High-pressure phase transition in 3-D printed nanolamellar high-entropy alloy by imaging and simulation insights.

We report on the high-resolution imaging and molecular dynamics simulations of a 3D-printed eutectic high-entropy alloy (EHEA) Ni40Co20Fe10Cr10Al18W2 consisting of nanolamellar BCC and FCC phases. The direct lattice imaging of 3D-printed samples shows the Kurdjumov-Sachs (K-S) orientation relation {111} FCC parallel to {110} BCC planes in the dual-phase lamellae. Unlike traditional iron and steels, this alloy shows an irreversible BCC-to-FCC phase transformation under high pressures. The nanolamellar morphology is maintained after pressure cycling to 30 GPa, and nano-diffraction studies show both layers to be in the FCC phase. The chemical compositions of the dual-phase lamellae after pressure recovery remain unchanged, suggesting a diffusion-less BCC-FCC transformation in this EHEA. The lattice imaging of the pressure-recovered sample does not show any specific orientation relation between the two resulting FCC phases, indicating that many grain orientations are produced during the BCC-FCC phase transformation. Molecular dynamics simulations on phase transformation in a nanolamellar BCC/FCC in K-S orientation show that phase transformation from BCC to FCC is completed under high pressures, and the FCC phase is retained on decompression aided by the stable interfaces. Our work elucidates the irreversible phase transformation under static compression, providing an understanding of the orientation relationships in 3-D printed EHEA under high pressures.

Serum Cartilage Oligomeric Matrix Protein Concentration Increases More After Running Than Swimming for Older People.

BACKGROUND: Knee osteoarthritis is common in older people. Serum cartilage oligomeric matrix protein (sCOMP) is a biomarker of knee articular cartilage metabolism. The purpose of this study was 2-fold: to (1) determine acute effects of running and swimming on sCOMP concentration in older people; and (2) investigate relationships between sCOMP concentration change due to running and swimming and measures of knee health in older people. HYPOTHESES: Running would result in greater increase in sCOMP concentration than swimming, and increase in sCOMP concentration due to running and swimming would associate positively with measures of poor knee health. STUDY DESIGN: Cross-sectional. LEVEL OF EVIDENCE: Level 3. METHODS: A total of 20 participants ran 5 km and 19 participants swam 1500 m. sCOMP concentration was measured immediately before, immediately after, and 15, 30, and 60 minutes after running or swimming. sCOMP concentration change due to running and swimming was compared. Correlations between sCOMP concentration change due to running and swimming, and other measures of knee health were evaluated, including the Tegner Activity Scale and Knee injury and Osteoarthritis Outcome Score. RESULTS: sCOMP concentration increased 29% immediately after running, relative to baseline, but only 6% immediately after swimming (P < 0.01). No significant relationship was observed between acute sCOMP change due to running and swimming, and observed measures of knee health (P > 0.05). Participants with clinically relevant knee symptoms exhibited greater sCOMP concentration before and after running and swimming (P = 0.03) and had greater body mass (P = 0.04). CONCLUSION: Running results in greater acute articular cartilage metabolism than swimming; however, the chronic effects of this are unclear. Older people with clinically relevant knee symptoms possess greater sCOMP concentration and are heavier, independent of exercise mode and physical activity level. CLINICAL RELEVANCE: These results describe the effects of exercise (running and swimming) for older physically active persons, with and without knee pain.

Effectiveness of a proficiency-based progression e-learning approach to training in communication in the context of clinically deteriorating patients: a multi-arm randomised controlled trial.

OBJECTIVE: To determine the effectiveness of proficiency-based progression (PBP) e-learning in training in communication concerning clinically deteriorating patients. DESIGN: Single-centre multi-arm randomised double-blind controlled trial with three parallel arms. RANDOMISATION, SETTING AND PARTICIPANTS: A computer-generated program randomised and allocated 120 final year medical students in an Irish University into three trial groups. INTERVENTION: Each group completed the standard Identification, Situation, Background, Assessment, Recommendation communication e-learning; group 1 Heath Service Executive course group (HSE) performed this alone; group 2 (PBP) performed additional e-learning using PBP scenarios with expert-determined proficiency benchmarks composed of weighted marking schemes of steps, errors and critical errors cut-offs; group 3 (S) (self-directed, no PBP) performed additional e-learning with identical scenarios to (PBP) without PBP. MAIN OUTCOME MEASURES: Primary analysis was based on 114 students, comparing ability to reach expert-determined predefined proficiency benchmark in standardised low-fidelity simulation assessment, before and after completion of each group's e-learning requirements. Performance was recorded and scored by two independent blinded assessors. RESULTS: Post-intervention, proficiency in each group in the low-fidelity simulation environment improved with statistically significant difference in proficiency between groups (p<0.001). Proficiency was highest in (PBP) (81.1%, 30/37). Post hoc pairwise comparisons revealed statistically significant differences between (PBP) and self-directed (S) (p<0.001) and (HSE) (p<0.001). No statistically significant difference existed between (S) and (HSE) (p=0.479). Changes in proficiency from pre-intervention to post-intervention were significantly different between the three groups (p=0.001). Post-intervention, an extra 67.6% (25/37) in (PBP) achieved proficiency in the low-fidelity simulation. Post hoc pairwise comparisons revealed statistically significant differences between (PBP) and both (S) (p=0.020) and (HSE) (p<0.001). No statistically significant difference was found between (S) and (HSE) (p=0.156). CONCLUSIONS: PBP e-learning is a more effective way to train in communication concerning clinically deteriorating patients than standard e-learning or e-learning without PBP. TRIAL REGISTRATION NUMBER: NCT02937597.

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.

Identification via a Parallel Hit Progression Strategy of Improved Small Molecule Inhibitors of the Malaria Purine Uptake Transporter that Inhibit Plasmodium falciparum Parasite Proliferation.

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

A High-Content Imaging Screen for Cellular Regulators of ß-Catenin Protein Abundance.

Abnormal accumulation of ß-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, ß-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the ß-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to ß-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/ß-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect ß-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated ß-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce ß-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of ß-catenin degradation. This study demonstrates that detecting cell-based ß-catenin protein stability is a viable approach to identifying novel mechanisms of ß-catenin regulation as well as small molecules of therapeutic potential.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

A novel approach applying a chemical biology strategy in phenotypic screening reveals pathway-selective regulators of histone 3 K27 tri-methylation.

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.

Does malrotation of components correlate with patient dissatisfaction following secondary patellar resurfacing?

BACKGROUND: The aim of our study was to identify whether there was any correlation between the outcome of secondary patellar resurfacing and malrotation of either the femoral or tibial component. METHODS: We identified patients that underwent secondary patellar resurfacing following previous primary total knee arthroplasty (TKA) at a single, large orthopaedic department. Patients were reviewed for range of movement, satisfaction, health status and knee function. CT scanning was performed, assessing rotational alignment of the components. RESULTS: Twenty-one patients (23 knees) were reviewed. Nine out of 21 (39%) were satisfied while 14 (61%) remained dissatisfied after the secondary patellar resurfacing. There were no complications after the secondary procedure. All knees were internally rotated. The mean femoral internal rotation in the satisfied group was 0.92°, and in the dissatisfied group was 2.88° of internal rotation. In the dissatisfied group eight out of 14 TKAs were in >3° femoral internal rotation compared with only one in nine TKAs in the satisfied group (p<0.05). CONCLUSIONS: Investigation for malrotation should be considered in patients with post-operative pain, especially anteriorly, causing significant dissatisfaction amongst patients following TKA. This is especially true if the patella has not been primarily resurfaced and secondary resurfacing is being considered. Patients with more than 3(°) of femoral internal rotation undergoing secondary patella resurfacing should be warned of the possibility of a poor outcome. It may well be that if the underlying problem is component malrotation, revision knee replacement may lead to a more satisfactory outcome than secondary resurfacing alone. LEVEL OF EVIDENCE: Level of Evidence III.

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Perspectives on the discovery of small-molecule modulators for epigenetic processes.

Epigenetic gene regulation is a critical process controlling differentiation and development, the malfunction of which may underpin a variety of diseases. In this article, we review the current landscape of small-molecule epigenetic modulators including drugs on the market, key compounds in clinical trials, and chemical probes being used in epigenetic mechanistic studies. Hit identification strategies for the discovery of small-molecule epigenetic modulators are summarized with respect to writers, erasers, and readers of histone marks. Perspectives are provided on opportunities for new hit discovery approaches, some of which may define the next generation of therapeutic intervention strategies for epigenetic processes.

Waiting for organ transplantation: results of an analysis by an Institute of Medicine Committee.

One of the most visible and contentious issues regarding the fairness of the original system of organ procurement and allocation is the argument that it resulted in great disparities in the total amount of time a patient waited for an organ (i.e. the time from registration at a transplantation center to transplant), depending on where he or she lived. In an attempt to resolve this debate, Congress charged the National Academy of Sciences, Institute of Medicine to perform an independent study of the original system and proposed rule changes. In an analysis of approximately 68,000 transplant waiting list records, the committee developed several conclusions and recommendations largely specific to liver transplantation policies. The purpose of this paper is to describe both the results of the study and the statistical foundations of the mixed-effects multinomial logistic regression model that led to the committee's conclusions.

Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis.

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.

Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase: design, conformation and conversion to alpha-ketoamide inhibitors.

Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide, as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the P1 and P1' alanines with a k(cat)/K(m) of 2.3x10(6) M(-1)s(-1) at pH 8.5. Insertion of proline at P1' converted substrates to competitive inhibitors, whilst the incorporation of an alpha-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB.