas de décès non censurés). La survie médiane du greffon était de 9,5 ans pour les patients ayant développé un SLPG alors qu'elle était de 16 ans pour les autres. L'intensité du traitement immunosuppresseur a pu être réduite pour la majorité des patients. Les traitements additionnels incluaient la monothérapie au rituximab, le R-CHOP, la radiation et la chirurgie. LIMITES DE L'ÉTUDE: La nature rétrospective de l'étude est un facteur limitant la portée de nos résultats, de même que l'absence de données sur l'adhérence des patients au traitement immunosuppressif. De plus, nous n'avons pu mesurer précisément les doses cumulatives d'immunosuppresseurs reçues, ni le degré de réduction de ces derniers dans la prise en charge du SLPG. CONCLUSION: En Colombie-Britannique, l'incidence du SL post-greffe rénale s'est avérée faible et cohérente avec les taux rapportés dans la littérature. L'incidence de l'apparition tardive du SLPG et son association à un taux et une durée de survie amoindris (à la fois pour le patient et pour le greffon) justifient une analyse plus poussée de l'immunosuppression à long terme dans la population en question.

Notch ligand delta-like1: X-ray crystal structure and binding affinity.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 µM for this interaction.

Evaluation of a Once-Daily Vancomycin Regimen in an Outpatient Leukemia/Bone Marrow Transplant Clinic (OD-VANCO Study).

BACKGROUND: The Leukemia/Bone Marrow Transplant Program of British Columbia manages patients with high-risk febrile neutropenia and those with non-neutropenic immunocompromised states in an outpatient clinic setting. Because the program treats outpatients only, once-daily administration of IV antibiotics is desirable. A high-dose, once-daily vancomycin nomogram was developed and implemented as part of the antibiotic treatment regimen. OBJECTIVE: To determine if therapeutic vancomycin trough levels could be achieved with a high-dose, once-daily regimen in this outpatient setting. METHODS: A prospective, single-centre, observational cohort study was conducted over a 7-month period. Outpatients in the Leukemia/Bone Marrow Transplant Program were started on IV vancomycin with the high-dose, once-daily vancomycin nomogram, and outcomes were assessed. RESULTS: Of 48 outpatients treated over the 7-month period, 10 (21%) had therapeutic vancomycin trough concentrations (i.e., greater than 10 mg/L). Thirty-five (90%) of the 39 patients with suspected clinical infection experienced clinical cure, and 6 (67%) of the 9 patients with documented microbiological infection experienced microbiological cure. Thirty (62%) of the 48 patients experienced symptoms of "red man syndrome", and 7 (15%) experienced some degree of nephrotoxicity. Two of 3 patients with laboratory-reported minimum inhibitory concentration (MIC) for identified pathogens had a calculated area under the curve to MIC ratio greater than or equal to 400. CONCLUSION: The high-dose, once-daily vancomycin nomogram was effective in attaining trough levels greater than 10 mg/L in only 21% of patients in this study. A substantial number of adverse drug reactions were observed. Given these results, high-dose, once-daily vancomycin is no longer recommended for outpatient therapy.

CONTEXTE: Le programme sur la leucémie et la greffe de moelle osseuse de la Colombie-Britannique (Leukemia/Bone Marrow Transplant Program of British Columbia) traite en consultation externe des patients avec une neutropénie fébrile à risque élevé et d'autres en états non neutropéniques d'immunovulnérabilité. Comme le programme s'adresse uniquement à des patients externes, une administration intraveineuse (IV) uniquotidienne d'antibiotiques est souhaitée. Pour cette raison, un nomogramme posologique pour la vancomycine à dose uniquotidienne élevée a été élaboré et mis en place dans le cadre du schéma d'antibiothérapie. OBJECTIF: Déterminer s'il est possible d'atteindre des concentrations minimales thérapeutiques de vancomycine à l'aide d'un schéma thérapeutique à dose uniquotidienne élevée dans ce service de consultation externe. MÉTHODES: Une étude de cohorte prospective observationnelle a été menée dans un seul centre sur une période de sept mois. Le nomogramme posologique a servi à commencer le traitement IV par la vancomycine à dose uniquotidienne élevée de patients externes participant au programme sur la leucémie et la greffe de moelle osseuse, et les résultats ont été évalués. RÉSULTATS: Parmi les quarante-huit patients externes traités pendant une période de sept mois, des concentrations minimales thérapeutiques de vancomycine (c.-à-d. plus de 10 mg/L) ont été atteintes chez dix (21 %) d'entre eux. Trente-cinq (90 %) des trente-neuf patients chez qui l'on soupçonnait une infection clinique ont obtenu une guérison clinique et une éradication microbiologique a été notée chez six (67 %) des neuf patients présentant une infection microbiologique attestée. Trente (62 %) des 48 patients ont présenté un syndrome de l'homme rouge et sept patients (15 %) ont manifesté un certain degré de néphrotoxicité. Deux des trois patients pour qui le laboratoire avait déterminé une concentration minimale inhibitrice (CMI) contre les agents pathogènes en cause avaient un rapport aire sous la courbe sur CMI égal ou supérieur à 400. CONCLUSION: Le nomogramme posologique pour la vancomycine à dose uniquotidienne élevée a permis d'atteindre des concentrations minimales de plus de 10 mg/L chez seulement 21 % des patients de cette étude. Un nombre considérable d'effets indésirables liés au médicament a été observé. Compte tenu de ces résultats, il n'est plus recommandé de donner des doses uniquotidiennes élevées de vancomycine à titre de traitement aux patients externes. [Traduction par l'éditeur].

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

Crystal structure of the entire ectodomain of gp130: insights into the molecular assembly of the tall cytokine receptor complexes.

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1-6, D1-D6) at 3.6 A resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4-D6) at 1.9 A. This represents the first atomic resolution structure of the complete ectodomain of any "tall" cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR.

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

An unusual cytokine:Ig-domain interaction revealed in the crystal structure of leukemia inhibitory factor (LIF) in complex with the LIF receptor.

Leukemia inhibitory factor (LIF) receptor is a cell surface receptor that mediates the actions of LIF and other IL-6 type cytokines through the formation of high-affinity signaling complexes with gp130. Here we present the crystal structure of a complex of mouse LIF receptor with human LIF at 4.0 A resolution. The structure is, to date, the largest cytokine receptor fragment determined by x-ray crystallography. The binding of LIF to its receptor via the central Ig-like domain is unlike other cytokine receptor complexes that bind ligand predominantly through their cytokine-binding modules. This structure, in combination with previous crystallographic studies, also provides a structural template to understand the formation and orientation of the high-affinity signaling complex between LIF, LIF receptor, and gp130.

Structure of a calcium-deficient form of influenza virus neuraminidase: implications for substrate binding.

The X-ray structure of influenza virus neuraminidase (NA) isolated from whale, subtype N9, has been determined at 2.2 A resolution and contains a tetrameric protein in the asymmetric unit. In structures of NA determined previously, a calcium ion is observed to coordinate amino acids near the substrate-binding site. In three of the NA monomers determined here this calcium is absent, resulting in structural alterations near the substrate-binding site. These changes affect the conformation of residues that participate in several key interactions between the enzyme and substrate and provide at a molecular level the basis of the structural and functional role of calcium in substrate and inhibitor binding. Several sulfate ions were identified in complex with the protein. These are located in the active site, occupying the space reserved for the substrate (sialic acid) carboxylate, and in positions leading away from the substrate-binding site. These sites offer a new opportunity for the design of inhibitors of influenza virus NA.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum.

The malaria parasite Plasmodium falciparum is responsible for about two million deaths annually, making it important to obtain information about enzymes from this organism that represent potential drug targets. The gene for P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) has been cloned and the protein expressed as a hexahistidine-tagged recombinant protein in Escherichia coli. The recombinant protein has been crystallized and its three-dimensional structure determined. One molecule of the cofactor NAD+ is bound to each of the four subunits in the tetrameric enzyme. The major structural feature distinguishing human GAPDH from PfGAPDH is the insertion of a dipeptide (-KG-) in the so-called S loop. This insert, together with other characteristic single-amino-acid substitutions, alters the chemical environment of the groove that encompasses the R dyad and that links adjacent cofactor-binding sites and may be responsible for the selective inhibition of the enzyme by ferriprotoporphyrin IX.