An induced thymic epithelial cell-based high throughput screen for thymus extracellular matrix mimetics.

Thymic epithelial cells (TECs) are key effectors of the thymic stroma and are critically required for T-cell development. TECs comprise a diverse set of related but functionally distinct cell types that are scarce and difficult to isolate and handle. This has precluded TEC-based screening assays. We previously described induced thymic epithelial cells (iTECs), an artificial cell type produced in vitro by direct reprogramming, raising the possibility that iTECs might provide the basis for functional screens related to TEC biology. Here, we present an iTEC-based three-stage medium/high-throughput in vitro assay for synthetic polymer mimics of thymic extracellular matrix (ECM). Using this assay, we identified, from a complex library, four polymers that bind iTEC as well as or better than gelatin but do not bind mesenchymal cells. We show that these four polymers also bind and maintain native mouse fetal TECs and native human fetal TECs. Finally, we show that the selected polymers do not interfere with iTEC function or T-cell development. Collectively, our data establish that iTECs can be used to screen for TEC-relevant compounds in at least some medium/high-throughput assays and identify synthetic polymer ECM mimics that can replace gelatin or ECM components in TEC culture protocols.

Alterations in cancer stem-cell marker CD44 expression predict oncologic outcome in soft-tissue sarcomas.

BACKGROUND: Cancer stem cells (CSCs) have been shown to resist chemotherapy and promote metastasis after cytotoxic therapies. We sought to determine if the expression of CSC markers (aldehyde dehydrogenase [ALDH], CD44, and epidermal growth factor receptor [EGFR]) predicted outcomes in soft-tissue sarcoma (STS) patients. METHODS: We queried an institutional database of 23 STS patients and evaluated immunohistochemical expression of CSC markers ALDH, CD44, and EGFR. The Cancer Genome Atlas (TCGA) was also queried for STS clinical and genomic data. Disease-specific (DSS) and overall survival (OS) were assessed by univariate and Kaplan-Meier analysis. RESULTS: Of the 23 institutional patients, the majority was female, had high-grade tumors and had extremity tumors. With a median follow-up of 27 months, nine patients (39%) experienced distant recurrence, and four (17%) died of disease. Mean H-scores at diagnosis (±standard error of the mean) for CD44, ALDH1, and EGFR were 169 ± 27, 77 ± 15, and 144 ± 23, respectively. On univariate analysis, there was a trend for increased CD44 score to predict both worse DSS and OS (hazard ratio = 1.01, 95% confidence interval 1-1.02, P = 0.056), whereas ALDH and EGFR scores did not. Analysis of 74 TCGA STS cases with complete clinical and genomic data revealed that CD44 copy number alterations predicted worse DSS (9.89 months versus 72.5 months, P = 0.007) and a trend for worse OS (14.03 months versus 38.6 months, P = 0.12), whereas ALDH1 and EGFR copy number alteration did not. Multivariate analysis of the combined data sets was consistent with worse DSS among patients with higher CD44 expression. CONCLUSIONS: Institutional and national TCGA data show the association of elevated baseline CD44 expression with worse STS outcomes. Further study of CD44 as a possible novel STS biomarker appears indicated.

Turning a blind eye: HTLV-1-associated uveitis in Indigenous adults from Central Australia.

PURPOSE: We describe the first two cases of HTLV-1 associated uveitis to be associated with HTLV-1c subtype infection. METHODS: Case report. RESULTS: Uveitis was demonstrated in two Indigenous Australian men, both of whom had high HTLV-1c proviral loads in peripheral blood. Visual outcomes were poor in each case. CONCLUSION: Clinicians should be aware of HTLV-1c infection as a cause of uveitis in Australia, and HTLV-1 serology should be included in the basic uveitis work-up in HTLV-1-endemic areas.

Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRASG12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

Effects of Brief Mental Skills Training on Emergency Medicine Residents' Stress Response During a Simulated Resuscitation: A Prospective Randomized Trial.

BACKGROUND: Acute stress impairs physician decision-making and clinical performance in resuscitations. Mental skills training, a component of the multistep, cognitive-behavioral technique of stress inoculation, modulates stress response in high-performance fields. OBJECTIVE: We assessed the effects of mental skills training on emergency medicine (EM) residents' stress response in simulated resuscitations as well as residents' perceptions of this intervention. METHODS: In this prospective, educational intervention trial, postgraduate year-2 EM residents in seven Chicago-area programs were randomly assigned to receive either stress inoculation training or not. One month prior to assessment, the intervention group received didactic training on the "Breathe, Talk, See, Focus" mental performance tool. A standardized, case-based simulation was used for assessment. We measured subjective stress response using the six-item short form of the Spielberger State-Trait Anxiety Inventory (STAI-6). Objective stress response was measured through heart rate (HR) and heart rate variability (HRV) monitoring. We measured subjects' perceptions of the training via survey. RESULTS: Of 92 eligible residents, 61 participated (25 intervention; 36 control). There were no significant differences in mean pre-/post-case STAI-6 scores (-1.7 intervention, 0.4 control; p = 0.38) or mean HRV (-3.8 milliseconds [ms] intervention, -3.8 ms control; p = 0.58). Post-assessment surveys indicated that residents found this training relevant and important. CONCLUSION: There was no difference in subjective or objective stress measures of EM resident stress response after a didactic, mental performance training session, although residents did value the training. More extensive or longitudinal stress inoculation curricula may provide benefit.

The Discovery of Two Novel Classes of 5,5-Bicyclic Nucleoside-Derived PRMT5 Inhibitors for the Treatment of Cancer.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Stereoselective formation of a functionalized dipeptide isostere by zinc carbenoid-mediated chain extension.

The application of a zinc carbenoid-mediated chain-extension reaction to a functionalized peptide isostere is reported. The cleavage site of human CVM protease was utilized as a target for testing the synthetic methodology. The utility of this chain-extension reaction is demonstrated in the preparation of an amino acid-derived alpha-unsubstituted gamma-keto ester, which is incorporated into a framework that mimics a tetrapeptide. The identification of a suitable protecting group strategy facilitated the application of a tandem reaction for the incorporation of an alpha-side chain, and the use of an oxazolidinone auxiliary provided excellent diastereocontrol in a tandem chain-extension-aldol reaction. Stereoselectivity of the tandem chain-extension-aldol reaction was determined through application of a CAN-mediated oxidative cleavage reaction.

Using GPS Technology to Understand Spatial and Temporal Activity of Kangaroos in a Peri-Urban Environment.

The increasing kangaroo occurrence in expanding peri-urban areas can be problematic when kangaroos become aggressive towards people and present a collision risk to motor vehicles. An improved understanding on kangaroo spatial and temporal activity patterns in the peri-urban environment is essential to manage kangaroo⁻human conflict. In this study, we used GPS telemetry to determine activity patterns of male Eastern Grey Kangaroos (Macropus giganteus) in a peri-urban community on the north-coast of New South Wales, Australia. Two types of GPS devices were employed; collars and cheaper alternative glue-on units. Kangaroos moved on average 2.39 km a day, with an average movement rate of 1.89 m/min, which was greatest at dawn. The GPS glue-on devices had short deployment lengths of one to 12 days. Despite limitations in attachment time, the glue-on devices were viable in obtaining daily spatial and temporal activity data. Our results aid towards alleviating conflict with kangaroos by providing new insights into kangaroo movements and activity within a peri-urban environment and introduces a potential cheap GPS alternative for obtaining this data relative to more expensive collars.

Chemical Transformations in Proto-Cytoplasmic Media. Phosphorus Coupling in the Silica Hydrogel Phase.

It has been proposed that prebiotic chemical studies on the emergence of primitive life would be most relevant when performed in a hydrogel, rather than an aqueous, environment. In this paper we describe the ambient temperature coupling of phosphorus oxyacids [Pi] mediated by Fe(II) under aerobic conditions within a silica hydrogel (SHG) environment. We have chosen to examine SHGs as they have considerable geological precedence as key phases in silicification en route to rock formation. Following a description of the preparation and characterization studies on our SHG formulations, coupling experiments between Pi species are described across multiple permutations of (i) Pi compound; (ii) gel formulation; (iii) metal salt additive; and (iv) pH-modifying agent. The results suggest that successful Pi coupling, indicated by observation of pyrophosphate [PPi(V)] via 31P-NMR spectroscopy, takes place when the following components are present: (i) a mixture of mixture of Pi(III) and Pi(V) or pure PPi(III-V); (ii) Fe(II); (iii) acetic or formic acid (not hydrochloric acid); (iv) aerobic conditions or the presence of H2O2 as an oxidant; and (v) the presence of a gel system. On the basis of these, and aqueous control reactions, we suggest mechanistic possibilities.

Interactions between circulating nanoengineered polymer particles and extracellular matrix components in vitro.

The extracellular matrix (ECM) that surrounds cells in vivo represents a biological barrier for nanomaterials in biomedicine. Herein, we present a system for investigating the interactions between circulating polymer particles and ECM components in vitro using a commercially available flow-based device. We use this system to show how material-dependent interactions of two different particle types-one assembled using poly(ethylene glycol) (PEG) and one prepared using poly(methacrylic acid) (PMA)-affect their interactions with basement membrane extracts during in vitro circulation, with PEG particles remaining in circulation longer than PMA particles. Further, by comparing macroporous hyaluronic acid gel constructs (typically used for tissue engineering) with basement membrane extracts, we show that scaffold-effects (porosity and surface chemistry) impact on circulation time in vitro. The presented system is simple and modular, and can be used to rapidly screen fundamental interactions of engineered particles with biologically relevant microenvironments under flow-conditions.

Identification of conserved regulatory RNA structures in prokaryotic metabolic pathway genes.

A combination of algorithms to search RNA sequence for the potential for secondary structure formation, and search large numbers of sequences for structural similarity, were used to search the 5'UTRs of annotated genes in the Escherichia coli genome for regulatory RNA structures. Using this approach, similar RNA structures that regulate genes in the thiamin metabolic pathway were identified. In addition, several putative regulatory structures were discovered upstream of genes involved in other metabolic pathways including glycerol metabolism and ethanol fermentation. The results demonstrate that this computational approach is a powerful tool for discovery of important RNA structures within prokaryotic organisms.

Formation and characterisation of a modifiable soft macro-porous hyaluronic acid cryogel platform.

A facile method for the synthesis of cell supportive, highly macro-porous hyaluronic acid (HA) hydrogels via cryogelation is presented. Unmodified HA was chemically cross-linked via EDC/NHS zero-length cross-linking at sub-zero temperatures to yield cryogels with high porosity and high pore interconnectivity. The physical properties of the HA cryogels including porosity, average pore size, elasticity and swelling properties were characterised as a function of cryogelation conditions and composition of the precursor solution. The HA cryogels swell extensively in water, with the average porosities observed being ~90% under all conditions explored. The morphology of the cryogels can be controlled, allowing scaffolds with an average pore size ranging from 18 ± 2 to 87 ± 5 µm to be formed. By varying the cross-linking degree and HA concentration, a wide range of bulk elastic properties can be achieved, ranging from ~1 kPa to above 10 kPa. Preliminary cell culture experiments, with NIH 3T3 and HEK 293 cell lines, performed on biochemically modified and unmodified gels show the cryogels support cell proliferation and cell interactions, illustrating the biomedical potential of the platform.

Cryogels for biomedical applications.

The use of hydrogels as support materials for the growth and proliferation of mammalian cells has been well documented as they closely mimic the gel-like properties - and in some cases also the chemical properties - of the extracellular matrix (ECM), which naturally surrounds the cells of any biological tissue. Macro-porous hydrogels set below the freezing point of the solvent, so-called 'cryogels', have recently gained significant interest in the fields of tissue engineering and in vitro cell culture, thanks to their inherent interconnected macro-porous structure and ease of formation in comparison to other macro-pore forming techniques. This review highlights recent advances in cryogelation techniques and starting materials that can be utilised to synthesise biocompatible and biologically relevant cryogels as well as discussing physicochemical characterisation techniques for these materials. Lastly, emerging trends in the application of cryogels, particularly as three-dimensional ECM mimicking scaffolds for cell culture and tissue engineering, are discussed.