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.

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.

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.

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.

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.

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.

Impact of Tumour Hypoxia on Evofosfamide Sensitivity in Head and Neck Squamous Cell Carcinoma Patient-Derived Xenograft Models.

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7-7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes-MKI67, POR, and SLFN11-did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

An improved method for the heterologous production of soluble human ribosomal proteins in Escherichia coli.

Human ribosomal proteins play important structural and functional roles in the ribosome and in protein synthesis. An efficient method to recombinantly produce and purify these proteins would enable their full characterisation. However, the production of human ribosomal proteins can be challenging. The only published method about the recombinant production of human ribosomal proteins involved the recovery of proteins from inclusion bodies, a process that is tedious and may lead to significant loss of yield. Herein, we explored the use of different Escherichia coli competent cells and fusion protein tags for the recombinant production of human ribosomal proteins. We found that, by using thioredoxin as a fusion protein, soluble ribosomal protein could be obtained directly from cell lysates, thus leading to an improved method to recombinantly produce these proteins.

Computational mapping of redox-switchable metal complexes based on ferrocene derivatives.

DFT calculations were used to capture the properties of redox-switchable metal complexes relevant to the ring-opening polymerisation of cyclic esters by varying the metals, donors, linkers, and substituents in both accessible ferrocene oxidation states. A map of this chemical space highlights that modifying the ligand architecture and the metal has a larger impact on structural changes than changing the oxidation state of the ferrocene backbone.

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.

Mental Health Service Use Across the Life Course Among Adults With Psychiatric Disorders and Prior Suicidal Behavior.

OBJECTIVE: Little is known about mental health service use by adults with prior suicidal behavior and current mood or anxiety disorders. This study determined nationally representative prevalence estimates of current mental health service use by these adults, examining racial-ethnic, age, and gender differences. METHODS: Service use across the life course was examined with Collaborative Psychiatric Epidemiology Survey data from 1,139 adults with a history of suicidal behavior and current mood or anxiety disorders. RESULTS: Overall service use was 47.3%. Across the life course, African Americans showed increasing service use that paralleled use by non-Hispanic whites, Hispanics, and others, whereas use by these three groups decreased in the latter half of the life course (p interaction=.01). CONCLUSIONS: Adults with prior suicidal behavior and current mood or anxiety disorders have low mental health service use. Findings of racial-ethnic disparities in use can help identify those in need of care.

Integration of EEG lead placement templates into traditional technologist-based staffing models reduces costs in continuous video-EEG monitoring service.

PURPOSE: The purpose of this study was to determine the relative cost reductions within different staffing models for continuous video-electroencephalography (cvEEG) service by introducing a template system for 10/20 lead application. METHODS: We compared six staffing models using decision tree modeling based on historical service line utilization data from the cvEEG service at our center. Templates were integrated into technologist-based service lines in six different ways. The six models studied were templates for all studies, templates for intensive care unit (ICU) studies, templates for on-call studies, templates for studies of ≤ 24-hour duration, technologists for on-call studies, and technologists for all studies. RESULTS: Cost was linearly related to the study volume for all models with the "templates for all" model incurring the lowest cost. The "technologists for all" model carried the greatest cost. Direct cost comparison shows that any introduction of templates results in cost savings, with the templates being used for patients located in the ICU being the second most cost efficient and the most practical of the combined models to implement. Cost difference between the highest and lowest cost models under the base case produced an annual estimated savings of $267,574. Implementation of the ICU template model at our institution under base case conditions would result in a $205,230 savings over our current "technologist for all" model. CONCLUSIONS: Any implementation of templates into a technologist-based cvEEG service line results in cost savings, with the most significant annual savings coming from using the templates for all studies, but the most practical implementation approach with the second highest cost reduction being the template used in the ICU. The lowered costs determined in this work suggest that a template-based cvEEG service could be supported at smaller centers with significantly reduced costs and could allow for broader use of cvEEG patient monitoring.

Discovery of 4-aryl-7-hydroxyindoline-based P2Y1 antagonists as novel antiplatelet agents.

Adenosine diphosphate (ADP)-mediated platelet aggregation is signaled through two distinct G protein-coupled receptors (GPCR) on the platelet surface: P2Y12 and P2Y1. Blocking P2Y12 receptor is a clinically well-validated strategy for antithrombotic therapy. P2Y1 antagonists have been shown to have the potential to provide equivalent antithrombotic efficacy as P2Y12 inhibitors with reduced bleeding in preclinical animal models. We have previously reported the discovery of a potent and orally bioavailable P2Y1 antagonist, 1. This paper describes further optimization of 1 by introducing 4-aryl groups at the hydroxylindoline in two series. In the neutral series, 10q was identified with excellent potency and desirable pharmacokinetic (PK) profile. It also demonstrated similar antithrombotic efficacy with less bleeding compared with the known P2Y12 antagonist prasugrel in rabbit efficacy/bleeding models. In the basic series, 20c (BMS-884775) was discovered with an improved PK and liability profile over 1. These results support P2Y1 antagonism as a promising new antiplatelet target.

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

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.

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.