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The ectoenzyme CD38 is highly expressed on the surface of mature immune cells, where they are a marker for cell activation, and also on the surface of multiple tumor cells such as multiple myeloma (MM). CD38-targeted monoclonal antibodies (MABs) such as daratumumab and isatuximab bind to CD38 and promote cancer cell death by stimulating the antitumor immune response. Although MABs are achieving unprecedented success in a percentage of cases, high rates of resistance limit their efficacy. Formation of the immunosuppressive intermediate adenosine is a major route by which this resistance is mediated. Thus there is an urgent need for small molecule agents that boost the immune response in T-cells. Importantly, CD38 is a dual-function enzyme, serving as a hydrolase and a nicotinamide adenine dinucleotide (NAD+) cyclase, and both of these activities promote immunosuppression. We have employed virtual and physical screening to identify novel compounds that are selective for either the hydrolase or the cyclase activity of CD38, and have demonstrated that these compounds activate T cells in vitro. We are currently optimizing these inhibitors for use in immunotherapy. These small molecule inhibitors of the CD38-hydrolase or cyclase activity can serve as chemical probes to determine the mechanism by which CD38 promotes resistance to MAB therapy, and could become novel and effective therapeutic agents that produce immunostimulatory effects. Our studies have identified the first small molecule inhibitors of CD38 specifically for use as immunostimulants.

Abdominal cerclage is an effective treatment for cervical incompetence in patients with a previously failed vaginal cerclage or with anatomic restrictions to a vaginal cerclage. Management of second trimester complications that warrant a delivery impose a complex clinical situation in patients with an abdominal cerclage. We report 3 cases of successful removal of an abdominal cerclage by posterior and anterior colpotomy in the second trimester of pregnancy. This new and minimally invasive surgical technique avoids the need for extensive dilation, laparoscopy, or laparotomy to remove the cerclage and allow a vaginal delivery.

A series of five benzimidazole-based compounds were identified using a machine learning algorithm as potential inhibitors of the respiratory syncytial virus (RSV) fusion protein. These compounds were synthesized, and compound 2 in particular exhibited excellent in vitro potency with an EC(50) value of 5 nM. This new scaffold was then further refined leading to the identification of compound 44, which exhibited a 10-fold improvement in activity with an EC(50) value of 0.5 nM.

Background Glioblastoma (GBM) is the most aggressive and common brain tumor. Despite multimodal therapy which consists of complete tumor removal, chemo- and radiation therapy in most patients, the tumor recurs, and the overall survival of only 14 months remains very poor. To identify tumor-induced immune evasion and treatment resistance mechanisms induced during GBM recurrence, we aimed at characterizing the immunological/molecular changes in patient-matched treatment-naïve and recurrent GBM samples. Material and Methods Proteins and total RNA were extracted from 16 patient-matched treatment-naïve (primary) and recurrent GBM fresh frozen tumor samples. The expression of genes involved in brain immune responses were compared using Nanostring panels encompassing Cancer and Neuroinflammation genes. Proteomic was conducted using 6 patient-matched primary and recurrent tumor samples. Immunostaining was performed on a tissue microarray containing matched primary and recurrent tumor samples to identify tumor cells and microglia in order to assess spatial transcriptome of these specific cell types in situ using GeoMX technology. Moreover, correlation analysis of differentially genes and proteins with time to relapse was performed. Results Genes significantly differentially expressed between primary and recurrent tumors revealed genes overexpressed in recurrent tumors belonging to “FCGR-mediated Phagocytosis” and “synapse pruning” pathways. Proteomic confirmed the synaptic signalling considering overexpressed proteins. Correlation studies with the time to relapse indicated that higher expression of FCG receptors, complement molecules and synaptic components correlate with shorter time to relapse, sharing similarities with neurodegenerative/cognitive disorders such as schizophrenia and Alzheimer disease. The source for these molecules is under evaluation with the spatial transcriptomic analysis. Conclusion Upregulation of specific proteins such as FCG receptors, complement and synaptic components may result in more favourable conditions for tumor regrowth and may therefore be identified as negative predictors patient outcome. Interfering with these molecules may represent novel therapeutic options after initial GBM surgery.

BACKGROUND: Literature suggests that patient-informing process prior to obtaining surgical informed consent (SIC) does not function well. This study aimed to provide insight into the current practice of SIC in the Netherlands.METHODS: This is a prospective, observational, and multicenter study, conducted in one academic and two non-academic teaching hospitals in the Netherlands. Audio recordings were made during outpatient consultations with patients presenting with Dupuytren Disease. The recorded informing process was scored according to a checklist. Written documentation of the SIC process in the patient's chart was compared to these scored checklists. Time spent on SIC during the consultations was also recorded.RESULTS: A total of 41 outpatient consultations were included in the study. Consultations were conducted by 25 plastic surgeons and their residents. Average time spent on SIC was 55.6% of the total consultation time. Considerable variation was observed concerning the amount and type of information given and discussed. In 59% of the consultations, discrepancies were observed between written documentation of consultations and audio recordings. Information on treatment risks, the postoperative period, and the operating surgeon was addressed the least.CONCLUSION: Despite a relatively large part of the consultation time being spent on SIC, patients received scarce information concerning treatment risks, postoperative period, and who their operating surgeon would be. Discrepancies were observed between the written documentation of SIC and information recorded on the audio recordings. This occurred predominantly in one hospital that used a pre-made list of 'discussed information' in its digital patient chart.

We have previously described the synthesis and evaluation of 3,5-diamino-1,2,4-triazole analogues as inhibitors of the flavin-dependent histone demethylase LSD1. These compounds are potent inhibitors of LSD1 without activity against monoamine oxidases A and B, and promote the elevation of H3K4me2 levels in tumor cells in vitro. We now report that the cytotoxicity of these analogues in pancreatic tumor cells correlates with the overexpression of LSD1 in each tumor type. In addition, we show that a subset of these 3,5-diamino-1,2,4-triazole analogues inhibit a related flavin-dependent oxidase, the polyamine catabolic enzyme spermine oxidase (SMOX) in vitro.

IntroductionDozens of medications have been re-evaluated as possible treatments for coronavirus 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Tocilizumab is a humanized monoclonal antibody directed against the interleukin-6 (IL-6) receptor that has been proposed as a therapy for patients with severe COVID-19 pneumonia. It is possible that the immunomodulatory effects of tocilizumab may also have the unintended consequence of predisposing to secondary infections. We sought to assess the risk of invasive fungal disease and therapeutic impact of tocilizumab on hospital and intensive care unit (ICU) outcomes in critically ill patients with severe COVID-19. MethodsRecords of critically-ill patients with COVID-19 over a 6 month period at our institution were reviewed, and medical histories, treatments, and clinical outcomes were extracted. The incidence of fungal infections in those that received tocilizumab in addition to standard COVID-19 treatments was assessed. ICU length of stay (LOS), duration of mechanical ventilation, and hospital LOS were compared between treatment groups. ResultsOne-hundred and thirty patients were identified, of which 56 met inclusion criteria. 16 patients received tocilizumab, and 40 did not. Invasive fungal infections occurred in 10.7% of all patients. Infection rates were significantly higher in the tocilizumab group than in the control group (31.2% v 2.5%, RR 12.5, p

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic ligand-gated glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Several neurological disorders may involve NMDAR hypofunction, which has driven therapeutic interest in positive allosteric modulators (PAMs) of NMDAR function. Here we describe modest changes to the tetrahydroisoquinoline scaffold of GluN2C/GluN2D-selective PAMs that expands activity to include GluN2A- and GluN2B-containing recombinant and synaptic NMDARs. These new analogues are distinct from GluN2C/GluN2D-selective compounds like (+)-(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ) by virtue of their subunit selectivity, molecular determinants of action, and allosteric regulation of agonist potency. The (S)-enantiomers of two analogues (EU1180-55, EU1180-154) showed activity at NMDARs containing all subunits (GluN2A, GluN2B, GluN2C, GluN2D), whereas the (R)-enantiomers were primarily active at GluN2C- and GluN2D-containing NMDARs. Determination of the actions of enantiomers on triheteromeric receptors confirms their unique pharmacology, with greater activity of (S) enantiomers at GluN2A/GluN2D and GluN2B/GluN2D subunit combinations than (R) enantiomers. Evaluation of the (S)-EU1180-55 and EU1180-154 response of chimeric kainate/NMDA receptors revealed structural determinants of action within the pore-forming region and associated linkers. Scanning mutagenesis identified structural determinants within the GluN1 pre-M1 and M1 regions that alter the activity of (S)-EU1180-55 but not (R)-EU1180-55. By contrast, mutations in pre-M1 and M1 regions of GluN2D perturb the actions of only the (R)-EU1180-55 but not the (S) enantiomer. Molecular modeling supports the idea that the (S) and (R) enantiomers interact distinctly with GluN1 and GluN2 pre-M1 regions, suggesting that two distinct sites exist for these NMDAR PAMs, each of which has different functional effects.

Mutations in the pre-M1 helix of NMDA receptor subunits are associated with neurological disease. McDaniel et al. reveal that the helix influences channel gating, potentially through interactions with a network of aromatic amino acids within the subunit’s pre-M1 and pore-forming M3 helices and the adjacent subunit’s M4 helix., The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

Background:In preaxial polydactyly of the foot, the choice for excision of the lateral or medial hallux is not straightforward, in particular with proximal phalangeal (type IV) and metatarsal (type VI) duplication, because of anatomical characteristics. We evaluated whether medial or lateral hallux excision gives better outcomes in these duplication types, to help clinical decision making.Methods:Children with type IV or type VI duplication (n=14, age: 4.4-17.2 years), who were operatively treated by excision of the lateral or medial hallux, were assessed for foot function using plantar pressure measurements and clinical examination. Foot aesthetics were scored by the child, an expert, and 10 laypersons, and additional patient-reported outcome questionnaires were obtained. Outcomes were compared between lateral and medial excision, per duplication type.Results:In type IV duplication (n=11), lateral excision showed a better distribution of peak pressure between the hallux and first metatarsal with significantly lower median first metatarsal peak pressure ( P = .008). Lateral excision showed more medial hallux deviation ( P = .017). Foot aesthetics were not different between excision sides. In type VI duplication (n=12), lateral excision showed a 59% higher hallux peak pressure, larger medial hallux deviation ( P = .004), and more reoperations. Foot aesthetics were scored significantly better after lateral excision by experts and laypersons.Conclusions:Foot function by virtue of plantar pressure was better after lateral hallux excision in type IV and after medial hallux excision in type VI duplication. Surgeons and laypersons perceived the foot as more normal after lateral excision in type VI, whereas children reported no differences. These outcomes can be used in clinical decision making.Level of Evidence:Level III, therapeutic, comparative study.

HUNK is a protein kinase that is implicated in HER2-positive (HER2+) breast cancer progression and resistance to HER2 inhibitors. Though prior studies suggest there is therapeutic potential for targeting HUNK in HER2+ breast cancer, pharmacological agents that target HUNK are yet to be identified. A recent study showed that the broad-spectrum kinase inhibitor staurosporine binds to the HUNK catalytic domain, but the effect of staurosporine on HUNK enzymatic activity was not tested. We now show that staurosporine inhibits the kinase activity of a full length HUNK protein. Our findings further suggest that inhibiting HUNK with staurosporine has a strong effect on suppressing cell viability of HER2/neu mammary and breast cancer cells, which express high levels of HUNK protein and are dependent on HUNK for survival. Significantly, we use in vitro and in vivo methods to show that staurosporine synergizes with the HER2 inhibitor lapatinib to restore sensitivity toward HER2 inhibition in a HER2 inhibitor resistant breast cancer model. Collectively, these studies indicate that pharmacological inhibition of HUNK kinase activity has therapeutic potential for HER2+ breast cancers, including HER2+ breast cancers that have developed drug resistance.

NMDA receptors (NMDARs) are tetrameric complexes comprising two glycine‐binding GluN1 and two glutamate‐binding GluN2 subunits. Four GluN2 subunits encoded by different genes can produce up to 10 different di‐ and triheteromeric receptors. In addition, some neurological patients contain a de novo mutation or inherited rare variant in only one subunit. There is currently no mechanistic framework to describe tetrameric receptor function that can be extended to receptors with two different GluN1 or GluN2 subunits. Here we use the structural features of glutamate receptors to develop a mechanism describing both single channel and macroscopic NMDAR currents. We propose that each agonist‐bound subunit undergoes some rate‐limiting conformational change after agonist binding, prior to channel opening. We hypothesize that this conformational change occurs within a triad of interactions between a short helix preceding the M1 transmembrane helix, the highly conserved M3 motif encoded by the residues SYTANLAAF, and the linker preceding the M4 transmembrane helix of the adjacent subunit. Molecular dynamics simulations suggest that pre‐M1 helix motion is uncorrelated between subunits, which we interpret to suggest independent subunit‐specific conformational changes may influence these pre‐gating steps. According to this interpretation, these conformational changes are the main determinants of the key kinetic properties of NMDA receptor activation following agonist binding, and so these steps sculpt their physiological role. We show that this structurally derived tetrameric model describes both single channel and macroscopic data, giving a new approach to interpreting functional properties of synaptic NMDARs that provides a logical framework to understanding receptors with non‐identical subunits.

Contains fulltext : 191468.pdf (Publisher’s version ) (Open Access) Background and purpose - Preaxial polydactyly of the foot is a rare malformation and clinicians are often unfamiliar with the associated malformations and syndromes. In order to give guidelines for diagnostics and referral to a clinical geneticist, we provide an overview of the presentation using a literature review and our own patient population. Patients and methods - The literature review was based on the Human Phenotype Ontology (HPO) project. From the HPO dataset, all phenotypes describing preaxial polydactyly were obtained and related diseases were identified and selected. An overview was generated in a heatmap, in which the phenotypic contribution of 12 anatomical groups to each disease is displayed. Clinical cases were obtained from our hospital database and were reviewed in terms of phenotype, genotype, heredity, and diagnosed syndromes. Results - From the HPO dataset, 21 diseases were related to preaxial polydactyly of the foot. The anatomical groups with the highest phenotypic contribution were lower limb, upper limb, and craniofacial. From our clinical database, we included 76 patients with 9 different diseases, of which 27 had a GLI3 mutation. Lower limb malformations (n = 55), upper limb malformations (n = 59), and craniofacial malformations (n = 32) were most frequently observed. Malformations in other anatomical groups were observed in 27 patients. Interpretation - Preaxial polydactyly of the foot often presents with other upper and lower limb malformations. In patients with isolated preaxial polydactyly of the foot, referral to a clinical geneticist is not mandatory. In patients with additional malformations, consultation with a clinical geneticist is recommended. When additional limb malformations are present, analysis of GLI3 is most feasible.

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo GRIN2A missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg2+, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient's observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association-approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.

Epilepsy and intellectual disability are associated with rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with essential roles in brain development and function. By assessing genetic variation across GluN2 domains, we determined that the agonist binding domain, transmembrane domain, and the linker regions between these domains were particularly intolerant to functional variation. Notably, the agonist binding domain of GluN2B exhibited significantly more variation intolerance than that of GluN2A. To understand the ramifications of missense variation in the agonist binding domain, we investigated the mechanisms by which 25 rare variants in the GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When introduced into recombinant human NMDARs, these rare variants identified in individuals with neurologic disease had complex, and sometimes opposing, consequences on agonist binding, channel gating, receptor biogenesis, and forward trafficking. Our approach combined quantitative assessments of these effects to estimate the overall impact on synaptic and non-synaptic NMDAR function. Interestingly, similar neurologic diseases were associated with both gain- and loss-of-function variants in the same gene. Most rare variants in GluN2A were associated with epilepsy, whereas GluN2B variants were associated with intellectual disability with or without seizures. Finally, discerning the mechanisms underlying NMDAR dysregulation by these rare variants allowed investigations of pharmacologic strategies to correct NMDAR function.

Machine learning continues to make strident advances in the prediction of desired properties concerning drug development. Problematically, the efficacy of machine learning in these arenas is reliant upon highly accurate and abundant data. These two limitations, high accuracy and abundance, are often taken together; however, insight into the dataset accuracy limitation of contemporary machine learning algorithms may yield insight into whether non-bench experimental sources of data may be used to generate useful machine learning models where there is a paucity of experimental data. We took highly accurate data across six kinase types, one GPCR, one polymerase, a human protease, and HIV protease, and intentionally introduced error at varying population proportions in the datasets for each target. With the generated error in the data, we explored how the retrospective accuracy of a Naïve Bayes Network, a Random Forest Model, and a Probabilistic Neural Network model decayed as a function of error. Additionally, we explored the ability of a training dataset with an error profile resembling that produced by the Free Energy Perturbation method (FEP+) to generate machine learning models with useful retrospective capabilities. The categorical error tolerance was quite high for a Naïve Bayes Network algorithm averaging 39% error in the training set required to lose predictivity on the test set. Additionally, a Random Forest tolerated a significant degree of categorical error introduced into the training set with an average error of 29% required to lose predictivity. However, we found the Probabilistic Neural Network algorithm did not tolerate as much categorical error requiring an average of 20% error to lose predictivity. Finally, we found that a Naïve Bayes Network and a Random Forest could both use datasets with an error profile resembling that of FEP+. This work demonstrates that computational methods of known error distribution like FEP+ may be useful in generating machine learning models not based on extensive and expensive in vitro-generated datasets.

We introduce the hydrological watershed, a watershed where water can penetrate the soil, and compare it with the topological watershed for a two-dimensional landscape. For this purpose, we measure the fractal dimension of the hydrological watershed for different penetration depths and different grid sizes. Through finite size scaling, we find that the fractal dimension is 1.31 ± 0.02 which is significantly higher than the fractal dimension of the topological watershed. This indicates that the hydrological watershed belongs to a new universality class. We also find that, as opposed to the topological watershed, the hydrodynamic watershed can exhibit disconnected islands.

Background In human fetuses with cardiac defects and increased nuchal translucency, abnormal ductus venosus flow velocity waveforms are observed. It is unknown whether abnormal ductus venosus flow velocity waveforms in fetuses with increased nuchal translucency are a reflection of altered cardiac function or are caused by local morphological alterations in the ductus venosus. Aim The aim of this study was to investigate if the observed increased nuchal translucency, cardiac defects and abnormal lymphatic development in the examined mouse models are associated with local changes in ductus venosus morphology. Study design Mouse embryos with anomalous lymphatic development and nuchal edema ( Ccbe1 −/− embryos), mouse embryos with cardiac defects and nuchal edema ( Fkbp12 −/− , Tbx1 −/− , Chd7 fl / fl ;Mesp1Cre , Jarid2 −/− NE+ embryos) and mouse embryos with cardiac defects without nuchal edema ( Tbx2 −/− , Fgf10 −/− , Jarid2 −/− NE − embryos) were examined. Embryos were analyzed from embryonic day (E) 11.5 to 15.5 using markers for endothelium, smooth muscle actin, nerve tissue and elastic fibers. Results All mutant and wild-type mouse embryos showed similar, positive endothelial and smooth muscle cell expression in the ductus venosus at E11.5–15.5. Nerve marker and elastic fiber expression were not identified in the ductus venosus in all investigated mutant and wild-type embryos. Local morphology and expression of the used markers were similar in the ductus venosus in all examined mutant and wild-type embryos. Conclusions Cardiac defects, nuchal edema and abnormal lymphatic development are not associated with morphological changes in the ductus venosus. Ductus venosus flow velocity waveforms most probably reflect intracardiac pressure.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

The C-X-C chemokine receptor type 4 (CXCR4) is a potential therapeutic target for HIV infection, metastatic cancer, and inflammatory autoimmune diseases. In this study, we screened the ZINC chemical database for novel CXCR4 modulators through a series of in silico guided processes. After evaluating the screened compounds for their binding affinities to CXCR4 and inhibitory activities against the chemoattractant CXCL12, we identified a hit compound (ZINC 72372983) showing 100 nM affinity and 69% chemotaxis inhibition at the same concentration (100 nM). To increase the potency of our hit compound, we explored the protein-ligand interactions at an atomic level using molecular dynamics simulation which enabled us to design and synthesize a novel compound (Z7R) with nanomolar affinity (IC50 = 1.25 nM) and improved chemotaxis inhibition (78.5%). Z7R displays promising anti-inflammatory activity (50%) in a mouse edema model by blocking CXCR4-expressed leukocytes, being supported by our immunohistochemistry study.

N-methyl-d-aspartate receptors (NMDARs) are an important receptor in the brain and have been implicated in multiple neurological disorders. Many non-selective NMDAR-targeting drugs are poorly tolerated, leading to efforts to target NMDAR subtypes to improve the therapeutic index. We describe here a series of negative allosteric NMDAR modulators with submaximal inhibition at saturating concentrations. Modest changes to the chemical structure interconvert negative and positive modulation. All modulators share the ability to enhance agonist potency and are use-dependent, requiring the binding of both agonists before modulators act with high potency. Data suggest that these modulators, including both enantiomers, bind to the same site on the receptor and share structural determinants of action. Due to the modulator properties, submaximal negative modulators in this series may spare NMDAR at the synapse, while augmenting the response of NMDAR in extrasynaptic spaces. These modulators could serve as useful tools to probe the role of extrasynaptic NMDARs., eLife digest The neurons in the brain form networks that can change in response to experience, causing new connections to form between certain neurons and breaking the connections between others. This remodeling process underlies learning and memory. However, in certain neurological disorders, such as schizophrenia and epilepsy, these networks are disrupted and no longer work correctly. A receptor protein called the NMDA receptor plays an important role in reshaping the networks of neurons. Chemicals called neurotransmitters that are released by one neuron bind to and activate NMDA receptors on a neighbouring neuron to communicate with it. This activation encourages new connections to form between neurons. Drugs that alter the activity of the NMDA receptor could potentially act as treatments for neurological conditions that disrupt how the networks of neurons work. However, few have been approved for use in patients because most of the potential drug compounds investigated so far produce severe side effects. Perszyk et al. have now identified a new group of compounds that can potentially alter the activity of NMDA receptors without fully blocking the response, potentially eliminating the unwanted side effects. The compounds were tested on frog egg cells and human embryonic kidney cells that had been engineered to produce NMDA receptors. Small changes to the chemical structure of these compounds could switch their effect from increasing to decreasing the activity of the receptor. The compounds only interact with active receptors that have a neurotransmitter bound to them, and compete with each other to bind to the receptors. Perszyk et al. also found that some compounds behaved differently depending on how active the NMDA receptor was. These compounds could potentially be used to sense the activity in a network of neurons, which opens up new options for treating neurological conditions that affect the networks. Further experiments are now required to see how these compounds affect the activity of NMDA receptors in neurons and living animals. The range of effects produced by the compounds studied by Perszyk et al. suggests that other related compounds may have different effects on receptor activity. Future work could investigate the properties of these compounds to see if they could treat a different set of neurological disorders.

Research purpose: The aim of this article was to evaluate the relevance of Global Management Accounting Principles (GMAPs) in the sustainability of a mechanised piggery in a South African context.Motivation for the study: Considering the pressure which commercial agriculture in South Africa is experiencing, as well as the growing nature of pork production in this sector, mechanised piggeries would have to consider ways in which to manage their operations in order to remain sustainable.Research approach, design and method: The research design took the form of an exploratory case study and contextual applied research with an inductivist approach to qualitative research.Main findings: The findings include that, without pre-knowledge of GMAPs, Piggery A employed a large number of the concepts and principles described by GMAPs as best practice.Practical and managerial implications: All commercial farming operations should consider GMAPs as a tool to establish best practice in the support of decision-making that promotes sustainability of farming operations.Contribution or value-add: The article’s contribution is to highlight that GMAPs can also be utilised as an effective decision-making tool in a mechanised piggery albeit it was designed for use in large corporations.

We study two new models of two particle species invading a surface from opposite sides. Collisions of particles of different species lead to the formation of congestion fronts. One of the models implements a reversible process whereas in the other model the congestion front forms irreversibly. For both models we find that the congestion fronts are self-affine but with different roughness exponents. For low densities the system does not congest and we find a phase transition between a phase of freely moving particles and a congestion phase.

Objective To assess whether cardiac failure, because of cardiac defects, and abnormal jugular lymphatic development are involved in nuchal edema (NE) – the morphological equivalent of increased nuchal translucency – in various euploid mutant mouse models. Method Mouse embryos with lymphatic abnormalities and NE (Ccbe1−/−), with cardiac defects and NE (Fkbp12−/−, Tbx1−/−, Chd7fl/fl;Mesp1Cre, Jarid2−/−NE+) and with cardiac malformations without NE (Tbx2−/−, Pitx2−/−, Fgf10−/−, Jarid2−/−NE−) were examined. Embryos were analyzed from embryonic day 11.5 to 15.5. Markers for lymphatic vessels, endothelium, smooth muscle cells and nerves were used to study the nuchal region. Hematoxylin–Azophloxine staining was performed to examine cardiac morphology. Results Mouse embryos with lymphatic abnormalities and NE (Ccbe1−/−) showed no formation of the jugular lymphatic sac but normal cardiac morphology. In mouse embryos with cardiac defects and NE (Fkbp12−/−, Tbx1−/−, Chd7fl/fl;Mesp1Cre, Jarid2−/−NE+) enlarged jugular lymphatic sacs or large nuchal cavities within the NE were found. In mouse embryos with a cardiac malformation without NE (Tbx2−/−, Pitx2−/−, Fgf10−/−, Jarid2−/−NE−) normal jugular lymphatic sacs were observed. Conclusion NE consistently coincides with abnormal jugular lymphatic development in euploid mouse embryos, independent of cardiac anatomy. NE is unlikely to be caused by temporary cardiac failure solely because of a cardiac defect. © 2015 John Wiley & Sons, Ltd.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated, cation-selective channels that mediate a slow component of excitatory synaptic transmission. Subunit-selective positive allosteric modulators of NMDA receptor function have therapeutically relevant effects on multiple processes in the brain. A series of pyrrolidinones, such as PYD-106, that selectively potentiate NMDA receptors that contain the GluN2C subunit have structural determinants of activity that reside between the GluN2C amino terminal domain and the GluN2C agonist binding domain, suggesting a unique site of action. Here we use molecular biology and homology modeling to identify residues that line a candidate binding pocket for GluN2C-selective pyrrolidinones. We also show that occupancy of only one site in diheteromeric receptors is required for potentiation. Both GluN2A and GluN2B can dominate the sensitivity of triheteromeric receptors to eliminate the actions of pyrrolidinones, thus rendering this series uniquely sensitive to subunit stoichiometry. We experimentally identified NMR-derived conformers in solution, which combined with molecular modeling allows the prediction of the bioactive binding pose for this series of GluN2C-selective positive allosteric modulators of NMDA receptors. These data advance our understanding of the site and nature of the ligand-protein interaction for GluN2C-selective positive allosteric modulators for NMDA receptors.

We have identified a series of positive allosteric NMDA receptor (NMDAR) modulators derived from a known class of GluN2C/D-selective tetrahydroisoquinoline analogues that includes CIQ. The prototypical compound of this series contains a single isopropoxy moiety in place of the two methoxy substituents present in CIQ. Modifications of this isopropoxy-containing scaffold led to the identification of analogues with enhanced activity at the GluN2B subunit. We identified molecules that potentiate the response of GluN2B/GluN2C/GluN2D, GluN2B/GluN2C, and GluN2C/GluN2D-containing NMDARs to maximally effective concentrations of agonist. Multiple compounds potentiate the response of NMDARs with submicromolar EC50 values. Analysis of enantiomeric pairs revealed that the S-(−) enantiomer is active at the GluN2B, GluN2C, and/or GluN2D subunits, whereas the R-(+) enantiomer is only active at GluN2C/D subunits. These results provide a starting point for the development of selective positive allosteric modulators for GluN2B-containing receptors.

Translated examples of verbatim comments for the indicated focus group topics. (DOC 1.10 mb)

Organisation flowchart of the whole project. (DOCX 44Â kb)

Individuals’ speech times In FG I and II. (DOCX 39 kb)

ObjectiveIncreased nuchal translucency originates from disturbed lymphatic development. Abnormal neural crest cell (NCC) migration may be involved in lymphatic development. Because both neuronal and lymphatic development share retinoic acid (RA) as a common factor, this study investigated the involvement of NCCs and RA in specific steps in lymphatic endothelial cell (LEC) differentiation and nuchal edema, which is the morphological equivalent of increased nuchal translucency. MethodsMouse embryos in which all NCCs were fluorescently labeled (Wnt1-Cre;Rosa26(eYfp)), reporter embryos for in vivo RA activity (DR5-luciferase) and embryos with absent (Raldh2(-/-)) or in utero inhibition of RA signaling (BMS493) were investigated. Immunofluorescence using markers for blood vessels, lymphatic endothelium and neurons was applied. Flow cytometry was performed to measure specific LEC populations. ResultsCranial nerves were consistently close to the jugular lymph sac (JLS), in which NCCs were identified. In the absence of RA synthesis, enlarged JLS and nuchal edema were observed. Inhibiting RA signaling in utero resulted in a significantly higher amount of precursor-LECs at the expense of mature LECs and caused nuchal edema. ConclusionsNeural crest cells are involved in lymphatic development. RA is required for differentiation into mature LECs. Blocking RA signaling in mouse embryos results in abnormal lymphatic development and nuchal edema. (c) 2014 John Wiley & Sons, Ltd. (Less)

Machine learning continues to make strident advances in the prediction of desired properties concerning drug development. Problematically, the efficacy of machine learning in these arenas is reliant upon highly accurate and abundant data. These two limitations, high accuracy and abundance, are often taken together, however, insight into the dataset accuracy limitation of contemporary machine learning algorithms may yield insight into whether non-bench experimental sources of data may be used to generate useful machine learning models where there is a paucity of experimental data. We took highly accurate data across six kinase types, one GPCR, one polymerase, a human protease, and HIV protease, and intentionally introduced error at varying population proportions in the datasets for each target. With the generated error in the data, we explored how the retrospective accuracy of a Na&iuml, ve Bayes Network, a Random Forest Model, and a Probabilistic Neural Network model decayed as a function of error. Additionally, we explored the ability of a training dataset with an error profile resembling that produced by the Free Energy Perturbation method (FEP+) to generate machine learning models with useful retrospective capabilities. The categorical error tolerance was quite high for a Na&iuml, ve Bayes Network algorithm averaging 39% error in the training set required to lose predictivity on the test set. Additionally, a Random Forest tolerated a significant degree of categorical error introduced into the training set with an average error of 29% required to lose predictivity. However, we found the Probabilistic Neural Network algorithm did not tolerate as much categorical error requiring an average of 20% error to lose predictivity. Finally, we found that a Na&iuml, ve Bayes Network and a Random Forest could both use datasets with an error profile resembling that of FEP+. This work demonstrates that computational methods of known error distribution like FEP+ may be useful in generating machine learning models not based on extensive and expensive in vitro-generated datasets.

Objectives. Doctors’ empathy towards their patients is considered important for treatment outcome. However, during medical school there might be a decline in empathy called “hardening of the heart.” This study evaluated the cognitive and emotional empathy in medical students and investigated the influence of a preference for a specialty and students attachment styles. Methods. 126 first-year medical students were included and completed the Reading the Mind in the Eyes Test revised version (RME-R), the Balanced Emotional Empathy Scale (BEES), and the Experiences in Close Relationships-Revised Adult Attachment Questionnaire (ECR-R). Results. Students identified of 36 photographs in the RME-R test correctly (norm: 26). The female students’ mean BEES total score was and the male students’ ; . The female students’ mean BEES score was significantly () below the female norm of 60. Students who preferred a specialty with continuity of patient care scored significantly higher in the BEES (). A more avoidant attachment style was associated with a lower BEES score (). Conclusion. The students showed low emotional and cognitive empathy scores and an avoidant attachment style. This supports the inclusion of specific training in cognitive and emotional empathy in medical education.

In 1957, Bergman proved that every natural number can be expressed uniquely as a sum of distinct, non-consecutive integral powers of φ=(1+5)/2. More recently, in 2009, Gerdemann showed how such a decomposition of n leads to the corresponding “Zeckendorf representation” of nFm, for all sufficiently large m, in which Fm denotes the mth Fibonacci number. Here we extend these results by replacing φ with an arbitrary quadratic irrational real number α. In this general setting, we will connect our new “base α” representation of n to Ostrowskiʼs decomposition of nqm, in which qm denotes the mth continuant associated with α.

This paper presents a real-time capable graphics processing unit (GPU)-based ultrasound simulator suitable for medical education. The main focus of the simulator is to synthesize realistic looking ultrasound images in real-time including artifacts, which are essential for the interpretation of this data. The simulation is based on a convolution-enhanced ray-tracing approach and uses a deformable mesh model. Deformations of the mesh model are calculated using the PhysX engine. Our method advances the state of the art for real-time capable ultrasound simulators by following the path of the ultrasound pulse, which enables better simulation of ultrasound-specific artifacts. An evaluation of our proposed method in comparison with recent generative slicing-based strategies as well as real ultrasound images is performed. Hereby, a gelatin ultrasound phantom containing syringes filled with different media is scanned with a real transducer. The obtained images are then compared to images which are simulated using a slicing-based technique and our proposed method. The particular benefit of our method is the accurate simulation of ultrasound-specific artifacts, like range distortion, refraction and acoustic shadowing. Several test scenarios are evaluated regarding simulation time, to show the performance and the bottleneck of our method. While being computationally more intensive than slicing techniques, our simulator is able to produce high-quality images in real-time, tracing over 5000 rays through mesh models with more than 2 000 000 triangles of which up to 200 000 may be deformed each frame.

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo GRIN2A missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg2+, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient’s observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association–approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.

A commercially available dry chemiluminescence (CI) instrument for fast and precise measurement of ozone (O3) is specified. The sensitivity is ~9000 counts s−1 per ppbv of ozone. Its precision is entirely determined by the number of photons reaching the detector (being a photomultiplier), i.e. is quantum-noise limited. The relative precision (ΔO3/O3 in %) thus follows Poisson statistics and scales with the square root of the measurement frequency f and with the inverse O3 mixing ratio: ΔO3/O3 ∝ f0.5 · O3−0.5. At typical O3 mixing ratios between 10 and 100 ppbv (and 1 bar), the precision is 0.3–1.0% at f = 10 Hz. The maximum measurement frequency is 50 Hz. The mechanical and electronic set-up as well as the instrument performance is described. Recommendations on the adequate inlet tube configuration (inlet tube length, sampling flow) and on the way of calibration at stationary ground-based platforms and onboard aircraft are given.

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of N-methyl-d-aspartate (NMDA) receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC(50) values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent and could not be surmounted by increasing concentrations of either coagonist, glutamate or glycine, consistent with a noncompetitive mechanism of action. DQP-1105 inhibited single-channel currents in excised outside-out patches without significantly changing mean open time or single-channel conductance, suggesting that DQP inhibits a pregating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist-binding domain.

The Millimeter Bolometer Array Camera (MBAC) will be installed on the 6-meter Atacama Cosmology Telescope (ACT) in late 2007. For the first season of observations, MBAC will comprise a 145 GHz diffraction-limited, 1024-pixel, focal plane array of Transition Edge Sensor (TES) Bolometers. This will be the largest array of pop-up-detector bolometers ever fielded as well as one of the largest arrays of TES bolometers. We discuss the design specifications for the array and pre-assembly testing procedures for the cryogenic components. We present dark measurements of the TES bolometer properties of numerous 32-pixel columns that have been assembled into the first kilopixel array for ACT, as well as optical measurements made with our 256-pixel prototype array, including first light measurements on ACT.

We have developed multi-channel electronics (MCE) which work in concert with time-domain multiplexors developed at NIST, to control and read signals from large format bolometer arrays of superconducting transition edge sensors (TESs). These electronics were developed as part of the Submillimeter Common-User Bolometer Array-2 (SCUBA2 ) camera, but are now used in several other instruments. The main advantages of these electronics compared to earlier versions is that they are multi-channel, fully programmable, suited for remote operations and provide a clean geometry, with no electrical cabling outside of the Faraday cage formed by the cryostat and the electronics chassis.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by chromosomal rearrangements possibly enforcing arrest at specific development stages. We studied the relationship between molecular- cytogenetic abnormalities and T-cell development stage to investigate whether arrest at specific stages can explain the prognostic significance of specific abnormalities. We extensively studied 72 pediatric T-ALL cases for genetic abnormalities and expression of transcription factors, NOTCH1 mutations and expression of specific CD markers. HOX11 cases were CD1 positive consistent with a cortical stage, but as 4/5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre- AB developmental stages, but 3/17 HOX11L2 mature cases were restricted to the gamma delta-lineage. TAL1 rearrangements were restricted to the alpha beta-lineage with most cases being TCR-alpha beta positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. CALM-AF10 was associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcomes, respectively. Also cases with high vs low TAL1 expression levels demonstrated a trend toward good outcome. Most cases with lower TAL1 levels were HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for outcome. Classification into T-cell developmental subgroups was not predictive for outcome.