Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Assessing racial/ethnic and nativity disparities in US cancer mortality using a new integrated platform.

BACKGROUND: Foreign-born (FB) populations in the US have significantly increased, yet cancer trends remain unexplored. Survey-based Population-Adjusted Rate Calculator (SPARC) is a new tool for evaluating nativity differences in cancer mortality. METHODS: Using SPARC, we calculated 3-year (2016-2018) age-adjusted mortality rates (AAMRs) and rate ratios (RRs) for common cancers by sex, age group, race/ethnicity, and nativity. Trends by nativity were examined for the first time for 2006-2018. Traditional cancer statistics draw populations from decennial censuses. However, nativity-stratified populations are from the American Community Surveys, thus involve sampling errors. To rectify this, SPARC employed bias-corrected estimators. Death counts came from the National Vital Statistics System. RESULTS: AAMRs were higher among US-born (UB) populations across nearly all cancer types, with the largest UB- FB difference observed in lung cancer among Black females (RR = 3.67, 95%CI = 3.37-4.00). The well-documented White-Black differences in breast cancer mortality existed mainly among UB women. For all cancers combined, descending trends were more accelerated for the UB compared to the FB in all race/ethnicity groups with changes ranging from -2.6% per year in UB Black males to stable (non-significant) among FB Black females. Pancreas and liver cancers were exceptions with increasing, stable, or decreasing trends depending on nativity and race/ethnicity. Notably, FB Black males and FB Hispanic males did not show a favorable decline in colorectal cancer mortality. CONCLUSIONS: While all groups show beneficial cancer mortality trends, those with higher rates in 2006 have experienced sharper declines. Persistent disparities between the UB and the FB, especially among Black people, necessitate further investigation.

A Structural Model for the Core Nup358-BicD2 Interface.

Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a "cargo recognition α-helix" upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development.

Bilobed flap reconstruction after en-bloc removal of solitary fibrous tumor of the lacrimal sac.

Purpose: To report a rare case of a solitary fibrous tumor (SFT) of the lacrimal sac and discuss considerations for management of similar cases. Observations: We present the case of a 41-year-old woman who presented with a primary lacrimal sac SFT for which she underwent en-bloc surgical resection. We discuss management options for SFTs and our surgical approach for this case: bilobed flap reconstruction of the medial canthus and inferior orbit. Conclusions: We present an uncommon presentation of a rare tumor and a successful one-stage reconstruction with a bilobed flap.

Apolipoprotein†E Recognizes Alzheimer's Disease Associated 3-O Sulfation of Heparan Sulfate.

Apolipoprotein E (ApoE)'s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk.

The effect of COVID-19 on self-reported safety incidents in aviation: An examination of the heterogeneous effects using causal machine learning.

INTRODUCTION: Disruptions to aviation operations occur daily on a micro-level with negligible impacts beyond the inconvenience of rebooking and changing aircrew schedules. The unprecedented disruption in global aviation due to COVID-19 highlighted a need to evaluate emergent safety issues rapidly. METHOD: This paper uses causal machine learning to examine the heterogeneous effects of COVID-19 on reported aircraft incursions/excursions. The analysis utilized self report data from NASA Aviation Safety Reporting System collected from 2018 to 2020. The report attributes include self identified group characteristics and expert categorization of factors and outcomes. The analysis identified attributes and subgroup characteristics that were most sensitive to COVID-19 in inducing incursions/excursions. The method included the generalized random forest and difference-in-difference techniques to explore causal effects. RESULTS: The analysis indicates first officers are more prone to experiencing incursion/excursion events during the pandemic. In addition, events categorized with the human factors confusion, distraction, and the causal factor fatigue increased incursion/excursion events. PRACTICAL APPLICATIONS: Understanding the attributes associated with the likelihood of incursion/excursion events provides policymakers and aviation organizations insights to improve prevention mechanisms for future pandemics or extended periods of reduced aviation operations.

Temperature responses vary between riffle beetles from contrasting aquatic environments.

Organisms living in environmentally stable ecosystems are hypothesized to exhibit narrow environmental tolerance ranges; however, previous experiments testing this prediction with invertebrates in spring habitats are equivocal. Here we examined the effects of elevated temperatures on four riffle beetle species (family: Elmidae) native to central and west Texas, USA. Two of these, Heterelmis comalensis and Heterelmis cf. glabra are known to occupy habitats immediately adjacent to spring openings and are thought to have stenothermal tolerance profiles. The other two, Heterelmis vulnerata and Microcylloepus pusillus are surface stream species with more cosmopolitan distributions and are assumed to be less sensitive to variation in environmental conditions. We examined performance and survival of elmids in response to increasing temperatures using dynamic and static assays. Additionally, changes in metabolic rate in response to thermal stress were assessed for all four species. Our results indicated that spring-associated H. comalensis is most sensitive while the more cosmopolitan elmid M. pusillus is least sensitive to thermal stress. However, there were differences in temperature tolerances among the two spring-associated species: H. comalensis had relatively narrow thermal tolerance in comparison to H. cf. glabra. This could be due to differences in the climatic and hydrological conditions in the geographical regions which the respective riffle beetle populations reside. However, despite these differences, H. comalensis and H. cf. glabra showed a dramatic increase in their metabolic rates with increasing temperatures indicating that these species are indeed spring specialists and likely have a stenothermal profile.

Proline-Rich Region II (PRR2) Plays an Important Role in Tau-Glycan Interaction: An NMR Study.

(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.

Motor Responses in Pediatric Pompe Disease in the ADVANCE Participant Cohort.

BACKGROUND: ADVANCE (NCT01526785) presented an opportunity to obtain a more nuanced understanding of motor function changes in treatment-experienced children with Pompe disease receiving 4000L-production-scale alglucosidase alfa for 52 weeks. OBJECTIVE: To estimate minimal detectable change (MDC) and effect size on Gross Motor Function Measure-88 (GMFM-88) after 52 weeks of 4000L alglucosidase alfa (complete data N =  90). METHODS: The GMFM-88 mean total % score changes, MDC, and effect size were analyzed post hoc by Pompe Motor Function Level at enrollment, age groups at enrollment, and fraction of life on pre-study 160L-production-scale alglucosidase alfa. RESULTS: Overall, participants aged < 2 years surpassed MDC at Week 52 (change [mean±standard deviation] 21.1±14.1, MDC range 5.7-13.3, effect size 1.1), whereas participants aged≥2 years did not attain this (change -0.9±15.3, MDC range 10.8-25.2, effect size -0.03). In participants aged < 2 years, improvements surpassed the MDC for walkers (change 17.1±13.3, MDC range 3.0-6.9, effect size 1.7), supported standers (change 35.2±18.0, MDC range 5.9-13.7, effect size 1.8) and sitters (change 24.1±12.1, MDC range 2.6-6.2, effect size 2.7). Age-independent MDC ranges were only attained by walkers (change 7.7±12.3, MDC range 6.4-15.0, effect size 0.4) and sitters (change 9.9±17.2, MDC range 3.3-7.7, effect size 0.9). CONCLUSIONS: These first GMFM-88 minimal-detectable-change estimates for alglucosidase alfa-treated Pompe disease offer utility for monitoring motor skills. TRIAL REGISTRATION: ClinicalTrials.gov; NCT01526785; Registered 6 February 2012; https://clinicaltrials.gov/ct2/show/NCT01526785.

Covalent fragment inhibits intramembrane proteolysis.

Alzheimer's disease (AD) is a serious public health crisis with only one current modifying treatment. The reduction of amyloid load by targeting γ-secretase (GS) has been a leading approach in AD drug discovery and development. Despite the focus on GS inhibition, multiple GS inhibitors (GSIs) have failed in clinical trials as a result of side effects including exacerbated cognitive decline. These side effects are largely attributable to inhibition of normal GS function. Standard enzyme inhibitors target catalytic or allosteric sites of the enzyme, including the active site presenilin, as previous GSIs did. To avoid issues observed from broad-spectrum GSIs we discovered that fragment 6H8 that covalently binds to the substrate of GS, the transmembrane domain of amyloid precursor protein (APPTM). Nuclear Magnetic Resonance (NMR) Spectroscopy combined with MALDI-TOF-MS established 6H8 covalently binds to APPTM. 6H8 acts as a Michael acceptor and covalently links to the side chain amines of lysine residues, specifically targeting a cluster of C-terminal lysines K53-K55. Through this modification, 6H8 can inhibit intramembrane proteolysis of an archaeal homolog of presenilin (the active subunit of GS) via substrate binding with a 2-4 µM IC50, determined by a gel-based cleavage assay. 6H8, while too small to be an effective drug candidate, can be combined with a specific non-covalent partner and function as an effective covalent warhead of a targeted covalent inhibitor (TCI). The future development of the 6H8 fragment into the covalent warhead of a TCI is, to our knowledge, a novel approach to AD drug discovery.

Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance titration and chemical exchange saturation transfer, mutagenesis, and circular dichroism spectroscopy, a Nup358 α-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an α-helical conformation upon BicD2 binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 through a 'cargo recognition α-helix,' a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.

PRUNE1 c.933G>A synonymous variant induces exon 7 skipping, disrupts the DHHA2 domain, and leads to an atypical NMIHBA syndrome presentation: Case report and review of the literature.

Prune exopolyphosphatase-1 (PRUNE1) encodes a member of the aspartic acid-histidine-histidine (DHH) phosphodiesterase superfamily that regulates cell migration and proliferation during brain development. In 2015, biallelic PRUNE1 loss-of-function variants were identified to cause the neurodevelopmental disorder with microcephaly, hypotonia, and variable brain abnormalities (NMIHBA, OMIM#617481). NMIHBA is characterized by the namesake features and structural brain anomalies including thinning of the corpus callosum, cerebral and cerebellar atrophy, and delayed myelination. To date, 47 individuals have been reported in the literature, but the phenotypic spectrum of PRUNE1-related disorders and their causative variants remains to be characterized fully. Here, we report a novel homozygous PRUNE1 NM_021222.2:c.933G>A synonymous variant identified in a 6-year-old boy with intellectual and developmental disabilities, hypotonia, and spastic diplegia, but with the absence of microcephaly, brain anomalies, or seizures. Fibroblast RNA sequencing revealed that the PRUNE1 NM_021222.1:c.933G>A variant resulted in an in-frame skipping of the penultimate exon 7, removing 53 amino acids from an important protein domain. This case represents the first synonymous variant and the third pathogenic variant known to date affecting the DHH-associated domain (DHHA2 domain). These findings extend the genotypic and phenotypic spectrums in PRUNE1-related disorders and highlight the importance of considering synonymous splice site variants in atypical presentations.

Small-molecule compound from AlphaScreen disrupts tau-glycan interface.

Tauopathies are neurodegenerative diseases characterized by intracellular abnormal tau deposits in the brain. Tau aggregates can propagate from one neuron to another in a prion-like manner, mediated by the interaction between tau and cell surface heparan sulfate proteoglycans. We developed an AlphaScreen assay, with His-tagged tau and biotinylated heparin, to represent the tau-HS interface to target the tau-glycan interface. Using our AlphaScreen assay, with a Z-factor of 0.65, we screened ∼300 compounds and discovered a small-molecule compound (herein referred to as A9), which can disrupt the tau-heparin interaction with micromolar efficacy. A9 also effectively inhibited heparin-induced tau aggregation in Thioflavin T fluorescence assays and attenuated tau internalization by H4 neuroglioma cells. These results strongly suggest that A9 can disrupt the tau-glycan interface in both in vitro molecular and cellular environments. We further determined that A9 interacts with heparin rather than tau and does so with micromolar binding affinity as shown by nuclear magnetic resonance and surface plasmon resonance experiments. A9 binds to heparin in a manner that blocks the sites where tau binds to heparin on the cell surface. These results demonstrate our AlphaScreen method as an effective method for targeting the tau-glycan interface in drug discovery and A9 as a promising lead compound for tauopathies, including Alzheimer's disease.

Cardiac responses in paediatric Pompe disease in the ADVANCE patient cohort.

Pompe disease results from lysosomal acid α-glucosidase deficiency, which leads to cardiomyopathy in all infantile-onset and occasional late-onset patients. Cardiac assessment is important for its diagnosis and management. This article presents unpublished cardiac findings, concomitant medications, and cardiac efficacy and safety outcomes from the ADVANCE study; trajectories of patients with abnormal left ventricular mass z score at enrolment; and post hoc analyses of on-treatment left ventricular mass and systolic blood pressure z scores by disease phenotype, GAA genotype, and "fraction of life" (defined as the fraction of life on pre-study 160 L production-scale alglucosidase alfa). ADVANCE evaluated 52 weeks' treatment with 4000 L production-scale alglucosidase alfa in ≥1-year-old United States of America patients with Pompe disease previously receiving 160 L production-scale alglucosidase alfa. M-mode echocardiography and 12-lead electrocardiography were performed at enrolment and Week 52. Sixty-seven patients had complete left ventricular mass z scores, decreasing at Week 52 (infantile-onset patients, change -0.8 ± 1.83; 95% confidence interval -1.3 to -0.2; all patients, change -0.5 ± 1.71; 95% confidence interval -1.0 to -0.1). Patients with "fraction of life" <0.79 had left ventricular mass z score decreasing (enrolment: +0.1 ± 3.0; Week 52: -1.1 ± 2.0); those with "fraction of life" ≥0.79 remained stable (enrolment: -0.9 ± 1.5; Week 52: -0.9 ± 1.4). Systolic blood pressure z scores were stable from enrolment to Week 52, and no cohort developed systemic hypertension. Eight patients had Wolff-Parkinson-White syndrome. Cardiac hypertrophy and dysrhythmia in ADVANCE patients at or before enrolment were typical of Pompe disease. Four-thousand L alglucosidase alfa therapy maintained fractional shortening, left ventricular posterior and septal end-diastolic thicknesses, and improved left ventricular mass z score.Trial registry: ClinicalTrials.gov Identifier: NCT01526785 https://clinicaltrials.gov/ct2/show/NCT01526785.Social Media Statement: Post hoc analyses of the ADVANCE study cohort of 113 children support ongoing cardiac monitoring and concomitant management of children with Pompe disease on long-term alglucosidase alfa to functionally improve cardiomyopathy and/or dysrhythmia.

Automated evaluation of psychotherapy skills using speech and language technologies.

With the growing prevalence of psychological interventions, it is vital to have measures which rate the effectiveness of psychological care to assist in training, supervision, and quality assurance of services. Traditionally, quality assessment is addressed by human raters who evaluate recorded sessions along specific dimensions, often codified through constructs relevant to the approach and domain. This is, however, a cost-prohibitive and time-consuming method that leads to poor feasibility and limited use in real-world settings. To facilitate this process, we have developed an automated competency rating tool able to process the raw recorded audio of a session, analyzing who spoke when, what they said, and how the health professional used language to provide therapy. Focusing on a use case of a specific type of psychotherapy called "motivational interviewing", our system gives comprehensive feedback to the therapist, including information about the dynamics of the session (e.g., therapist's vs. client's talking time), low-level psychological language descriptors (e.g., type of questions asked), as well as other high-level behavioral constructs (e.g., the extent to which the therapist understands the clients' perspective). We describe our platform and its performance using a dataset of more than 5000 recordings drawn from its deployment in a real-world clinical setting used to assist training of new therapists. Widespread use of automated psychotherapy rating tools may augment experts' capabilities by providing an avenue for more effective training and skill improvement, eventually leading to more positive clinical outcomes.

Multi-label Multi-task Deep Learning for Behavioral Coding.

We propose a methodology for estimating human behaviors in psychotherapy sessions using mutli-label and multi-task learning paradigms. We discuss the problem of behavioral coding in which data of human interactions is the annotated with labels to describe relevant human behaviors of interest. We describe two related, yet distinct, corpora consisting of therapist client interactions in psychotherapy sessions. We experimentally compare the proposed learning approaches for estimating behaviors of interest in these datasets. Specifically, we compare single and multiple label learning approaches, single and multiple task learning approaches, and evaluate the performance of these approaches when incorporating turn context. We demonstrate the prediction performance gains which can be achieved by using the proposed paradigms and discuss the insights these models provide into these complex interactions.

Leading cancers contributing to educational disparities in cancer mortality in the US, 2017.

PURPOSE: To inform prevention efforts, we sought to determine which cancer types contribute the most to cancer mortality disparities by individual-level education using national death certificate data for 2017. METHODS: Information on all US deaths occurring in 2017 among 25-84-year-olds was ascertained from national death certificate data, which include cause of death and educational attainment. Education was classified as high school or less (≤ 12 years), some college or diploma (13-15 years), and Bachelor's degree or higher (≥ 16 years). Cancer mortality rate differences (RD) were calculated by subtracting age-adjusted mortality rates (AMR) among those with ≥ 16 years of education from AMR among those with ≤ 12 years. RESULTS: The cancer mortality rate difference between those with a Bachelor's degree or more vs. high school or less education was 72 deaths per 100,000 person-years. Lung cancer deaths account for over half (53%) of the RD for cancer mortality by education in the US. CONCLUSION: Efforts to reduce smoking, particularly among persons with less education, would contribute substantially to reducing educational disparities in lung cancer and overall cancer mortality.

Inhibition of Staphylococcus aureus biofilm-forming functional amyloid by molecular tweezers.

Biofilms are rigid and largely impenetrable three-dimensional matrices constituting virulence determinants of various pathogenic bacteria. Here, we demonstrate that molecular tweezers, unique supramolecular artificial receptors, modulate biofilm formation of Staphylococcus aureus. In particular, the tweezers affect the structural and assembly properties of phenol-soluble modulin α1 (PSMα1), a biofilm-scaffolding functional amyloid peptide secreted by S. aureus. The data reveal that CLR01, a diphosphate tweezer, exhibits significant S. aureus biofilm inhibition and disrupts PSMα1 self-assembly and fibrillation, likely through inclusion of lysine side chains of the peptide. In comparison, different peptide binding occurs in the case of CLR05, a tweezer containing methylenecarboxylate units, which exhibits lower affinity for the lysine residues yet disrupts S. aureus biofilm more strongly than CLR01. Our study points to a possible role for molecular tweezers as potent biofilm inhibitors and antibacterial agents, particularly against untreatable biofilm-forming and PSM-producing bacteria, such as methicillin-resistant S. aureus.