ABC and ABAB Block Copolymers by Electrochemically Controlled Ring-Opening Polymerization.

An electrochemically controlled synthesis of multiblock copolymers by alternating the redox states of (salfan)Zr(OtBu)2 (salfan = 1,1'-di(2-tert-butyl-6-N-methylmethylenephenoxy)ferrocene) is reported. Aided by electrochemistry with a glassy carbon working electrode, an in situ potential switch alters the catalyst's oxidation state and its subsequent monomer (l-lactide, ß-butyrolactone, or cyclohexene oxide) selectivity in one pot. Various multiblock copolymers were prepared, including an ABAB tetrablock copolymer, poly(cyclohexene oxide-b-lactide-b-cyclohexene oxide-b-lactide), and an ABC triblock copolymer, poly(hydroxybutyrate-b-cyclohexene oxide-b-lactide). The polymers produced using this technique are similar to those produced via a chemical redox reagent method, displaying moderately narrow dispersities (1.1-1.5) and molecular weights ranging from 7 to 26 kDa.

Adapting ACMG/AMP sequence variant classification guidelines for single-gene copy number variants.

PURPOSE: The ability of a single technology, next-generation sequencing, to provide both sequence and copy number variant (CNV) results has driven the merger of clinical cytogenetics and molecular genetics. Consequently, the distinction between the definition of a sequence variant and a CNV is blurry. As the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for interpretation of sequence variants address CNV classification only sparingly, this study focused on adapting ACMG/AMP criteria for single-gene CNV interpretation. METHODS: CNV-specific modifications of the 2015 ACMG/AMP criteria were developed and their utility was independently tested by three diagnostic laboratories. Each laboratory team interpreted the same 12 single-gene CNVs using three systems: (1) without ACMG/AMP guidance, (2) with ACMG/AMP criteria, and (3) with new modifications. A replication study of 12 different CNVs validated the modified criteria. RESULTS: The adapted criteria system presented here showed improved concordance and usability for single-gene CNVs compared with using the ACMG/AMP interpretation guidelines focused on sequence variants. CONCLUSION: These single-gene CNV criteria modifications could be used as a supplement to the ACMG/AMP guidelines for sequence variants, allowing for a streamlined workflow and a step toward a uniform classification system for both sequence and copy number alterations.

Correction: Adapting ACMG/AMP sequence variant classification guidelines for single-gene copy-number variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High Occurrence of Psychiatric Disorders and Suicidal Behavior Across Dementia Subtypes.

OBJECTIVE: To compare occurrence of clinically diagnosed psychiatric disorders and suicidal behavior (mental health disorders) across dementia subtypes in the largest healthcare system in the United States. METHODS: We aggregated two national databases (Department of Veterans Affairs [VA] National Patient Care Database, National Suicide Prevention Applications Network [SPAN]) and estimated 2-year prevalence of mental health disorders across five dementia subtypes during fiscal years 2012-2013. Using VA healthcare systems throughout the United States, the sample included 56,296 older patients (≥50 years) with Alzheimer's disease (AD; n = 30,578), vascular dementia (VD; n = 17,924), frontotemporal dementia (FTD; n = 1,181), Lewy body dementia (LBD; n = 3,194), and mixed dementia (MD; n = 3,419). Mental health disorders were determined by International Classification of Diseases, Ninth Revision, Clinical Modification codes and the National SPAN. RESULTS: Roughly 25% of patients had at least one mental health disorder, with 2-year prevalence reaching 30%-45% in FTD, VD, LBD, and MD. Compared with other subtypes, patients with FTD had the highest prevalence of mood (19%), anxiety (20%), and substance use (19%) disorders, as well as suicidal behavior (4%), with nearly 0.5% with a suicidal plan/attempt. Those with VD also showed a high prevalence of these disorders (14%-17%). Although patients with LBD and MD had a slightly lower prevalence of mood and anxiety disorders (12%-15%), they had a much lower prevalence of substance use disorders (9%) and suicidal behavior (2%). Patients with AD had the lowest 2-year prevalence of all mental health disorders (<7%). CONCLUSION: Occurrence of mental health disorders is high and differs across dementia subtypes, highlighting the importance of reducing the burden of mental health disorders in dementia subtypes.

Predictors of Mental Health Services Use Across the Life Course among Racially-Ethnically Diverse Adults.

OBJECTIVE: Little is known about key factors associated with use of mental health services across the life course. This study determined key socioeconomic, social support, psychiatric, and medical predictors of services use in younger, middle, and older age. DESIGN, SETTING, PARTICIPANTS, MEASUREMENTS: The sample included 3,708 adults with DSM-IV-based mood, anxiety, and substance use disorders in the Collaborative Psychiatric Epidemiology Surveys. Key predictors of mental health services use for each age group were systematically determined by multivariable models, and exploratory analyses examining potential effect modification by race-ethnicity and sex were assessed by interaction terms. Statistical analyses included complex design-corrected and weighted logistic regression analyses that provide results generalizable to the United States. RESULTS: Psychiatric and medical issues such as prior suicidal behavior, comorbid psychiatric disorders, and perceived cognitive impairment increased odds of mental health services use in younger, middle, and older age. Chronic medical conditions also influenced services use in younger and older age, with their impact on use across age potentially modified by racial-ethnic disparities (p interaction = 0.01). Moreover, socioeconomic factors like marital status influenced use in middle and older age, where being divorced, separated, widowed, or never married encouraged use. The effect of marital status on use across age was also potentially modified by racial-ethnic disparities (p interaction = 0.02). CONCLUSIONS: Key socioeconomic, social support, psychiatric, and medical predictors uniquely influence use of mental health services across the life course. These findings will help inform efforts to encourage greater services use by adults across the life course in need of care.

Macrocyclic inhibitors of Factor XIa: Discovery of alkyl-substituted macrocyclic amide linkers with improved potency.

Optimization of macrocyclic inhibitors of FXIa is described which focused on modifications to both the macrocyclic linker and the P1 group. Increases in potency were discovered through interactions with a key hydrophobic region near the S1 prime pocket by substitution of the macrocyclic linker with small alkyl groups. Both the position of substitution and the absolute stereochemistry of the alkyl groups on the macrocyclic linker which led to improved potency varied depending on the ring size of the macrocycle. Replacement of the chlorophenyltetrazole cinnamide P1 in these optimized macrocycles reduced the polar surface area and improved the oral bioavailability for the series, albeit at the cost of a decrease in potency.

Partitioning of lysolipids, fatty acids and their mixtures in aqueous lipid bilayers: solute concentration/composition effects.

Distributions of lysopalmitoylphosphatidylcholine (LPPC), palmitic acid (PA) and their 1:1 mixtures between water and dipalmitoylphosphatidylcholine (DPPC) bilayer were determined using a fluorescence probe that selectively detects only the solutes in water. Water solute concentrations were obtained at each of several lipid concentrations. Dynamic Light Scattering experiments confirmed that the lipid/solute aggregates were vesicles in the concentration range investigated. Lipid concentration dependence of the solute component in water was fit to a thermodynamic model of solute distribution between two coexisting solvents. Water/bilayer partition coefficient and the free energy of transfer, for each of these solutes were determined from the fit. Main findings are: (1) Water/bilayer partition coefficient of solute is greater for 2 to 10% solute mole fraction than for 0 to 2%, signaling solute induced bilayer perturbation that increases bilayer solubility, beginning at 2% solute mole fraction. (2) Partition coefficients are in the order LPPC

Clinical relevance and therapeutic predictive ability of hypoxia biomarkers in head and neck cancer tumour models.

Tumour hypoxia promotes poor patient outcomes, with particularly strong evidence for head and neck squamous cell carcinoma (HNSCC). To effectively target hypoxia, therapies require selection biomarkers and preclinical models that can accurately model tumour hypoxia. We established 20 patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of HNSCC that we characterised for their fidelity to represent clinical HNSCC in gene expression, hypoxia status and proliferation and that were evaluated for their sensitivity to hypoxia-activated prodrugs (HAPs). PDX models showed greater fidelity in gene expression to clinical HNSCC than cell lines, as did CDX models relative to their paired cell lines. PDX models were significantly more hypoxic than CDX models, as assessed by hypoxia gene signatures and pimonidazole immunohistochemistry, and showed similar hypoxia gene expression to clinical HNSCC tumours. Hypoxia or proliferation status alone could not determine HAP sensitivity across our 20 HNSCC and two non-HNSCC tumour models by either tumour growth inhibition or killing of hypoxia cells in an ex vivo clonogenic assay. In summary, our tumour models provide clinically relevant HNSCC models that are suitable for evaluating hypoxia-targeting therapies; however, additional biomarkers to hypoxia are required to accurately predict drug sensitivity.

Topical application of aminopeptidase N-neutralizing antibody accelerates wound closure.

Upon release from keratinocytes, 14-3-3 sigma (also known as stratifin) acts on the dermal fibroblast and modulates its production of extracellular matrix proteins. Subsequent to the recent identification as a receptor responsible for stratifin-mediated matrix turnover in dermal fibroblasts, aminopeptidase N has been implicated in the regulation of epidermal-dermal communication and expression of key matrix proteases and adhesion molecules. In light of the growing importance of aminopeptidase N in modulation of the fibroblast phenotype, the present study evaluates the potential of targeting the ectoenzyme in cutaneous repair, and demonstrates that neutralization of aminopeptidase N led to acceleration of wound closure. This was attributed to at least in part an increase of collagen deposition and fibroblast contractility in the granulation tissue. These findings confirmed the important role of aminopeptidase N in post-injury tissue remodeling and wound contraction.

Microarray-based identification of aminopeptidase N target genes in keratinocyte conditioned medium-stimulated dermal fibroblasts.

Of the many processes that affect the outcome of wound repair, epidermal-dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell-surface receptor for stratifin and is responsible for the stratifin-mediated matrix metalloproteinase-1 (MMP-1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte-derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin-C, MMP-1, MMP-3, and MMP-12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor-mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin-C expression and enhances the contraction of fibroblast-populated collagen gel, suggesting an activity-dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin-C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte-mediated ECM remodeling and fibroblast contractile activity.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts.

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.

Inflammatory effects of ex vivo human Th17 cells are suppressed by regulatory T cells.

Th17 cells are proinflammatory cells associated with many immune-mediated diseases. Major factors limiting the study of human Th17 cells are the lack of an accepted method for their in vitro differentiation or for isolation of a homogenous population of Th17 cells that do not cosecrete IFN-gamma. To overcome these hurdles, we established a novel method to isolate in vivo differentiated Th17 cells from peripheral blood by sorting CD161(+)CCR4(+)CCR6(+)CXCR3(-)CD4(+) T cells. The resulting cells produce high levels of IL-17 but not IFN-gamma, express high levels of retinoic acid-related orphan receptor variant 2, and maintain this phenotype upon expansion. Ex vivo Th17 cells exhibit a low cytotoxic potential and are hyporesponsive to polyclonal anti-CD3/anti-CD28 stimulation. Importantly, ex vivo Th17 cells were susceptible to suppression by both naive and memory regulatory T cells (Tregs), which inhibited production of IL-17, IL-22, and CXCL8. Moreover, Tregs suppressed the antifibrotic effects of Th17 cells in a wound-healing model. These findings provide new tools for the study of normal and pathological functions of bona fide Th17 cells in humans. They also provide new insight into the cross-talk between Th17 cells and immune and nonimmune cells, and they establish the paradigm that adoptive Treg-based therapies may effectively limit Th17-mediated inflammation.

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin.

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.

Dermal fibroblasts influence the expression profile of 14-3-3 proteins in human keratinocytes.

We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (ß, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.

Highly efficient stable expression of indoleamine 2,3 dioxygenase gene in primary fibroblasts.

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

Inhibitory effect of anti-aminopeptidase N/CD13 antibodies on fibroblast migration.

Aminopeptidase N (APN)/CD13 is a widely expressed transmembrane ectoenzyme and has been implicated in a myriad of physiological processes that are specific to cell type and tissue origin, including cancer cell metastasis, angiogenesis, cholesterol uptake, apoptosis, and cell migration. Skin cells, in particular fibroblasts have a relatively high level of APN/CD13 expression. The migratory capacity of skin cells is critical for the outcome of wound repair, as successful wound healing requires timely re-epithelialization which involves reformation of epithelium over wound surface by migrating keratinocytes. While failure of keratinocytes to undergo proper migration leads to chronic non-healing wounds, the presence of excess fibroblasts may contribute to formation of hypertrophic scars and keloids. The aim of this study was to investigate the role of APN/CD13 in skin cell migration and explore its potential as a therapeutic target in wound healing. Our results show an elevated expression of APN/CD13 in fibroblasts on the edge of the wound compared to unwounded cells. The presence of anti-APN/CD13 antibodies WM15, 3D8, and H300 reduces the migratory activity of human dermal fibroblasts in a dose-dependent manner by 42, 21, and 28%, respectively. However, the antibodies have no effect on keratinocyte migration. Further, none of the anti-APN/CD13 antibodies used in this study has any antiproliferative and cytotoxic effect on primary human keratinocytes or fibroblasts when used at 10 µg/ml in vitro. The differential inhibition on the migratory capacity of fibroblasts and keratinocytes presents an opportunity for anti-APN/CD13 antibodies to be used as a therapeutic agent for high fibroblast cellularity seen in fibroproliferative disorders.

Patient-Derived Xenograft and Organoid Models for Precision Medicine Targeting of the Tumour Microenvironment in Head and Neck Cancer.

Patient survival from head and neck squamous cell carcinoma (HNSCC), the seventh most common cause of cancer, has not markedly improved in recent years despite the approval of targeted therapies and immunotherapy agents. Precision medicine approaches that seek to individualise therapy through the use of predictive biomarkers and stratification strategies offer opportunities to improve therapeutic success in HNSCC. To enable precision medicine of HNSCC, an understanding of the microenvironment that influences tumour growth and response to therapy is required alongside research tools that recapitulate the features of human tumours. In this review, we highlight the importance of the tumour microenvironment in HNSCC, with a focus on tumour hypoxia, and discuss the fidelity of patient-derived xenograft and organoids for modelling human HNSCC and response to therapy. We describe the benefits of patient-derived models over alternative preclinical models and their limitations in clinical relevance and how these impact their utility in precision medicine in HNSCC for the discovery of new therapeutic agents, as well as predictive biomarkers to identify patients' most likely to respond to therapy.

Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species.

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

Potent, Orally Bioavailable, and Efficacious Macrocyclic Inhibitors of Factor XIa. Discovery of Pyridine-Based Macrocycles Possessing Phenylazole Carboxamide P1 Groups.

Factor XIa (FXIa) inhibitors are promising novel anticoagulants, which show excellent efficacy in preclinical thrombosis models with minimal effects on hemostasis. The discovery of potent and selective FXIa inhibitors which are also orally bioavailable has been a challenge. Here, we describe optimization of the imidazole-based macrocyclic series and our initial progress toward meeting this challenge. A two-pronged strategy, which focused on replacement of the imidazole scaffold and the design of new P1 groups, led to the discovery of potent, orally bioavailable pyridine-based macrocyclic FXIa inhibitors. Moreover, pyridine-based macrocycle 19, possessing the phenylimidazole carboxamide P1, exhibited excellent selectivity against relevant blood coagulation enzymes and displayed antithrombotic efficacy in a rabbit thrombosis model.