External Trigger Free Charge Switchable Cationic Ligands in the Design of Safe and Effective Universal Heparin Antidote.

Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin-based anticoagulants such as unfractionated heparin, low-molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA-approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin-binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies.

Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal.

International cancer registries make real-world genomic and clinical data available, but their joint analysis remains a challenge. AACR Project GENIE, an international cancer registry collecting data from 19 cancer centers, makes data from >130,000 patients publicly available through the cBioPortal for Cancer Genomics (https://genie.cbioportal.org). For 25,000 patients, additional real-world longitudinal clinical data, including treatment and outcome data, are being collected by the AACR Project GENIE Biopharma Collaborative using the PRISSMM data curation model. Several thousand of these cases are now also available in cBioPortal. We have significantly enhanced the functionalities of cBioPortal to support the visualization and analysis of this rich clinico-genomic linked dataset, as well as datasets generated by other centers and consortia. Examples of these enhancements include (i) visualization of the longitudinal clinical and genomic data at the patient level, including timelines for diagnoses, treatments, and outcomes; (ii) the ability to select samples based on treatment status, facilitating a comparison of molecular and clinical attributes between samples before and after a specific treatment; and (iii) survival analysis estimates based on individual treatment regimens received. Together, these features provide cBioPortal users with a toolkit to interactively investigate complex clinico-genomic data to generate hypotheses and make discoveries about the impact of specific genomic variants on prognosis and therapeutic sensitivities in cancer. SIGNIFICANCE: Enhanced cBioPortal features allow clinicians and researchers to effectively investigate longitudinal clinico-genomic data from patients with cancer, which will improve exploration of data from the AACR Project GENIE Biopharma Collaborative and similar datasets.

Characterization of the phonological system in low-income Brazilian preschoolers.

PURPOSE: The purpose of this study is to characterize the phonological skills of low-income preschool children in a city in the Natal in the Northeast, Brazil. METHODS: The researchers assessed the phonological skills of 90 children (from 5 to 6:11) in early childhood education in three public schools located in regions of social and economic vulnerability. The evaluators used the phonology subtests of the Test of Childhood language (ABFW) children's language test. In addition to performing the standard analysis they examined the following: Phonological Processes (PP), Percentage of Correct Consonants (PCC), Percentage of Correct Consonants Revised (PCC-R), and Process Density Index (PDI). The Spearman's Correlation Coefficient test was used to analyze for correlations among the PCC, PCC-R, and PDI. RESULTS: According to the cutoff values of children who speak Brazilian Portuguese (BP), adequacy of the PCC and PCC-R values was observed in most participants (PCC: 82 children - 91.1%; PCC-R: 87 children - 94.6%). The processes of liquid simplification (LS), consonant clusters simplification (CCS), final consonant simplification (FCS) were productive of which the CCS (32.2%) and FCS (20%) are still expected for age and LS are not. There was a robust negative correlation between the variables PCC x PDI and PCC-R x PDI. CONCLUSION: Most children showed adequate phonological development. Variations were observed in syllabic segments, especially in the coda, which reflect the influence of regional linguistic differences. The evidence obtained regarding the phonological performance of children within this region contributes to a more accurate speech-language diagnosis.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Characterization of the phonological system in low-income Brazilian preschoolers / Caracterização do sistema fonológico em pré-escolares de baixa renda

ABSTRACT Purpose The purpose of this study is to characterize the phonological skills of low-income preschool children in a city in the Natal in the Northeast, Brazil. Methods The researchers assessed the phonological skills of 90 children (from 5 to 6:11) in early childhood education in three public schools located in regions of social and economic vulnerability. The evaluators used the phonology subtests of the Test of Childhood language (ABFW) children's language test. In addition to performing the standard analysis they examined the following: Phonological Processes (PP), Percentage of Correct Consonants (PCC), Percentage of Correct Consonants Revised (PCC-R), and Process Density Index (PDI). The Spearman's Correlation Coefficient test was used to analyze for correlations among the PCC, PCC-R, and PDI. Results According to the cutoff values of children who speak Brazilian Portuguese (BP), adequacy of the PCC and PCC-R values was observed in most participants (PCC: 82 children - 91.1%; PCC-R: 87 children - 94.6%). The processes of liquid simplification (LS), consonant clusters simplification (CCS), final consonant simplification (FCS) were productive of which the CCS (32.2%) and FCS (20%) are still expected for age and LS are not. There was a robust negative correlation between the variables PCC x PDI and PCC-R x PDI. Conclusion Most children showed adequate phonological development. Variations were observed in syllabic segments, especially in the coda, which reflect the influence of regional linguistic differences. The evidence obtained regarding the phonological performance of children within this region contributes to a more accurate speech-language diagnosis.

RESUMO Objetivo Caracterizar o sistema fonológico de crianças pré-escolares de baixa renda da cidade de Natal, Nordeste do Brasil. Método Foi analisado o nível fonológico de 90 crianças da educação infantil de três escolas públicas localizadas em regiões de vulnerabilidade social. Foi utilizado o instrumento ABFW, nomeação e imitação, por meio da análise tradicional e das medidas de Porcentagem de Consoantes Corretas (PCC), Porcentagem de Consoantes Corretas Revisado (PCC-R) e Índice de Ocorrência de Processos (PDI). Para análise estatística inferencial foi utilizado o teste do Coeficiente de Correlação de Spearman para analisar a correlação entre as variáveis PCC, PCC-R e PDI. Resultados De acordo com os valores de corte das crianças que falam Português Brasileiro (PB), a adequação dos valores da PCC e PCC-R foi observada na maioria dos participantes (PCC: 82 crianças - 91,1%; PCC R: 87 crianças - 94,6%). Os processos de simplificação de líquidas (SL), simplificação de encontros consonantais (SEC), simplificação de consoante final (SCF) foram produtivos dos quais o CCS (32,2%) e FCS (20%) ainda são esperados para a idade e SL não. Houve correlação negativa muito forte entre as variáveis PCC x PDI e PCC-R x PDI. Conclusão A maioria das crianças apresentou desenvolvimento fonológico adequado. Foram observadas variações nos segmentos silábicos, principalmente na coda, que refletem a influência das diferenças linguísticas regionais. As evidências obtidas sobre o desempenho fonológico das crianças dessa região contribuem para um diagnóstico fonoaudiológico mais preciso.

Studying the Interactions of U24 from HHV-6 in Order to Further Elucidate Its Potential Role in MS.

A number of studies have suggested that human herpesvirus 6A (HHV-6A) may play a role in multiple sclerosis (MS). Three possible hypotheses have been investigated: (1) U24 from HHV-6A (U24-6A) mimics myelin basic protein (MBP) through analogous phosphorylation and interaction with Fyn-SH3; (2) U24-6A affects endocytic recycling by binding human neural precursor cell (NPC) expressed developmentally down-regulated protein 4-like WW3* domain (hNedd4L-WW3*); and (3) MS patients who express Killer Cell Immunoglobulin Like Receptor 2DL2 (KIR2DL2) on natural killer (NK) cells are more susceptible to HHV-6 infection. In this contribution, we examined the validity of these propositions by investigating the interactions of U24 from HHV-6B (U24-6B), a variant less commonly linked to MS, with Fyn-SH3 and hNedd4L-WW3* using heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) titrations and isothermal titration calorimetry (ITC). In addition, the importance of phosphorylation and the specific role of U24 in NK cell activation in MS patients were examined. Overall, the findings allowed us to shed light into the models linking HHV-6 to MS and the involvement of U24.

Aptamers used for molecular imaging and theranostics - recent developments.

Aptamers are single stranded oligonucleotides that fold into three dimensional structures and are able to recognize a variety of molecular targets. Due to the similarity to antibodies with regards to specificity and affinity and their chemical versatility, aptamers are increasingly used to create targeted probes for in vivo molecular imaging and therapy. Hence, aptamer-based probes have been utilized in practically all major imaging modalities such as nuclear imaging, magnetic resonance imaging, x-ray computed tomography, echography and fluorescence imaging, as well as newer modalities such as surface enhanced Raman spectroscopy. Aside from targeting, aptamers have been used for the creation of sensors that allow the localized detection of cellular markers such as ATP in vivo. This review focuses on in vivo studies of aptamer-based probes for imaging and theranostics since the comprehensive overview by Bouvier-Müller and Ducongé in 2018.

Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750 UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750 UHRA-8 and 32 in mPEG750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

The effect of cue type on directive-following in children with moderate to severe autism spectrum disorder.

For this study, 11 children with moderate to severe autism spectrum disorder (ASD) were given directives containing prepositions in three cue conditions: (a) spoken alone, (b) a short video clip along with spoken cues, and (c) a sequence of three graphic symbols accompanied by spoken cues. Participants followed directives significantly more accurately with the video clip than with spoken cues only, and significantly more accurately with spoken cues only relative to the sequence of graphic symbols. Results suggest that the short video clip along with spoken cues may be an optimal mode for enhancing learners' ability to follow directives containing prepositions. In addition, results reveal three statistically significant correlations between participants' preexisting skills and directive-following accuracy: a positive correlation between spoken preposition preassessment total score and accuracy in the spoken-alone condition; a positive correlation between spoken noun preassessment total score and accuracy in the video-clip condition; and a positive correlation between ASD severity and the need for repetition in the video-clip condition. Results also suggested that, for children with more severe ASD symptoms, the video clips require repetitions so that the relationships illustrated within it can gain more semantic salience. Implications for clinical practice and future research are discussed.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Do caregiver proxy reports and congruence of client-proxy activity participation goals relate to quality of life in people with aphasia?

BACKGROUND: Achieving activity participation goals is a key factor in quality of life (QOL) for people with aphasia (PWA), but expressing participation goals can be difficult for many of them. Proxy reports by caregivers may not accurately reflect the interests and participation goals of PWA, and discrepancies in these goals between PWA and their caregivers may affect QOL, based on the assumption that caregivers' awareness of their loved ones' unique participation goals may be important to increasing PWA activity participation. AIMS: To examine everyday activities valued by PWA using the Life Interests and Values (LIV) Cards; to measure congruence between PWA and their caregivers on life participation goals; and to measure how congruence of PWA-caregiver participation goals related to QOL. METHODS & PROCEDURES: A convenience sample of 25 PWA completed the LIV Card assessment and the Stroke Aphasia Quality of Life Scale-39 to assess participation goals and QOL. Participation goals were also evaluated with respect to age, time post-onset and aphasia severity. A total of 12 caregivers were administered the LIV Cards to calculate agreement between PWA-proxy activity reports and the relationship between agreement and QOL. OUTCOMES & RESULTS: PWA endorsed wanting to participate more in a wide range of activities, with common interests in walking/running, going to the beach and eating out, among others. PWA-caregiver activity agreement was fair to moderate with point-to-point agreement averaging 70%. However, no relationship between degree of congruence in PWA-proxy pairs and QOL was found. CONCLUSIONS & IMPLICATIONS: PWA have a variety of activity participation goals that can be integrated into intervention plans. Dependence on proxy respondents should be reduced as much as possible to support self-determination for PWA. What this paper adds What is already known on the subject Achieving activity participation goals is a key factor in QOL for PWA, but communicating about participation goals can be difficult for many of them. Because proxy reports by caregivers may not accurately reflect the interests and participation goals of PWA, this study examined how both PWA and their caregivers responded to an aphasia-friendly assessment for determining participation goals, and then compared level of agreement about these goals to QOL. Because activity participation is known to be an important factor in QOL, the reason for investigating how agreement relates to QOL is that caregivers' awareness of their loved ones' unique participation goals likely facilitates increased participation by PWA in their ongoing desired activities. The relationship between PWA-caregiver agreement regarding participation goals and QOL in PWA had not yet been investigated before this study. What this paper adds to existing knowledge This study adds additional as well as confirmatory information to the existing literature about life participation goals of community-dwelling individuals with chronic aphasia. Top activities endorsed by a group of 25 PWA are reported within four activity domains (home and community activities, creative and relaxing activities, physical activities, and social activities). Results indicated that agreement between PWA and their caregiver proxies on PWA's most desired activities was < 50%. However, the level of agreement between caregivers and proxies on participation goals was not significantly related to QOL in this sample. What are the potential or actual clinical implications of this work? PWA have a variety of participation goals that can be integrated into intervention plans to be carried out with clinicians, caregivers and family members. The use of proxy respondents when determining participation goals should be reduced as much as possible to support self-determination for PWA. Use of the LIV Cards, a picture-based sorting-task assessment, reduces the need for proxy responders and guesswork about the specific participation goals of PWA.

Comparison of reversal activity and mechanism of action of UHRA, andexanet, and PER977 on heparin and oral FXa inhibitors.

Anticoagulants such as unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and direct oral anticoagulants (DOACs) targeting thrombin (IIa) or factor Xa (FXa) are widely used in prevention and treatment of thromboembolic disorders. However, anticoagulant-associated bleeding is a concern that demands monitoring and neutralization. Protamine, the UFH antidote, has limitations, while there is no antidote available for certain direct FXa inhibitors. Improved antidotes in development include UHRA (Universal Heparin Reversal Agent) for all heparin anticoagulants; andexanet alfa (andexanet), a recombinant antidote for both direct FXa inhibitors and LMWHs; and ciraparantag (PER977), a small-molecule antidote for UFH, LMWHs, and certain DOACs. The binding affinities of these antidotes for their presumed anticoagulant targets have not been compared. Here, isothermal titration calorimetry (ITC) was used to determine the affinity of each antidote for its putative targets. Clotting and chromogenic FXa assays were used to characterize neutralization activity, and electron microscopy was used to visualize the effect of each antidote on clot morphology in the absence or presence of anticoagulant. ITC confirmed binding of UHRA to all heparins, and binding of andexanet to edoxaban and rivaroxaban, and to the antithrombin-enoxaparin complex. PER977 was found to bind heparins weakly, but not the direct FXa inhibitors studied. For UHRA and andexanet, an affinity at or below the micromolar level was found to correlate with neutralization activity, while no reversal activity was observed for the PER977/anticoagulant systems. Standard metrics of clot structure were found to correlate weakly with PER977's activity. This is the first study comparing 3 antidotes in development, with each exerting activity through a distinct mechanism.

Highly Efficient and Reliable DNA Aptamer Selection Using the Partitioning Capabilities of ddPCR: The Hi-Fi SELEX Method.

In addition to its growing use in detecting and quantifying genes and larger genomic events, the partitioning used in digital PCR can serve as a powerful tool for high-fidelity amplification of synthetic combinatorial libraries of single-stranded DNA. Sequence-diverse libraries of this type are used as a basis for selecting tight-binding aptamers against a specific target. Here we provide a detailed description of the Hi-Fi SELEX protocol for rapid and efficient DNA aptamer selection. As part of that methodology, we describe how Hi-Fi SELEX gains advantages over other aptamer selection methods in part through the use of the massive partitioning capability of digital PCR.

Design of Polyphosphate Inhibitors: A Molecular Dynamics Investigation on Polyethylene Glycol-Linked Cationic Binding Groups.

Inorganic polyphosphate (polyP) released by human platelets has recently been shown to activate blood clotting and identified as a potential target for the development of novel antithrombotics. Recent studies have shown that polymers with cationic binding groups (CBGs) inhibit polyP and attenuate thrombosis. However, a good molecular-level understanding of the binding mechanism is lacking for further drug development. While molecular dynamics (MD) simulation can provide molecule-level information, the time scale required to simulate these large biomacromolecules makes classical MD simulation impractical. To overcome this challenge, we employed metadynamics simulations with both all-atom and coarse-grained force fields. The force field parameters for polyethylene glycol (PEG) conjugated CBGs and polyP were developed to carry out coarse-grained MD simulations, which enabled simulations of these large biomacromolecules in a reasonable time scale. We found that the length of the PEG tail does not impact the interaction between the (PEG) n-CBG and polyP. As expected, increasing the number of the charged tertiary amine groups in the head group strengthens its binding to polyP. Our simulation shows that (PEG) n-CBG initially form aggregates, mostly with the PEG in the core and the hydrophilic CBG groups pointing toward water; then the aggregates approach the polyP and sandwich the polyP to form a complex. We found that the binding of (PEG) n-CBG remains intact against various lengths of polyP. Binding thermodynamics for two of the (PEG) n-CBG/polyP systems simulated were measured by isothermal titration calorimetry to confirm the key finding of the simulations that the length PEG tail does not influence ligand binding to polyP.

Approaches to prevent bleeding associated with anticoagulants: current status and recent developments.

Anticoagulants are widely used for the prophylaxis and treatment of cardiovascular disorders and to prevent blood clotting during surgeries. However, the major limitation associated with anticoagulant therapy is bleeding; all the current anticoagulants do have a bleeding risk. The propensity to bleed is much higher among the elderly population and patients with renal insufficiency. Therefore, there is an utmost and urgent clinical need for a highly efficient, nontoxic antidote with excellent anticoagulant reversal activity. This will significantly improve the safety of anticoagulation therapy. This review summarizes the current options and approaches to reverse anticoagulation activity of clinically used anticoagulants. We start with an introduction to thrombosis and then summarize the details of current clinically available anticoagulants and their mechanisms of action and limitations. This is followed by current practices in anticoagulant neutralization including the details of the only clinically approved unfractionated heparin antidote, protamine; recent advances in the development of antidotes against heparin-based drugs; and direct oral anticoagulants (DOACs).

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism.

Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of Fel and Fsteric). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.

Interactions of U24 from Roseolovirus with WW domains: canonical vs noncanonical.

U24 is a C-terminal membrane-anchored protein found in both human herpes virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminal segment that is rich in prolines (PPxY motif in both HHV-6A and 7; PxxP motif in HHV-6A). Previous work has shown that U24 interacts strongly with Nedd4 WW domains, in particular, hNedd4L-WW3*. It was also shown that this interaction depends strongly on the nature of the amino acids that are upstream from the PY motif in U24. In this contribution, data was obtained from pull-downs, isothermal titration calorimetry, and NMR to further determine what modulates U24:WW domain interactions. Specifically, 3 non-canonical WW domains from human Smad ubiquitination regulatory factor (Smurf), namely hSmurf2-WW2, hSmurf2-WW3, and a tandem construct hSmurf2-WW2 + 3, were studied. Overall, the interactions between U24 and these Smurf WW domains were found to be weaker than those in U24:Nedd4 WW domain pairs, suggesting that U24 function is tightly linked to specific E3 ubiqitin ligases.

U24 from Roseolovirus interacts strongly with Nedd4 WW Domains.

U24 is a protein found in both roseoloviruses Human Herpes Virus type 6 and 7 (HHV-6 and HHV-7), with an N-terminus that is rich in prolines (PY motif in both HHV-6A and 7; PxxP motif in HHV-6A). Previous work has shown that the interaction between U24 and WW domains is important for endocytic recycling of T-cell receptors, but a cognate ligand was never identified. In this contribution, data was obtained from pull-downs, ITC, NMR and molecular dynamics simulations to show that a specific interaction exists between U24 and Nedd4 WW domains. ITC experiments were also carried out for U24 from HHV-6A phosphorylated at Thr6 (pU24-6A) and a peptide containing the PY motif from Nogo-A, a protein implicated in both the initial inflammatory and the neurodegenerative phases of multiple sclerosis (MS). The results suggest that phosphorylation of U24 from HHV-6A may be crucial for its potential role in MS.

Iron Binding and Iron Removal Efficiency of Desferrioxamine Based Polymeric Iron Chelators: Influence of Molecular Size and Chelator Density.

Desferrioxamine (DFO) is a clinically approved, high affinity iron chelator used for the treatment of iron overload. Due to its short half-life and toxicity, DFO is administered for 8-12 h per day, 5-7 d per week. In this manuscript, the influence of molecular properties of hyperbranched polyglycerol (HPG)-DFO conjugates on their iron binding by isothermal titration calorimetry, iron removal efficiency from ferritin in presence and absence of a low molecular weight (MW) iron chelator, and protection against iron mediated oxidation of proteins is reported. The iron binding properties of HPG-DFO are slightly altered with size and DFO density of conjugates. The lower MW conjugate shows greater iron removal efficiency at room temperature, however, the efficacy of high MW conjugates increases at physiological temperature. The iron removal from ferritin by HPG-DFO conjugates increases significantly in presence of a low MW chelator, suggesting the potential of combination therapy. The molecular properties of the polymer scaffold also have influence on the prevention of iron mediated oxidation of proteins by the conjugates. The results therefore help to define the iron binding thermodynamics of HPG-DFO and their dependence on MW, and can be extended to improve the general understanding of polymeric chelator-iron interactions in situ.