Increasing the versatility of the biphenyl-fused-dioxacyclodecyne class of strained alkynes.

Biphenyl-fused-dioxacyclodecynes are a promising class of strained alkyne for use in Cu-free 'click' reactions. In this paper, a series of functionalised derivatives of this class of reagent, containing fluorescent groups, are described. Studies aimed at understanding and increasing the reactivity of the alkynes are also presented, together with an investigation of the bioconjugation of the reagents with an azide-labelled protein.

Sex-Based Mechanisms of Cardiac Development and Function: Applications for Induced-Pluripotent Stem Cell Derived-Cardiomyocytes.

Males and females exhibit intrinsic differences in the structure and function of the heart, while the prevalence and severity of cardiovascular disease vary in the two sexes. However, the mechanisms of this sex-based dimorphism are yet to be elucidated. Sex chromosomes and sex hormones are the main contributors to sex-based differences in cardiac physiology and pathophysiology. In recent years, the advances in induced pluripotent stem cell-derived cardiac models and multi-omic approaches have enabled a more comprehensive understanding of the sex-specific differences in the human heart. Here, we provide an overview of the roles of these two factors throughout cardiac development and explore the sex hormone signaling pathways involved. We will also discuss how the employment of stem cell-based cardiac models and single-cell RNA sequencing help us further investigate sex differences in healthy and diseased hearts.

Koopman analysis of the periodic Korteweg-de Vries equation.

The eigenspectrum of the Koopman operator enables the decomposition of nonlinear dynamics into a sum of nonlinear functions of the state space with purely exponential and sinusoidal time dependence. For a limited number of dynamical systems, it is possible to find these Koopman eigenfunctions exactly and analytically. Here, this is done for the Korteweg-de Vries equation on a periodic interval using the periodic inverse scattering transform and some concepts of algebraic geometry. To the authors' knowledge, this is the first complete Koopman analysis of a partial differential equation, which does not have a trivial global attractor. The results are shown to match the frequencies computed by the data-driven method of dynamic mode decomposition (DMD). We demonstrate that in general, DMD gives a large number of eigenvalues near the imaginary axis and show how these should be interpreted in this setting.

Predicting chaotic statistics with unstable invariant tori.

It has recently been speculated that long-time average quantities of hyperchaotic dissipative systems may be approximated by weighted sums over unstable invariant tori embedded in the attractor, analogous to equivalent sums over periodic orbits, which are inspired by the rigorous periodic orbit theory and which have shown much promise in fluid dynamics. Using a new numerical method for converging unstable invariant two-tori in a chaotic partial differential equation (PDE), and exploiting symmetry breaking of relative periodic orbits to detect those tori, we identify many quasiperiodic, unstable, invariant two-torus solutions of a modified Kuramoto-Sivashinsky equation. The set of tori covers significant parts of the chaotic attractor and weighted averages of the properties of the tori-with weights computed based on their respective stability eigenvalues-approximate average quantities for the chaotic dynamics. These results are a step toward exploiting higher-dimensional invariant sets to describe general hyperchaotic systems, including dissipative spatiotemporally chaotic PDEs.

Altered microRNA expression links IL6 and TNF-induced inflammaging with myeloid malignancy in humans and mice.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging ("inflammaging") has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening.

OBJECTIVES: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. METHODS: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. RESULTS: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. CONCLUSIONS: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Adverse events in the digital age and where to find them.

Exponential growth of health-related data collected by digital tools is a reality within pharmaceutical and medical device research and development. Data generated through digital tools may be categorized as relevant to efficacy and/or safety. The enormity of these data requires the adoption of new approaches for processing and evaluation. Recognition of patterns within the safety data is vital for sponsors seeking regulatory approval for their new products. Nontraditional data sources may contain relevant safety information; early evaluation of these data will help to determine the product safety profile. Advanced technologies have allowed the development of digital tools to screen these data, which in some situations are classified as software as a medical devices and subject to clinical evaluation and post-marketing surveillance. Artificial intelligence may help to reduce or even eliminate noise from within these data, allowing safety experts to focus on the most pertinent evidence. We propose a data typology and provide considerations on how to define adverse events within different types of data, even where no human reporter exists. Proposals are made for the automation of screening processes. We consider validation aspects to support solutions that are proven to produce reliable results, and to deliver trusted outputs to stakeholders.

Invariant tori in dissipative hyperchaos.

One approach to understand the chaotic dynamics of nonlinear dissipative systems is the study of non-chaotic yet dynamically unstable invariant solutions embedded in the system's chaotic attractor. The significance of zero-dimensional unstable fixed points and one-dimensional unstable periodic orbits capturing time-periodic dynamics is widely accepted for high-dimensional chaotic systems, including fluid turbulence, while higher-dimensional invariant tori representing quasiperiodic dynamics have rarely been considered. We demonstrate that unstable 2-tori are generically embedded in the hyperchaotic attractor of a dissipative system of ordinary differential equations; tori can be numerically identified via bifurcations of unstable periodic orbits and their parameteric continuation and characterization of stability properties are feasible. As higher-dimensional tori are expected to be structurally unstable, 2-tori together with periodic orbits and equilibria form a complete set of relevant invariant solutions on which to base a dynamical description of chaos.

Site-selective modification strategies in antibody-drug conjugates.

Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.

Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications.

This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.

General dual functionalisation of biomacromolecules via a cysteine bridging strategy.

Site-selective modification of peptides and proteins has resulted in the development of a host of novel tools for the study of cellular systems or the synthesis of enhanced biotherapeutics. There is a need for useful methodologies that enable site-selective modification of native peptides or proteins, which is even more prevalent when modification of the biomolecule with multiple payloads is desired. Herein, we report the development of a novel dual functional divinylpyrimidine (dfDVP) platform that enables robust and modular modification of peptides, antibody fragments and antibodies. These biomacromolecules could be easily functionalised with a range of functional payloads (e.g. fluorescent dyes, cytotoxic warheads or cell-penetrating tags). Importantly, the dual functionalised peptides and antibodies demonstrated exquisite bioactivity in a range of in vitro cellular assays, showcasing the enhanced utility of these bioactive conjugates.

Cleavable linkers in antibody-drug conjugates.

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.

An Alternative Focus for Route Design for the Synthesis of Antibody-Drug Conjugate Payloads.

An analysis of Antibody-Drug Conjugate Payload manufacturing has revealed that the majority of the cost is associated with the use of high-containment facilities for the latter stages of the synthesis. To make a significant reduction in the Cost of Goods (CoGs), a new approach to route design has been introduced which focuses on minimizing the number of steps that require high containment. This approach has been exemplified in a new synthesis of tesirine, including the first application of a ring-closing copper(I)/TEMPO aerobic oxidation to the pyrrolobenzodiazepine ring system, affording a 60% reduction in CoGs.

Use of ultrasound enhancing agents in transesophageal echocardiography to improve interpretive confidence of left atrial appendage thrombus.

BACKGROUND: The value of ultrasound enhancing agents (UEA) in patients undergoing transesophageal echocardiography (TEE) for the exclusion of left atrial appendage (LAA) thrombi prior to direct current cardioversion (DCCV) is evolving. METHODS: We retrospectively identified 88 consecutive TEEs, where a commercial UEA was used during LAA interrogation. De-identified non-enhanced (pre-UEA) and enhanced cine loop images (post-UEA) from the same subjects were randomly reviewed by four expert readers in a blinded fashion. RESULTS: In 33% of the cases, UEA use was associated with a statistically insignificant improvement in physician confidence (scale, 0-3) in determining the presence or absence of a LAA thrombus (P = 0.071). In instances where non-enhanced images yielded an uncertain interpretation or when the left atrium contained spontaneous echo contrast (SEC), UEA use was associated with an improvement in interpretive confidence in 49% (P < 0.001) and 41% of the cases (P = 0.001), respectively. Overall, the absolute rate of hypothetical decision to proceed with DCCV rose by 9% with the application of UEA (P = 0.004). In instances where non-enhanced images were interpreted with limited confidence or when SEC was present, there were absolute increases of 16% (P < 0.001) and 21% (P < 0.001) in hypothetical procession to DCCV, respectively. In cases of a combination of limited interpretive confidence and SEC, UEA use was associated with a 29% absolute increase in the rate of procession to DCCV (P < 0.001). CONCLUSIONS: In patients undergoing TEE interrogation of the LAA, the use of UEA is associated with an increase in the level of interpretive confidence and higher rates of theoretical procession to DCCV.

Synthesis and cycloaddition reactions of strained alkynes derived from 2,2'-dihydroxy-1,1'-biaryls.

A series of strained alkynes, based on the 2,2'-dihydroxy-1,1'-biaryl structure, were prepared in a short sequence from readily-available starting materials. These compounds can be readily converted into further derivatives including examples containing fluorescent groups with potential for use as labelling reagents. The alkynes are able to react in cycloadditions with a range of azides without the requirement for a copper catalyst, in clean reactions with no observable side reactions.

Deep profiling of multitube flow cytometry data.

MOTIVATION: Deep profiling the phenotypic landscape of tissues using high-throughput flow cytometry (FCM) can provide important new insights into the interplay of cells in both healthy and diseased tissue. But often, especially in clinical settings, the cytometer cannot measure all the desired markers in a single aliquot. In these cases, tissue is separated into independently analysed samples, leaving a need to electronically recombine these to increase dimensionality. Nearest-neighbour (NN) based imputation fulfils this need but can produce artificial subpopulations. Clustering-based NNs can reduce these, but requires prior domain knowledge to be able to parameterize the clustering, so is unsuited to discovery settings. RESULTS: We present flowBin, a parameterization-free method for combining multitube FCM data into a higher-dimensional form suitable for deep profiling and discovery. FlowBin allocates cells to bins defined by the common markers across tubes in a multitube experiment, then computes aggregate expression for each bin within each tube, to create a matrix of expression of all markers assayed in each tube. We show, using simulated multitube data, that flowType analysis of flowBin output reproduces the results of that same analysis on the original data for cell types of >10% abundance. We used flowBin in conjunction with classifiers to distinguish normal from cancerous cells. We used flowBin together with flowType and RchyOptimyx to profile the immunophenotypic landscape of NPM1-mutated acute myeloid leukemia, and present a series of novel cell types associated with that mutation.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

Aggregate analysis of regulatory authority assessors' comments to improve the quality of periodic safety update reports.

PURPOSE: Marketing authorization holders (MAHs) are expected to provide high-quality periodic safety update reports (PSURs) on their pharmaceutical products to health authorities (HAs). We present a novel instrument aiming at improving quality of PSURs based on standardized analysis of PSUR assessment reports (ARs) received from the European Union HAs across products and therapeutic areas. METHODS: All HA comments were classified into one of three categories: "Request for regulatory actions," "Request for medical and scientific information," or "Data deficiencies." The comments were graded according to their impact on patients' safety, the drug's benefit-risk profile, and the MAH's pharmacovigilance system. RESULTS: A total of 476 comments were identified through the analysis of 63 PSUR HA ARs received in 2013 and 2014; 47 (10%) were classified as "Requests for regulatory actions," 309 (65%) as "Requests for medical and scientific information," and 118 (25%) comments were related to "Data deficiencies." The most frequent comments were requests for labeling changes (35 HA comments in 19 ARs). The aggregate analysis revealed commonly raised issues and prompted changes of the MAH's procedures related to the preparation of PSURs. CONCLUSION: The authors believe that this novel instrument based on the evaluation of PSUR HA ARs serves as a valuable mechanism to enhance the quality of PSURs and decisions about optimization of the use of the products and, therefore, contributes to improve further the MAH's pharmacovigilance system and patient safety.

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo.

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Height and heart disease.

Height has a relationship with a number of medical conditions, including heart disease. Atrial fibrillation has been observed to be more common in taller individuals. Marfan syndrome, with its high prevalence of mitral valve disease and abnormalities of the aorta, is associated with increased height. Mitral valve prolapse in patients without Marfan syndrome may be more common in taller people. Conversely, congestive heart failure, coronary artery disease, and possibly aortic valve calcification are less prevalent with increasing height. The relationship between height and health will be of increasing importance as the population grows taller.