Emerging applications and regulatory strategies for advanced medicines manufacturing - Towards the development of a platform approach.

The last decade has seen Advanced Medicines Manufacturing (AMM) progress from isolated product developments to the creation of industry-academic centres of excellence, regulatory innovation progressing leading to new standards, and product commercialisation across multiple product formats. This paper examines these developments focusing on successful applications and strategies presented at the 2023 Symposium of the International Consortium for Advanced Medicines Manufacturing (ICAMM). Despite these exemplar applications, there remain significant challenges to the sector-wide adoption of AMM technologies. Drawing on Symposium delegate expert responses to open-ended questions, our coding-based thematic analysis suggest three primary enablers drive successful adoption of AMM technologies at scale, namely: the ability to leverage pre-competitive collaborations to challenge-based problem solving; information and knowledge sharing through centres of excellence; and the development of AMM specific regulatory standards. Further analysis of expert responses identified the emergence of a 'Platform creation' approach to AMM innovation; characterised by: i) New collaboration modes; ii) Exploration of common product-process platforms for new dosage forms and therapy areas; iii) Development of modular equipment assets that enable scale-out, and offer more decentralized or distributed manufacturing models; iv) Standards based on product-process platform archetypes; v) Implementation strategies where platform-thinking and AMM technologies can significantly reduce timelines between discovery, approval and GMP readiness. We provide a definition of the Platform creation concept for AMM and discuss the requirements for its systematic development.

Reliability and validity of the medial standing overhead arm reach (SOAR) test as a measure of functional hip adduction motion.

OBJECTIVE: The Posterior Standing Overhead Arm Reach (SOAR) test has been previously reported as a reliable clinical measure of closed chain hip extension motion. The proposed Medial SOAR test expands on that testing approach to provide a similar measure of functional hip adduction motion. This was a preliminary intrarater and interrater reliability and validity study of the Medial SOAR test as a measure of functional hip adduction. DESIGN: Cross-sectional. SETTING: University motion analysis laboratory. PARTICIPANTS: Fifty hips were assessed in 25 (22 female) asymptomatic participants (mean age = 23.4 years, SD = 0.8). MAIN MEASURES: Maximum hip adduction during the Medial SOAR test was measured with a standard goniometer independently by two examiners. The test was also performed using three-dimensional motion capture. The intrarater and interrater reliability of the goniometric measure was determined using intraclass correlation coefficients, and the relationship between measures obtained via goniometry and three-dimensional motion capture was assessed with Pearson correlations and Bland-Altman analysis. RESULTS: Intrarater reliability (ICC2,3) was 0.88 (95% CI = 0.80-0.92) for Examiner 1 and 0.87 (95% CI = 0.79-0.92) for Examiner 2. The standard error of measurement and minimal detectable change were less than 3.0°. Interrater reliability demonstrated an intraclass correlation coefficient = 0.62 (95% CI = 0.28-0.79). Pearson correlations were significant with low-to-moderate associations (r = 0.49, P < 0.001; r = 0.24, P = 0.045). CONCLUSIONS: Similar to the previously reported Posterior SOAR test, the Medial SOAR test demonstrated acceptable intrarater and interrater reliability, along with low-to-moderate associations with three-dimensional motion capture. The Medial SOAR test has the potential to provide a reliable and accurate assessment of closed chain hip adduction.

Admission Pa o2 and Mortality Among PICU Patients and Select Diagnostic Subgroups.

OBJECTIVES: Evaluate the relationship between admission Pa o2 and mortality in a large multicenter dataset and among diagnostic subgroups. DESIGN: Retrospective cohort study. SETTING: North American PICUs participating in Virtual Pediatric Systems, LLC (VPS), 2015-2019. PATIENTS: Noncardiac patients 18 years or younger admitted to a VPS PICU with admission Pa o2 . INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirteen thousand seventy-one patient encounters were included with an overall mortality of 13.52%. Age categories were equally distributed among survivors and nonsurvivors with the exception of small differences among neonates and adolescents. Importantly, there was a tightly fitting quadratic relationship between admission Pa o2 and mortality, with the highest mortality rates seen among hypoxemic and hyperoxemic patients (likelihood-ratio test p < 0.001). This relationship persisted after adjustment for illness severity using modified Pediatric Index of Mortality 3 scores. A similar U-shaped relationship was demonstrated among patients with diagnoses of trauma, head trauma, sepsis, renal failure, hemorrhagic shock, and drowning. However, among the 1,500 patients admitted following cardiac arrest, there was no clear relationship between admission Pa o2 and mortality. CONCLUSIONS: In a large multicenter pediatric cohort, admission Pa o2 demonstrates a tightly fitting quadratic relationship with mortality. The persistence of this relationship among some but not all diagnostic subgroups suggests the pathophysiology of certain disease states may modify the hyperoxemia association.

Communicating With Unconscious Patients: An Overview.

BACKGROUND: Nurses are told to speak to their unconscious patients because hearing is said to be the last sense to depart. There was little reliable evidence before the 1990s that patients in an unconscious state could hear and understand what was being said. That led to reluctance on the part of health professionals to communicate with these unresponsive patients. OBJECTIVE: This historical overview aims to present researched evidence from the 1990s to the present detailing awareness that occurs in unconscious patients, when that awareness increases, and how to detect that awareness. It also includes research about the benefits of communicating with unconscious patients and descriptions of how registered nurses and other health care professionals, from a postsurvey after a continuing education course on experiences of unconscious patients, plan to communicate with unconscious patients. METHODS: A literature search was conducted, which included more than 150 articles and books about experiences of unconscious patients in several electronic databases, including PubMed, CINAHL, and the British Nursing Index. In addition, an analysis of 105 postcourse responses by registered nurses (89%) and other health professionals (11%), licensed practical nurses, emergency medical technicians, and cardiac technicians after taking a continuing education course on experiences of previously unconscious patients were analyzed. RESULTS: The Glasgow Coma Scale and the Full Outline of Unresponsiveness scale are helpful behavioral tools to identify levels of coma but miss detecting awareness in patients who can hear and understand but cannot move. The estimates are that 25% to 40% (J Trauma. 1975;15:94-98; J Neurosci Nurs. 1988;20:223-228; J Neurosci Nurs. 1990;22(1):52-53; Am J Crit Care. 1995;3:227-232) of patients diagnosed with a disorder of consciousness can hear and understand what is being said in their environment. Substantial evidence supports that isolation and loneliness, such as experienced by some patients perceived to be unaware, can be physically and psychologically harmful. CONCLUSIONS: Strong evidence shows that some patients diagnosed as being in a vegetative state can hear and understand what is being said in their environment. Interviews with previously unconscious patients and electrophysiological methods show that awareness can be detected in patients perceived to be unconscious. There is documented evidence that patients experience awareness when going into unconsciousness, even when they appear unaware and when moved. To our knowledge, these times have not been researched using electrophysiological devices but established from interviews.

Observing many researchers using the same data and hypothesis reveals a hidden universe of uncertainty.

This study explores how researchers' analytical choices affect the reliability of scientific findings. Most discussions of reliability problems in science focus on systematic biases. We broaden the lens to emphasize the idiosyncrasy of conscious and unconscious decisions that researchers make during data analysis. We coordinated 161 researchers in 73 research teams and observed their research decisions as they used the same data to independently test the same prominent social science hypothesis: that greater immigration reduces support for social policies among the public. In this typical case of social science research, research teams reported both widely diverging numerical findings and substantive conclusions despite identical start conditions. Researchers' expertise, prior beliefs, and expectations barely predict the wide variation in research outcomes. More than 95% of the total variance in numerical results remains unexplained even after qualitative coding of all identifiable decisions in each team's workflow. This reveals a universe of uncertainty that remains hidden when considering a single study in isolation. The idiosyncratic nature of how researchers' results and conclusions varied is a previously underappreciated explanation for why many scientific hypotheses remain contested. These results call for greater epistemic humility and clarity in reporting scientific findings.

Scalable Constant pH Molecular Dynamics in GROMACS.

Molecular dynamics (MD) computer simulations are used routinely to compute atomistic trajectories of complex systems. Systems are simulated in various ensembles, depending on the experimental conditions one aims to mimic. While constant energy, temperature, volume, and pressure are rather straightforward to model, pH, which is an equally important parameter in experiments, is more difficult to account for in simulations. Although a constant pH algorithm based on the λ-dynamics approach by Brooks and co-workers [Kong, X.; Brooks III, C. L. J. Chem. Phys.1996, 105, 2414-2423] was implemented in a fork of the GROMACS molecular dynamics program, uptake has been rather limited, presumably due to the poor scaling of that code with respect to the number of titratable sites. To overcome this limitation, we implemented an alternative scheme for interpolating the Hamiltonians of the protonation states that makes the constant pH molecular dynamics simulations almost as fast as a normal MD simulation with GROMACS. In addition, we implemented a simpler scheme, called multisite representation, for modeling side chains with multiple titratable sites, such as imidazole rings. This scheme, which is based on constraining the sum of the λ-coordinates, not only reduces the complexity associated with parametrizing the intramolecular interactions between the sites but also is easily extendable to other molecules with multiple titratable sites. With the combination of a more efficient interpolation scheme and multisite representation of titratable groups, we anticipate a rapid uptake of constant pH molecular dynamics simulations within the GROMACS user community.

Best Practices in Constant pH MD Simulations: Accuracy and Sampling.

Various approaches have been proposed to include the effect of pH in molecular dynamics (MD) simulations. Among these, the λ-dynamics approach proposed by Brooks and co-workers [Kong, X.; Brooks III, C. L. J. Chem. Phys. 1996, 105, 2414-2423] can be performed with little computational overhead and hfor each typeence be used to routinely perform MD simulations at microsecond time scales, as shown in the accompanying paper [Aho, N. et al. J. Chem. Theory Comput. 2022, DOI: 10.1021/acs.jctc.2c00516]. At such time scales, however, the accuracy of the molecular mechanics force field and the parametrization becomes critical. Here, we address these issues and provide the community with guidelines on how to set up and perform long time scale constant pH MD simulations. We found that barriers associated with the torsions of side chains in the CHARMM36m force field are too high for reaching convergence in constant pH MD simulations on microsecond time scales. To avoid the high computational cost of extending the sampling, we propose small modifications to the force field to selectively reduce the torsional barriers. We demonstrate that with such modifications we obtain converged distributions of both protonation and torsional degrees of freedom and hence consistent pKa estimates, while the sampling of the overall configurational space accessible to proteins is unaffected as compared to normal MD simulations. We also show that the results of constant pH MD depend on the accuracy of the correction potentials. While these potentials are typically obtained by fitting a low-order polynomial to calculated free energy profiles, we find that higher order fits are essential to provide accurate and consistent results. By resolving problems in accuracy and sampling, the work described in this and the accompanying paper paves the way to the widespread application of constant pH MD beyond pKa prediction.

A preliminary study of the reliability and validity of the Posterior Standing Overhead Arm Reach (SOAR) test as a measure of functional hip extension motion.

BACKGROUND: Current clinical tests do not provide a method to reliably measure closed chain hip extension. We developed the Posterior Standing Overhead Arm Reach (SOAR) test for this purpose. OBJECTIVES: This was a preliminary intrarater and interrater reliability and validity study of the Posterior SOAR test as a measure of functional hip extension. DESIGN: Cross-sectional. METHOD: Hip extension on the Posterior SOAR test was measured with a standard goniometer independently by two examiners. The test was then repeated using three-dimensional (3D) motion capture. Intraclass correlation coefficients (ICC) were used to determine the intrarater and interrater reliability of the goniometric measure and Pearson correlations were used to assess the relationship between measures obtained via goniometry and 3D motion capture. RESULTS: Fifty hips were assessed in 25 (14 female, 11 male) asymptomatic participants (mean age = 24.0 years, SD = 1.1). Intrarater reliability (ICC2,3) was 0.80 (95% CI = 0.68-0.88) for Examiner 1 and 0.77 (95% CI = 0.64-0.86) for Examiner 2, indicating excellent reliability. The standard error of the measure (SEM90) ranged from 2.5° to 3.0° with a minimal detectable change (MDC90) of 3.5° to 4.2°. Interrater reliability was good with ICC = 0.65 (95% CI = 0.36-0.80). Pearson correlations were significant with low to moderate associations (r = 0.36, P = 0.009; r = 0.51, P < 0.001). CONCLUSIONS: The Posterior SOAR test demonstrated excellent intrarater reliability, good interrater reliability, and low to moderate associations with 3D motion capture. The Posterior SOAR test has the potential to provide a reliable and accurate assessment of closed chain hip extension.

Heterogeneous parallelization and acceleration of molecular dynamics simulations in GROMACS.

The introduction of accelerator devices such as graphics processing units (GPUs) has had profound impact on molecular dynamics simulations and has enabled order-of-magnitude performance advances using commodity hardware. To fully reap these benefits, it has been necessary to reformulate some of the most fundamental algorithms, including the Verlet list, pair searching, and cutoffs. Here, we present the heterogeneous parallelization and acceleration design of molecular dynamics implemented in the GROMACS codebase over the last decade. The setup involves a general cluster-based approach to pair lists and non-bonded pair interactions that utilizes both GPU and central processing unit (CPU) single instruction, multiple data acceleration efficiently, including the ability to load-balance tasks between CPUs and GPUs. The algorithm work efficiency is tuned for each type of hardware, and to use accelerators more efficiently, we introduce dual pair lists with rolling pruning updates. Combined with new direct GPU-GPU communication and GPU integration, this enables excellent performance from single GPU simulations through strong scaling across multiple GPUs and efficient multi-node parallelization.

Knowledge Base of Nurses Before and After a Human Trafficking Continuing Education Course.

BACKGROUND: Recent research shows up to 88% of human trafficking victims are seen in a health care setting sometime during their coerced servitude. Nurses are in key positions to identify trafficked victims but often lack sufficient information about how to assess and report trafficked victims. METHOD: This article includes participant data from one presurvey and two postsurveys from a human trafficking continuing education course. RESULTS: Data demonstrate a precourse knowledge base deficit about human trafficking. The postcourse surveys indicated that participants had increased knowledge and desire to implement the course information to identify trafficked individuals. CONCLUSION: Nurses learning about human trafficking through continuing education courses can be invaluable in identifying and helping trafficked victims. [J Contin Educ Nurs. 2020;51(7):316-321.].

Does suffering suffice? An experimental assessment of desert retributivism.

Michael S. Moore is among the most prominent normative theorists to argue that retributive justice, understood as the deserved suffering of offenders, justifies punishment. Moore claims that the principle of retributive justice is pervasively supported by our judgments of justice and sufficient to ground punishment. We offer an experimental assessment of these two claims, (1) the pervasiveness claim, according to which people are widely prone to endorse retributive judgments, and (2) the sufficiency claim, according to which no non-retributive principle is necessary for justifying punishment. We test these two claims in a survey and a related survey experiment in which we present participants (N = ~900) with the stylized description of a criminal case. Our results seem to invalidate claim (1) and provide mixed results concerning claim (2). We conclude that retributive justice theories which advance either of these two claims need to reassess their evidential support.

Identification and Surgical Management of Upper Arm and Forearm Compartment Syndrome.

Extremity muscles are grouped and divided by strong fascial membranes into compartments. Multiple pathological processes can result in an increase in the pressure within a muscle compartment. An increase in the compartment pressure beyond the adequate perfusion pressure has the potential to cause extremity compartment syndrome. There are multiple sites where compartment syndrome can occur. In this article, an arm and forearm compartment syndrome ensued secondary to a minor crushing injury that lead to supracondylar and medial epicondylar non-displaced fractures. A pure motor radial and ulnar nerve deficits noted on presentation, worsened with progression of the compartment syndrome. Ultimately, a surgical fasciotomy was carried out to release all compartments of the right upper arm and forearm.

Sharing Data from Molecular Simulations.

Given the need for modern researchers to produce open, reproducible scientific output, the lack of standards and best practices for sharing data and workflows used to produce and analyze molecular dynamics (MD) simulations has become an important issue in the field. There are now multiple well-established packages to perform molecular dynamics simulations, often highly tuned for exploiting specific classes of hardware, each with strong communities surrounding them, but with very limited interoperability/transferability options. Thus, the choice of the software package often dictates the workflow for both simulation production and analysis. The level of detail in documenting the workflows and analysis code varies greatly in published work, hindering reproducibility of the reported results and the ability for other researchers to build on these studies. An increasing number of researchers are motivated to make their data available, but many challenges remain in order to effectively share and reuse simulation data. To discuss these and other issues related to best practices in the field in general, we organized a workshop in November 2018 ( https://bioexcel.eu/events/workshop-on-sharing-data-from-molecular-simulations/ ). Here, we present a brief overview of this workshop and topics discussed. We hope this effort will spark further conversation in the MD community to pave the way toward more open, interoperable, and reproducible outputs coming from research studies using MD simulations.

Trust and cooperative behavior: Evidence from the realm of data-sharing.

Trust is praised by many social scientists as the foundation of functioning social systems owing to its assumed connection to cooperative behavior. The existence of such a link is still subject to debate. In the present study, we first highlight important conceptual issues within this debate. Second, we examine previous evidence, highlighting several issues. Third, we present findings from an original experiment, in which we tried to identify a "real" situation that allowed us to measure both trust and cooperation. People's expectations and behavior when they decide to share (or not) their data represents such a situation, and we make use of corresponding data. We found that there is no relationship between trust and cooperation. This non-relationship may be rationalized in different ways which, in turn, provides important lessons for the study of the trust-behavior nexus beyond the particular situation we study empirically.

Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies.

Human liver glycerol 3-phosphate dehydrogenase ( hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzyme-substrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E·NAD, and ternary E·NAD·DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion-activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains were likewise examined by double-mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

The evolution of multiple active site configurations in a designed enzyme.

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase.

Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrate-Schiff base to the product-Schiff base, which includes the rate-limiting cleavage of the Cα-H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the Cα-H bond involves direct hydride transfer to the topaquinone cofactor-a mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu2+ cation to the reaction site, lower the activation barrier to drive the chemical reaction.

Epoxide hydrolysis as a model system for understanding flux through a branched reaction scheme.

The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl-propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure-activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

Laboratory-Evolved Enzymes Provide Snapshots of the Development of Enantioconvergence in Enzyme-Catalyzed Epoxide Hydrolysis.

Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only a consequence of changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. In order to probe the structural origin of these differences in substrate selectivity and catalytic regiopreference, we solved the crystal structures for the evolved StEH1 variants. We used these structures as a starting point for molecular docking studies of the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking analysis, the apparent preferred binding modes appear to rationalize the experimentally determined regioselectivities. The analysis also identifies an active site residue (F33) as a potentially important interaction partner, a role that could explain the high conservation of this residue during evolution. Overall, our experimental, structural, and computational studies provide snapshots into the evolution of enantioconvergence in StEH1-catalyzed epoxide hydrolysis.