Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.

Annual Meeting Content Analysis: Leveraging Annual Meetings to Promote Diversity, Equity, Inclusion, and Belonging in the Academy of Consultation-Liaison Psychiatry.

BACKGROUND: There is an increasing need to promote diversity, equity, and inclusion (DEI) in all aspects of academic medicine, including through continuing medical education. Although professional medical organizations' annual meetings play an instrumental role in continuing medical education for physicians, there are no studies describing DEI content in the annual meeting programming of professional medical organizations, including the Academy of Consultation-Liaison Psychiatry (ACLP), the primary professional organization for consultation-liaison psychiatrists. OBJECTIVE: To examine the ACLP annual meeting titles using Content Analysis. METHODS: We examined the publicly available ACLP annual meeting content titles on the ACLP website from 2010 to 2021. National DEI leaders from ACLP's DEI subcommittee iteratively generated keywords that covered a broad scope of DEI-related themes. Each annual meeting's content was independently coded by 2 members of the DEI subcommittee with discrepancies adjudicated by 2 additional members. Descriptive statistics were used to characterize the content of the annual meeting. RESULTS: Of the 2615 annual meeting titles from 2010 to 2021 that were analyzed, 2531 were not coded to have DEI themes. Three percent (n = 84) of titles were coded to have a DEI theme as follows: Culture/diversity (n = 20, 24%), bias/disparities (n = 17, 20%), race/racism (n = 17, 20%), social justice (n = 12, 14%), gender/sexism (n = 10, 12%), and LGBTQ+ (n = 8, 10%). The frequency of DEI titles each year ranged from 1% (2010, 2018) to 17% (2021) with an increase in DEI content in 2021 (n = 24, 17%). CONCLUSIONS: Although professional medical organizations like the ACLP are poised to leverage their continuing medical education platforms embedded in annual meeting programming to train consultation-liaison psychiatrists on DEI topics, our findings suggest more work is needed to develop and promote DEI-focused educational programming for their annual meetings.

A dual action coumarin-camptothecin polymer for light responsive drug release and photodynamic therapy.

A light-responsive polymer allowing the controlled release of camptothecin and the generation of reactive oxygen species (ROS) is reported. The polymer was prepared by controlled copolymerisation of water-soluble N,N-dimethyl acrylamide with a bromocoumarin methacrylate monomer. The lipophilic chemotherapy agent camptothecin was caged onto the coumarin unit via a photo-cleavable carbonate ester enabling light-triggered cargo release. The polymer showed good biocompatibility in the dark, and high cancer cell killing activity mediated both by the photo-release of camptothecin and ROS generation.

Development of hydrogel-based standards and phantoms for non-linear imaging at depth.

Significance: Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques. Aim: We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth. Approach: Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers. Results: These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and 1240 µm, respectively. Once fabricated, the phantoms were found to be stable for many months. Conclusions: The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.

A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to increase its water solubility and activation kinetics by addition of a long PEG chain (5 kDa) at the C-terminus. A BODIPY fluorophore allowed detection of Cathepsin D across a wide pH range, important as the protease is active both at the low pH found in lysosomes and also in higher pH phagolysosomes, and in the cytosol. The probe was successfully used to detect Cathepsin D activity in macrophages challenged by exposure to bacteria.

Riboflavin-Vancomycin Conjugate Enables Simultaneous Antibiotic Photo-Release and Photodynamic Killing against Resistant Gram-Positive Pathogens.

Decades of antibiotic misuse have led to alarming levels of antimicrobial resistance, and the development of alternative diagnostic and therapeutic strategies to delineate and treat infections is a global priority. In particular, the nosocomial, multidrug-resistant "ESKAPE" pathogens such as Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp (VRE) urgently require alternative treatments. Here, we developed light-activated molecules based on the conjugation of the FDA-approved photosensitizer riboflavin to the Gram-positive specific ligand vancomycin to enable targeted antimicrobial photodynamic therapy. The riboflavin-vancomycin conjugate proved to be a potent and versatile antibacterial agent, enabling the rapid, light-mediated, killing of MRSA and VRE with no significant off-target effects. The attachment of riboflavin on vancomycin also led to an increase in antibiotic activity against S. aureus and VRE. Simultaneously, we evidenced for the first time that the flavin subunit undergoes an efficient photoinduced bond cleavage reaction to release vancomycin, thereby acting as a photoremovable protecting group with potential applications in drug delivery.

Erratum to "Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease".

[This corrects the article DOI: 10.34133/2021/9834163.].

A niche-mimicking polymer hydrogel-based approach to identify molecular targets for tackling human pancreatic cancer stem cells.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most fatal human cancers, but effective therapies remain to be established. Cancer stem cells (CSCs) are highly resistant to anti-cancer drugs and a deeper understanding of their microenvironmental niche has been considered important to provide understanding and solutions to cancer eradication. However, as the CSC niche is composed of a wide variety of biological and physicochemical factors, the development of multidisciplinary tools that recapitulate their complex features is indispensable. Synthetic polymers have been studied as attractive biomaterials due to their tunable biofunctionalities, while hydrogelation technique further renders upon them a diversity of physical properties, making them an attractive tool for analysis of the CSC niche. METHODS: To develop innovative materials that recapitulate the CSC niche in pancreatic cancers, we performed polymer microarray analysis to identify niche-mimicking scaffolds that preferentially supported the growth of CSCs. The niche-mimicking activity of the identified polymers was further optimized by polyethylene glycol (PEG)-based hydrogelation. To reveal the biological mechanisms behind the activity of the optimized hydrogels towards CSCs, proteins binding onto the hydrogel were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and the potential therapeutic targets were validated by looking at gene expression and patients' outcome in the TCGA database. RESULTS: PA531, a heteropolymer composed of 2-methoxyethyl methacrylate (MEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) (5.5:4.5) that specifically supports the growth and maintenance of CSCs was identified by polymer microarray screening using the human PAAD cell line KLM1. The polymer PA531 was converted into five hydrogels (PA531-HG1 to HG5) and developed to give an optimized scaffold with the highest CSC niche-mimicking activities. From this polymer that recapitulated CSC binding and control, the proteins fetuin-B and angiotensinogen were identified as candidate target molecules with clinical significance due to the correlation between gene expression levels and prognosis in PAAD patients and the proteins associated with the niche-mimicking polymer. CONCLUSION: This study screened for biofunctional polymers suitable for recapitulation of the pancreatic CSC niche and one hydrogel with high niche-mimicking abilities was successfully fabricated. Two soluble factors with clinical significance were identified as potential candidates for biomarkers and therapeutic targets in pancreatic cancers. Such a biomaterial-based approach could be a new platform in drug discovery and therapy development against CSCs, via targeting of their niche.

Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.

The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells.

Size-Controlled Ammonium-Based Homopolymers as Broad-Spectrum Antibacterials.

Ammonium group containing polymers possess inherent antimicrobial properties, effectively eliminating or preventing infections caused by harmful microorganisms. Here, homopolymers based on monomers containing ammonium groups were synthesized via Reversible Addition Fragmentation Chain Transfer Polymerization (RAFT) and evaluated as potential antibacterial agents. The antimicrobial activity was evaluated against Gram-positive (M. luteus and B. subtilis) and Gram-negative bacteria (E. coli and S. typhimurium). Three polymers, poly(diallyl dimethyl ammonium chloride), poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), and poly(vinyl benzyl trimethylammonium chloride), were examined to explore the effect of molecular weight (10 kDa, 20 kDa, and 40 kDa) on their antimicrobial activity and toxicity to mammalian cells. The mechanisms of action of the polymers were investigated with dye-based assays, while Scanning Electron Microscopy (SEM) showed collapsed and fused bacterial morphologies due to the interactions between the polymers and components of the bacterial cell envelope, with some polymers proving to be bactericidal and others bacteriostatic, while being non-hemolytic. Among all the homopolymers, the most active, non-Gram-specific polymer was poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), with a molecular weight of 40 kDa, with minimum inhibitory concentrations between 16 and 64 µg/mL, showing a bactericidal mode of action mediated by disruption of the cytoplasmic membrane. This homopolymer could be useful in biomedical applications such as surface dressings and in areas such as eye infections.

A signal amplification for Trp isomers electrochemical recognition based on PEDOT:PSS and CS/PAA multilayers.

In this work, enhanced tryptophan (Trp) isomers recognition was successfully demonstrated on (CS/PAA)3.5@PEDOT:PSS/GCE, a multilayer chiral sensor with good stability and reproducibility. The (CS/PAA)n multilayers chiral interface was first fabricated via alternating self-assembly of chiral chitosan (CS) and achiral polyacrylic acid (PAA). Conductive PEDOT:PSS was then compounded with (CS/PAA)n multilayers to obtain the chiral sensor for the electrochemical recognition of Trp isomers. The structure of the sensor and its chirality properties for Trp isomers were characterized by fourier transform infrared spectroscopy (FT-IR)，scanning electron microscopy (SEM) and electrochemical methods. The SEM images showed uniform distribution of PEDOT:PSS in the multilayer films, which changed the internal structure of the (CS/PAA)3.5. Consequently, (CS/PAA)3.5@PEDOT:PSS multilayers rendered more chiral centers in addition to improved good conductivity, which significantly amplified the oxidation peak current ratio of D-Trp to L-Trp (ID/IL) up to 6.71 at 25 °C. In addition, a linear relationship was observed between the peak current and Trp enantiomer concentration in the range of 0.002-0.15 mM, and the detection limits of D-Trp and L-Trp were 0.33 and 0.67 µM, respectively. More importantly, the percentage of D-Trp in non-racemic Trp enantiomers mixture solutions were successfully determined on the chiral interface, showing its effectiveness and promising potential in practical applications.

pH-Activated Dissolvable Polymeric Coatings to Reduce Biofouling on Electrochemical Sensors.

Implantable electrochemical sensors that enable the real-time detection of significant biomarkers offer huge potential for the enhancement and personalisation of therapies; however, biofouling is a key challenge encountered by any implantable system. This is particularly an issue immediately after implantation, when the foreign body response and associated biofouling processes are at their most active in passivating a foreign object. Here, we present the development of a sensor protection and activation strategy against biofouling, based on coatings consisting of a pH-triggered, dissolvable polymer, that covered a functionalised electrode surface. We demonstrate that reproducible delayed sensor activation can be achieved, and that the length of this delay can be controlled by the optimisation of coating thickness, homogeneity and density through tuning of the coating method and temperature. Comparative evaluation of the polymer-coated and uncoated probe-modified electrodes in biological media revealed significant improvements in their anti-biofouling characteristics, demonstrating that this offers a promising approach to the design of enhanced sensing devices.

Novel class of photochromic molecules exhibiting photo-switching in the solid state.

Photo-switching compounds are widely used as super-resolution imaging agents, anti-counterfeiting dyes, and molecules that are able to control drug-receptor interactions. However, advancement of this field has been limited by the number of classes of molecules that exhibit this phenomenon, and thus there are growing activities to discover new photo-switching compounds that diversify and improve current applications and include the so-called donor-acceptor Stenhouse adducts. Herein, a new class of compounds, phenylindole alkene dimers, are presented as a novel class of photochromic molecules that exhibit photo-switching in the solid state. The synthesis of a small library of these compounds allowed the tuning of their optical properties. Surfaces coated with these photo-switches can be used as writable materials in a variety of applications.

The Color of Autonomy: Examining Racial Inequity in Coercive Institutional Practices.

Two articles recently published in this journal identified racial inequities in routine psychiatric practice. This Open Forum discusses the need for a paradigm shift in inequities research. The two articles reviewed here, one by Shea and colleagues on racial-ethnic inequities in inpatient psychiatric civil commitment and one by Garrett and colleagues on racial-ethnic disparities in psychiatric decisional capacity consultations, are examples of the new research gaze. Four topics are identified for enhancing understanding of racism and other forms of structural exclusion in psychiatric practice: medical authority and power imbalance between providers and patients, involuntary psychiatric commitment and requests for decisional capacity consultations as strategic research events, limited use of theory, and limitations of the literature on psychiatric inequities.

Approved and investigational fluorescent optical imaging agents for disease detection in surgery.

Fluorescent optical imaging is becoming an increasingly attractive imaging tool that physicians can utilise as it can detect previously 'unseen' changes in tissue at a cellular level that are consistent with disease. This is possible using a range of fluorescently labelled imaging agents that, once excited by specific wavelengths of light, can illuminate damaged and diseased tissues. For surgeons, such agents can permit dynamic, intraoperative imaging providing a real-time guide as they resect diseased tissue.

Solid-Phase Synthesis of s-Tetrazines.

An efficient synthesis of s-tetrazines by solid-phase methods is described. This synthesis route was compatible with different solid-phase resins and linkers and did not require metal catalysts or high temperatures. Monosubstituted tetrazines were routinely synthesized using thiol-promoted chemistry, using dichloromethane as a carbon source, while disubstituted unsymmetrical aryl or alkyl tetrazines were synthesized using readily available nitriles. This efficient approach enabled the synthesis of s-tetrazines in high yields (70-94%), eliminating the classical solution-phase problems of mixtures of symmetrical and unsymmetrical tetrazines, with only a single final purification step required, and paves the way to the rapid synthesis of s-tetrazines with various applications in bioorthogonal chemistry and beyond.

Development of biodegradable nanogels for lipase accelerated drug release of 5-aminolevulinic acid.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is an important approach for the treatment of some skin diseases and cancers. A major defect of this approach is that it is difficult for 5-ALA to accumulate around lesions in deeper regions of tissue, resulting in poor conversion to the active fluorophore and photodynamic efficiencies. Because of their targeting and controlled release abilities, nanogel carriers could solve this problem. In this paper, nanogels were prepared by using micro-emulsion polymerization with various biodegradable polyester crosslinkers (L-lactide and ε-caprolactone). The swelling and degradation properties and entrapment efficiency, drug loading and drug release ability of the nanogels were investigated. Nanogels co-cultured with skin cancer cells (A2058) allowed the efficiency of the PDT in vitro to be demonstrated. The results showed that the swelling rate of hydrogels reduced with increasing crosslinker levels, which caused a slow-down in the release of 5-ALA, but lipase accelerated degradation of nanogels increased 5-ALA concentrations in tumor cells and leading to higher PDT efficiency. It was proved by in vivo experiment indicating that the development of skin cancer tissues were efficiently inhibited by the 5-ALA loaded nanogels.

SERS microsensors for pH measurements in the lumen and ECM of stem cell derived human airway organoids.

Patient derived organoids have the potential to improve the physiological relevance of in vitro disease models. However, the 3D architecture of these self-assembled cellular structures makes probing their biochemistry more complex than in traditional 2D culture. We explore the application of surface enhanced Raman scattering microsensors (SERS-MS) to probe local pH gradients within patient derived airway organoid cultures. SERS-MS consist of solid polymer cores decorated with surface immobilised gold nanoparticles which are functionalised with pH sensitive reporter molecule 4-mercaptobenzoic acid (MBA). We demonstrate that by mixing SERS-MS into the extracellular matrix (ECM) of airway organoid cultures the probes can be engulfed by expanding organoids and report on local pH in the organoid lumen and ECM.

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.

Rapid fabrication and screening of tailored functional 3D biomaterials: Validation in bone tissue repair - Part II.

Regenerative medicine strategies place increasingly sophisticated demands on 3D biomaterials to promote tissue formation at sites where tissue would otherwise not form. Ideally, the discovery/fabrication of the 3D scaffolds needs to be high-throughput and uniform to ensure quick and in-depth analysis in order to pinpoint appropriate chemical and mechanical properties of a biomaterial. Herein we present a versatile technique to screen new potential biocompatible acrylate-based 3D scaffolds with the ultimate aim of application in tissue repair. As part of this process, we identified an acrylate-based 3D porous scaffold that promoted cell proliferation followed by accelerated tissue formation, pre-requisites for tissue repair. Scaffolds were fabricated by a facile freeze-casting and an in-situ photo-polymerization route, embracing a high-throughput synthesis, screening and characterization protocol. The current studies demonstrate the dependence of cellular growth and vascularization on the porosity and intrinsic chemical nature of the scaffolds, with tuneable 3D scaffolds generated with large, interconnected pores suitable for cellular growth applied to skeletal reparation. Our studies showed increased cell proliferation, collagen and ALP expression, while chorioallantoic membrane assays indicated biocompatibility and demonstrated the angiogenic nature of the scaffolds. VEGRF2 expression in vivo observed throughout the 3D scaffolds in the absence of growth factor supplementation demonstrates a potential for angiogenesis. This novel platform provides an innovative approach to 3D scanning of synthetic biomaterials for tissue regeneration.