Ca 2+ -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

Bidirectional Neuronal Actuation by Uncaging with Violet and Green Light.

We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402 nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel. When partnered with coumarin-102 caged glutamate, we could use whole cell violet and green irradiation to fire and block neuronal action potentials with complete orthogonality. Localized irradiation of different dendritic segments, each connected to a neuronal cell body, in concert with 3-dimenional Ca2+ imaging, revealed that such inputs could function independently. Chemical signaling in living cells always involves a complex balance of multiple pathways, use of (thio)-coumarin-102 caged compounds will enable arbitrarily timed flashes of green and violet light to interrogate two independent pathways simultaneously.

A spatial human thymus cell atlas mapped to a continuous tissue axis.

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassall's Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

Discovery and pharmacophoric characterization of chemokine network inhibitors using phage-display, saturation mutagenesis and computational modelling.

CC and CXC-chemokines are the primary drivers of chemotaxis in inflammation, but chemokine network redundancy thwarts pharmacological intervention. Tick evasins promiscuously bind CC and CXC-chemokines, overcoming redundancy. Here we show that short peptides that promiscuously bind both chemokine classes can be identified from evasins by phage-display screening performed with multiple chemokines in parallel. We identify two conserved motifs within these peptides and show using saturation-mutagenesis phage-display and chemotaxis studies of an exemplar peptide that an anionic patch in the first motif and hydrophobic, aromatic and cysteine residues in the second are functionally necessary. AlphaFold2-Multimer modelling suggests that the peptide occludes distinct receptor-binding regions in CC and in CXC-chemokines, with the first and second motifs contributing ionic and hydrophobic interactions respectively. Our results indicate that peptides with broad-spectrum anti-chemokine activity and therapeutic potential may be identified from evasins, and the pharmacophore characterised by phage display, saturation mutagenesis and computational modelling.

The role of microneedles in the healing of chronic wounds.

Chronic wounds occur for several reasons, such as trauma, accidents, and diseases. Diabetes has been one of the primary causes of non-healing wounds, and the number of people with diabetes is increasing in most countries. Wounds in diabetic people have a complex and prolonged treatment process, with high treatment costs to both healthcare providers and patients. They often have severe consequences, such as pain, wound infection, tissue necrosis, and even limb amputation. Various methods have been used to treat chronic wounds, but clinical success has been limited due to inefficient delivery to the wound bed. Microneedles (MNs), as new platforms, can offer an effective treatment, easy to use and non-invasive with less tissue damage, capable of delivering a wide range of drugs to accelerate the wound healing process. Different methods and materials can be used for this technique, and there are different geometric parameters such as needle length, tip angle, shape and radius, together with needle array density to optimize for the most effective treatment. This review paper will investigate the role of MNs in healing chronic wounds and discuss the most recent development in MN-based devices in the field and their effectiveness. The manuscript will also discuss the various types of MNs and their potential applications for delivering therapeutic agents. Finally, the challenges associated with using MNs to heal chronic wounds and future directions in this field are discussed.

Reverse Engineering Caged Compounds: Design Principles for their Application in Biology.

Light passes through biological tissue, and so it is used for imaging biological processes in situ. Such observation is part of the very essence of science, but mechanistic understanding requires intervention. For more than 50 years a "second function" for light has emerged; namely, that of photochemical control. Caged compounds are biologically inert signaling molecules that are activated by light. These optical probes enable external instruction of biological processes by stimulation of an individual element in complex signaling cascades in its native environment. Cause and effect are linked directly in spatial, temporal, and frequency domains in a quantitative manner by their use. I provide a guide to the basic properties required to make effective caged compounds for the biological sciences.

Implementation of a medicine management plan (MMP) to reduce medication-related harm (MRH) in older people post-hospital discharge: a randomised controlled trial.

BACKGROUND: Medication-related harm (MRH) is an escalating global challenge especially among older adults. The period following hospital discharge carries high-risk for MRH due to medication discrepancies, limited patient/carer education and support, and poor communication between hospital and community professionals. Discharge Medical Service (DMS), a newly introduced NHS scheme, aims to reduce post-discharge MRH through an electronic communication between hospital and community pharmacists. Our study team has previously developed a risk-prediction tool (RPT) for MRH in the 8-weeks period post discharge from a UK hospital cohort of 1280 patients. In this study, we aim to find out if a Medicines Management Plan (MMP) linked to the DMS is more effective than the DMS alone in reducing rates of MRH. METHOD: Using a randomized control trial design, 682 older adults ≥ 65 years due to be discharged from hospital will be recruited from 4 sites. Participants will be randomized to an intervention arm (individualised medicine management plan (MMP) plus DMS) or a control arm (DMS only) using a 1:1 ratio stratification. Baseline data will include patients' clinical and social demographics, and admission and discharge medications. At 8-weeks post-discharge, a telephone interview and review of GP records by the study pharmacist will verify MRH in both arms. An economic and process evaluation will assess the cost and acceptability of the study methods. DATA ANALYSIS: Univariate analysis will be done for baseline variables comparing the intervention and control arms. A multivariate logistic regression will be done incorporating these variables. Economic evaluation will compare the cost-of-service use among the study arms and modelled to provide national estimates. Qualitative data from focus-group interviews will explore practitioners' understanding, and acceptance of the MMP, DMS and the RPT. CONCLUSION: This study will inform the use of an objective, validated RPT for MRH among older adults after hospital discharge, and provide a clinical, economic, and service evaluation of a specific medicines management plan alongside the DMS in the National Health Service (UK).

Fabrication and testing of polymer microneedles for transdermal drug delivery.

Microneedle (MN) patches have considerable potential for medical applications such as transdermal drug delivery, point-of-care diagnostics, and vaccination. These miniature microdevices should successfully pierce the skin tissues while having enough stiffness to withstand the forces imposed by penetration. Developing low-cost and simple manufacturing processes for MNs is of considerable interest. This study reports a simple fabrication process for thermoplastic MNs from cycloolefin polymers (COP) using hot embossing on polydimethylsiloxane (PDMS) soft molds. COP has gained interest due to its high molding performance and low cost. The resin master MN arrays (9 × 9) were fabricated using two-photon polymerization (TPP). A previous gap in the detailed characterization of the embossing process was investigated, showing an average of 4.99 ± 0.35% longitudinal shrinkage and 2.15 ± 0.96% lateral enlargement in the molded MN replicas. The effects of bending, buckling, and tip blunting were then examined using compression tests and also theoretically. MN array insertion performance was studied in vitro on porcine back skin using both a prototype custom-made applicator and a commercial device. An adjustable skin stretcher mechanism was designed and manufactured to address current limitations for mimicking skin in vivo conditions. Finite element analysis (FEA) was developed to simulate single MN insertion into a multilayered skin model and validated experimentally using a commercial Pen Needle as a model for the thermoplastic MNs. Margins of safety for the current MN design demonstrated its potential for transdermal drug delivery and fluid sampling. Experimental results indicated significant penetration improvements using the prototype applicator, which produced array penetration efficiencies as high as >92%, depending on the impact velocity setting.

GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits.

GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

An overview of microneedle applications, materials, and fabrication methods.

Microneedle-based microdevices promise to expand the scope for delivery of vaccines and therapeutic agents through the skin and withdrawing biofluids for point-of-care diagnostics - so-called theranostics. Unskilled and painless applications of microneedle patches for blood collection or drug delivery are two of the advantages of microneedle arrays over hypodermic needles. Developing the necessary microneedle fabrication processes has the potential to dramatically impact the health care delivery system by changing the landscape of fluid sampling and subcutaneous drug delivery. Microneedle designs which range from sub-micron to millimetre feature sizes are fabricated using the tools of the microelectronics industry from metals, silicon, and polymers. Various types of subtractive and additive manufacturing processes have been used to manufacture microneedles, but the development of microneedle-based systems using conventional subtractive methods has been constrained by the limitations and high cost of microfabrication technology. Additive manufacturing processes such as 3D printing and two-photon polymerization fabrication are promising transformative technologies developed in recent years. The present article provides an overview of microneedle systems applications, designs, material selection, and manufacturing methods.

High-resolution two-photon polymerization: the most versatile technique for the fabrication of microneedle arrays.

Microneedle patches have received much interest in the last two decades as drug/vaccine delivery or fluid sampling systems for diagnostic and monitoring purposes. Microneedles are manufactured using a variety of additive and subtractive micromanufacturing techniques. In the last decade, much attention has been paid to using additive manufacturing techniques in both research and industry, such as 3D printing, fused deposition modeling, inkjet printing, and two-photon polymerization (2PP), with 2PP being the most flexible method for the fabrication of microneedle arrays. 2PP is one of the most versatile and precise additive manufacturing processes, which enables the fabrication of arbitrary three-dimensional (3D) prototypes directly from computer-aided-design (CAD) models with a resolution down to 100 nm. Due to its unprecedented flexibility and high spatial resolution, the use of this technology has been widespread for the fabrication of bio-microdevices and bio-nanodevices such as microneedles and microfluidic devices. This is a pioneering transformative technology that facilitates the fabrication of complex miniaturized structures that cannot be fabricated with established multistep manufacturing methods such as injection molding, photolithography, and etching. Thus, microstructures are designed according to structural and fluid dynamics considerations rather than the manufacturing constraints imposed by methods such as machining or etching processes. This article presents the fundamentals of 2PP and the recent development of microneedle array fabrication through 2PP as a precise and unique method for the manufacture of microstructures, which may overcome the shortcomings of conventional manufacturing processes.

Local recovery of cardiac calcium-induced calcium release interrogated by ultra-effective, two-photon uncaging of calcium.

KEY POINTS: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. ABSTRACT: In cardiac myocytes Ca2+ -induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca2+ compound BIST-2EGTA and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

Transcriptomic Analysis of Inflammatory Cardiomyopathy Identifies Molecular Signatures of Disease and Informs in silico Prediction of a Network-Based Rationale for Therapy.

Inflammatory cardiomyopathy covers a group of diseases characterized by inflammation and dysfunction of the heart muscle. The immunosuppressive agents such as prednisolone, azathioprine and cyclosporine are modestly effective treatments, but a molecular rationale underpinning such therapy or the development of new therapeutic strategies is lacking. We aimed to develop a network-based approach to identify therapeutic targets for inflammatory cardiomyopathy from the evolving myocardial transcriptome in a mouse model of the disease. We performed bulk RNA sequencing of hearts at early, mid and late time points from mice with experimental autoimmune myocarditis. We identified a cascade of pathway-level events involving early activation of cytokine and chemokine-signaling pathways that precede leucocyte infiltration and are followed by innate immune, antigen-presentation, complement and cell-adhesion pathway activation. We integrated these pathway events into a network-like representation from which we further identified a 50-gene subnetwork that is predominantly induced during the course of autoimmune myocardial inflammation. We developed a combinatorial attack strategy where we quantify network tolerance to combinatorial node removal to determine target-specific therapeutic potential. We find that combinatorial attack of Traf2, Nfkb1, Rac1, and Vav1 disconnects 80% of nodes from the largest network component. Two of these nodes, Nfkb1 and Rac1, are directly targeted by prednisolone and azathioprine respectively, supporting the idea that the methodology developed here can identify valid therapeutic targets. Whereas Nfkb1 and Rac1 removal disconnects 56% of nodes, we show that additional removal of Btk and Pik3cd causes 72% node disconnection. In conclusion, transcriptome profiling, pathway integration, and network identification of autoimmune myocardial inflammation provide a molecular signature applicable to the diagnosis of inflammatory cardiomyopathy. Combinatorial attack provides a rationale for immunosuppressive therapy of inflammatory cardiomyopathy and provides an in silico prediction that the approved therapeutics, ibrutinib and idelalisib targeting Btk and Pik3cd respectively, could potentially be re-purposed as adjuncts to immunosuppression.

Photochemical control of drug efficacy - a comparison of uncaging and photoswitching ifenprodil on NMDA receptors.

Ifenprodil is an important negative allosteric modulator of the N-methyl-D-aspartate (NMDA) receptors. We have synthesized caged and photoswitchable derivatives of this small molecule drug. Caged ifenprodil was biologically inert before photolysis, UV irradiation efficiently released the drug allowing selective inhibition of GluN2B-containing NMDA receptors. Azobenzene-modified ifenprodil, on the other hand, is inert in both its trans and cis configurations, although in silico modeling predicted the trans form to be able to bind to the receptor. The disparity in effectiveness between the two compounds reflects, in part, the inherent ability of each method in manipulating the binding properties of drugs. With appropriate structure-activity relationship uncaging enables binary control of effector binding, whereas photoswitching using feely diffusable chromophores shifts the dose-response curve of drug-receptor interaction. Our data suggest that the efficacy of pharmacophores having a confined binding site such as ifenprodil can be controlled more easily by uncaging in comparison to photoswitching.

Intracellular photoswitchable neuropharmacology driven by luminescence from upconverting nanoparticles.

Photoswitchable drugs are small-molecule optical probes that undergo chromatically selective control of drug efficacy using, most often, UV-visible light. Here we report that luminescence produced by near-infrared stimulation of NaYF4:TmYb nanoparticles can be used for "remote control" of an azobenzene-based photochromic ion channel blocker of neurons in living brain slices.

Useful Caged Compounds for Cell Physiology.

Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells. However, over the past 50 years, a "second use" of light has emerged in cell physiology, namely one of rational control. The seminal method for this emerged in late 1970s with the invention of caged compounds. This was the point when "caged compounds" were defined as optical probes in which the active functionality of a physiological signaling molecule was blocked with a photochemical protecting group. Caged compounds are analogous to prodrugs; in both, the activity of the effector is latent. However, caged compounds, unlike prodrugs, use a trigger that confers the power of full temporal and spatial manipulation of the effects of release of its latent biological cargo. Light is distinct because it is bio-orthogonal, passes through living tissue (even into the cell interior), and initiates rapid release of the "caged" biomolecule. Further, because light can be directed to broad areas or focused to small points, caged compounds offer an array of timing scenarios for physiologists to dissect virtually any type of cellular process.The collaborative interaction between chemists and physiologists plays a fundamental role in the development of caged compounds. First, the physiologists must define the problem to be addressed; then, with the help of chemists, decide if a caged compound would be useful. For this, structure-activity relationships of the potential optical probe and receptor must be determined. If rational targets seem feasible, synthetic organic chemistry is used to make the caged compound. The crucial property of prephotolysis bio-inertness relies on physiological or biochemical assays. Second, detailed optical characterization of the caged compound requires the skill of photochemists because the rate and efficiency of uncaging are also crucial properties for a useful caged compound. Often, these studies reveal limitations in the caged compound which has been developed; thus, chemists and physiologists use their abilities for iterative development of even more powerful optical probes. A similar dynamic will be familiar to scientists in the pharmaceutical industry. Therefore, caged compound development provides an excellent training framework for (young) chemists both intellectually and professionally. In this Account, I draw on my long experience in the field of making useful caged compounds for cell physiology by showing how each probe I have developed has been defined by an important physiological problem. Fundamental to this process has been my initial training by the pioneers in aromatic photochemistry, Derek Bryce-Smith and Andrew Gilbert. I discuss making a range of "caged calcium" probes, ones which went on to be the most widely used of all caged compounds. Then, I describe the development of caged neurotransmitters for two-photon uncaging microscopy. Finally, I survey recent work on making new photochemical protecting groups for wavelength orthogonal, two-color, and ultraefficient two-photon uncaging.

Engineered anti-inflammatory peptides inspired by mapping an evasin-chemokine interaction.

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen-deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.