Data-driven approach to mitigate quality impact of hygroscopic pharmaceutical raw materials throughout the supply chain.

The challenges of working with hygroscopic pharmaceutical raw materials can have a significant impact on the industry's ability to make high-quality medicines. In order to mitigate the impact to the manufacturing process or product quality it is critical to understand the hygroscopicity of the raw materials across the entire supply chain so that the proper management strategies can be implemented, from the raw material manufacturing to the use of the raw material in the drug manufacturing process. Employing suitable controls protects these materials from physical and chemical changes due to moisture uptake such as caking or hydrolysis. We have developed a fit for purpose and data-driven approach to hygroscopicity classifications of over 200 commonly used chemicals, excipients, media and resins in drug manufacturing processes. Dynamic vapor sorption data is presented with supporting thermal gravimetric analysis and X-ray powder diffraction data where pertinent. Approximately 60% of all raw materials tested were determined to be hygroscopic. Strategies for applying this data to reduce the potential impact of hygroscopic materials on the manufacturing of pharmaceuticals are discussed with examples.

Parents' and Service Providers' Experiences of Accessing Health Services from an Intellectual Disability Health Team.

A pediatric assessment service for children with Intellectual Disability (ID) was established in a culturally diverse region of Sydney, Australia, to meet the health needs of children and young people with ID. This paper reports on parents' and providers' experiences using qualitative and quantitative analysis of surveys. The survey responses from the parents' enumerated their key concerns and the practical help they received from the service. Responses from service providers reported a high level of satisfaction with the services and valued the quality of assessment reports. The service facilitated inter-agency collaboration and enhanced the access to quality health care.

Atomic Resolution Cryo-EM Structure of ß-Galactosidase.

The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for ß-galactosidase bound to the inhibitor phenylethyl ß-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.

Cryo-EM: beyond the microscope.

The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

Resolution advances in cryo-EM enable application to drug discovery.

The prospect that the structures of protein assemblies, small and large, can be determined using cryo-electron microscopy (cryo-EM) is beginning to transform the landscape of structural biology and cell biology. Great progress is being made in determining 3D structures of biological assemblies ranging from icosahedral viruses and helical arrays to small membrane proteins and protein complexes. Here, we review recent advances in this field, focusing especially on the emerging use of cryo-EM in mapping the binding of drugs and inhibitors to protein targets, an application that requires structure determination at the highest possible resolutions. We discuss methods used to evaluate the information contained in cryo-EM density maps and consider strengths and weaknesses of approaches currently used to measure map resolution.

Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Strengthening the capacity of nursing leaders through multifaceted professional development initiatives: A mixed method evaluation of the 'Take The Lead' program.

BACKGROUND: Effective nursing leadership is necessary for the delivery of safe, high quality healthcare. Yet experience and research tells us that nursing leaders are commonly unprepared for their roles. Take The Lead (TTL), a large-scale, multifaceted professional development program was initiated in New South Wales, Australia, to strengthen the capacity of Nursing/Midwifery Unit Managers (N/MUMs). The aim of this study was to examine the effects of TTL on job performance, nursing leadership and patient experience. METHODS: Nursing/Midwifery Unit Managers (n = 30) and managers of N/MUMs (n = 30) who had completed the TTL program were interviewed between August and December 2010. The semi-structured interviews included a combination of open-ended questions and questions that required respondents to rate statements using a Likert scale. Data from the open-ended questions were thematically analysed to identify and categorise key concepts. The responses to the Likert items were analysed via descriptive statistics. RESULTS: Nursing/Midwifery Unit Managers' participation in TTL engendered improvements in job performance and leadership skills, as well as some improvement in patients' experiences of care. The program facilitated role clarification and helped foster peer-support and learning networks, which were perceived to provide ongoing professional and personal benefits to participants. CONCLUSIONS: Our study revealed a consensus about the beneficial outcomes of TTL among those involved with the program. It supports the significant and ongoing value of widely implemented, multifaceted nursing leadership development programs and demonstrates that participants value their informal interactions as highly as they do the formal content. These findings have implications for delivery mode of similar professional development programs.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

2.2 Å resolution cryo-EM structure of ß-galactosidase in complex with a cell-permeant inhibitor.

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli ß-galactosidase and the cell-permeant inhibitor phenylethyl ß-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM.

An ethnographic investigation of junior doctors' capacities to practice interprofessionally in three teaching hospitals.

Collaborative practice among early career staff is at the bedrock of interprofessional care. This study investigated factors influencing the enactment of interprofessional practice by using the day-to-day role of six junior doctors in three teaching hospitals as a gateway to understand the various professions' interactive behaviours. The contextual framework used for the study was Strauss' theory of negotiated order. Ethnographic techniques were applied to observe the actions and interactions of participants on typical working days in their hospital environments. Field notes were created and thematic analysis was applied to the data. Three themes explored were culture, communication, and collaboration. Issues identified highlight the bounded organisational and professional cultures within which junior doctors work, and systemic problems in interprofessional interaction and communication in the wards of hospitals. There are indications that early career doctors are interprofessional isolates. The constraints of short training terms and pressure from multi-faceted demands on junior doctors can interfere with the establishment of meaningful relationships with nurses and other health professionals. The realisation of sustained interprofessional practice is, therefore, practically and structurally difficult. Enabling factors supporting the sharing of expertise are outweighed by barriers associated with professional and hospital organisational cultures, poor interprofessional communication, and the pressure of competing individual task demands in the course of daily practice.

Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy.

The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target-cell membranes. Here, we present the cryo-EM structure, at subnanometer resolution (~6 Å at 0.143 FSC), of the 'closed', prefusion state of trimeric HIV-1 Env complexed to the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the 'open' quaternary conformation. The architecture of trimeric HIV-1 Env in the prefusion state and in the activated intermediate state resembles the corresponding states of influenza hemagglutinin trimers, thus providing direct evidence for the similarity in entry mechanisms used by HIV-1, influenza and related enveloped viruses.

HIV-1 envelope glycoprotein trimers display open quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibody Z13e1.

We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.

Cryo-electron microscopy--a primer for the non-microscopist.

Cryo-electron microscopy (cryo-EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, are poised to further advance the effectiveness of cryo-EM methods. These developments promise to increase the speed and extent of automation, and to improve the resolutions that may be achieved, making this technology useful to determine a wide variety of biological structures. Additionally, established modalities for structure determination, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo-EM density maps to achieve atomic-resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo-EM methodology, we provide an overview of emerging themes in the application of this technology to investigate diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo-EM is deposited and archived in a publicly accessible database.

Application of two-dimensional selective-TOCSY HMBC for structure elucidation of impurities in mixture without separation.

In the pharmaceutical industry, regulatory expectations driven by patient safety considerations make structure elucidation of impurities at levels greater than 0.1% in the active pharmaceutical ingredient (API) of primary interest. Impurities can be generated from isomers in starting materials, or produced from different process steps toward the final API. Proton peaks belonging to different impurities could be potentially identified in the one-dimensional (1)H NMR spectrum, when evaluated in combination with two-dimensional (2-D) COSY and HSQC data. However, in 2-D HMBC data, correlation responses from different impurities may overlap with those from the major component, causing uncertainty of long-range proton to carbon correlations and quaternary carbon assignments. This observation prompts us to design the 2-D selective-TOCSY HMBC experiment to distinguish responses from different impurities in mixtures to obtain 2-D NMR data for each impurity, thus eliminating the use of a chromatographic isolation step to obtain material for NMR analysis. This methodology is demonstrated for structure elucidation of impurities ranging from 8.2% in the raw material to 0.4% in the API in this study, and would be particularly useful for industrial samples in which the solubility and availability of material are not an issue.

Structural mechanism of trimeric HIV-1 envelope glycoprotein activation.

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.

Iodide-catalyzed reductions: development of a synthesis of phenylacetic acids.

A new convenient and scalable synthesis of phenylacetic acids has been developed via the iodide catalyzed reduction of mandelic acids. The procedure relies on in situ generation of hydroiodic acid from catalytic sodium iodide, employing phosphorus acid as the stoichiometric reductant.

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography.

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures.

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response.

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.