Sorting the Wheat From the Chaff: Programmed Cell Death as a Marker of Stress Tolerance in Agriculturally Important Cereals

Conventional methods for screening for stress-tolerant cereal varieties rely on expensive, labour-intensive field testing and molecular biology techniques. Here, we use the root hair assay (RHA) as a rapid screening tool to identify stress-tolerant varieties at the early seedling stage. Wheat and barley seedlings had stress applied, and the response quantified in terms of programmed cell death (PCD), viability and necrosis. Heat shock experiments of seven barley varieties showed that winter and spring barley varieties could be partitioned into their two distinct seasonal groups based on their PCD susceptibility, allowing quick data-driven evaluation of their thermotolerance at an early seedling stage. In addition, evaluating the response of eight wheat varieties to heat and salt stress allowed identification of their PCD inflection points (35°C and 150 mM NaCl), where the largest differences in PCD levels arise. Using the PCD inflection points as a reference, we compared different stress effects and found that heat-susceptible wheat varieties displayed similar vulnerabilities to salt stress. Stress-induced PCD levels also facilitated the assessment of the basal, induced and cross-stress tolerance of wheat varieties using single, combined and multiple individual stress exposures by applying concurrent heat and salt stress in a time-course experiment. Two stress-susceptible varieties were found to have low constitutive resistance as illustrated by their high PCD levels in response to single and combined stress exposure. However, both varieties had a fast, adaptive response as PCD levels declined at the other time-points, showing that even with low constitutive resistance, the initial stress cue primes cross-stress tolerance adaptations for enhanced resistance even to a second, different stress type. Here, we demonstrate the RHA’s suitability for high-throughput analysis (~4 days from germination to data collection) of multiple cereal varieties and stress treatments. We also showed the versatility of using stress-induced PCD levels to investigate the role of constitutive and adaptive resistance by exploring the temporal progression of cross-stress tolerance. Our results show that by identifying suboptimal PCD levels in vivo in a laboratory setting, we can preliminarily identify stress-susceptible cereal varieties and this information can guide further, more efficiently targeted, field-scale experimental testing

Hyaluronic Acid: Its Versatile Use in Ocular Drug Delivery with a Specific Focus on Hyaluronic Acid-Based Polyelectrolyte Complexes.

Extensive research is currently being conducted into novel ocular drug delivery systems (ODDS) that are capable of surpassing the limitations associated with conventional intraocular anterior and posterior segment treatments. Nanoformulations, including those synthesised from the natural, hydrophilic glycosaminoglycan, hyaluronic acid (HA), have gained significant traction due to their enhanced intraocular permeation, longer retention times, high physiological stability, inherent biocompatibility, and biodegradability. However, conventional nanoformulation preparation methods often require large volumes of organic solvent, chemical cross-linkers, and surfactants, which can pose significant toxicity risks. We present a comprehensive, critical review of the use of HA in the field of ophthalmology and ocular drug delivery, with a discussion of the physicochemical and biological properties of HA that render it a suitable excipient for drug delivery to both the anterior and posterior segments of the eye. The pivotal focus of this review is a discussion of the formation of HA-based nanoparticles via polyelectrolyte complexation, a mild method of preparation driven primarily by electrostatic interaction between opposing polyelectrolytes. To the best of our knowledge, despite the growing number of publications centred around the development of HA-based polyelectrolyte complexes (HA-PECs) for ocular drug delivery, no review articles have been published in this area. This review aims to bridge the identified gap in the literature by (1) reviewing recent advances in the area of HA-PECs for anterior and posterior ODD, (2) describing the mechanism and thermodynamics of polyelectrolyte complexation, and (3) critically evaluating the intrinsic and extrinsic formulation parameters that must be considered when designing HA-PECs for ocular application

Posterior Segment Ophthalmic Drug Delivery: Role of Muco-Adhesion with a Special Focus on Chitosan.

Posterior segment eye diseases (PSEDs) including age macular degeneration (AMD) and diabetic retinopathy (DR) are amongst the major causes of irreversible blindness worldwide. Due to the numerous barriers encountered, highly invasive intravitreal (IVT) injections represent the primary route to deliver drugs to the posterior eye tissues. Thus, the potential of a more patient friendly topical route has been widely investigated. Mucoadhesive formulations can decrease precorneal clearance while prolonging precorneal residence. Thus, they are expected to enhance the chances of adherence to corneal and conjunctival surfaces and as such, enable increased delivery to the posterior eye segment. Among the mucoadhesive polymers available, chitosan is the most widely explored due to its outstanding mucoadhesive characteristics. In this review, the major PSEDs, their treatments, barriers to topical delivery, and routes of topical drug absorption to the posterior eye are presented. To enable the successful design of mucoadhesive ophthalmic drug delivery systems (DDSs), an overview of mucoadhesion, its theory, characterization, and considerations for ocular mucoadhesion is given. Furthermore, chitosan-based DDs that have been explored to promote topical drug delivery to the posterior eye segment are reviewed. Finally, challenges of successful preclinical to clinical translation of these DDSs for posterior eye drug delivery are discussed

Development of Molecularly Imprinted Polymers for Corticosteroids

The aim of this research was to prepare a series of molecularly imprinted polymers (MIPs) for corticosteroid templates using bulk and precipitation polymerisation strategies. Initially, a systematic study was undertaken, investigating the factors affecting the synthesis of MIPs by precipitation polymerisation. Careful optimisation of polymerisation parameters, including the monomer concentration, initiator type and solvent, allowed for a high degree of control over particle size and morphology. Preequilibrium interactions between the primary target budesonide (BDN) and functional monomers were investigated using NMR studies and showed weak interactions with dissociation constants, kdiss, ranging from 0.534-1.47 M Due to this weak functional monomer-template interaction, polymers were prepared in bulk format using a target analogue approach utilising hydrocortisone-17-butyrate (HCB) as a low-cost alternative to BDN. The HCB imprinted ground bulk polymers demonstrated imprinting factors (IF) as high as 7.5 for the template. Selectivity studies on six steroidal analogues showed that recognition was driven by shape and size selectivity as well as hydrophobic interactions, with IF values ranging from 1.2-9.5 in both organic and aqueous based systems. Applications of the imprinted polymers were demonstrated by solid-phase extraction (SPE) and analytical column chromatography. SPE studies utilising HCB imprinted polymers displayed a seven-fold increase in the specific retention of BDN. Precipitated prednisolone imprinted polymers achieved chromatographic separation between the template and hydrocortisone, a close structural analogue. Selectivity factors as high as 1.4 and a resolution value of 0.75 were achieved. Two custom functional monomer strategies were utilised towards enhancing MIP performance. The synthesis of tailor-made monomers based on derivatised cyclodextrins (CD) achieved a substitution of 2.5 polymerisable vinyl groups per CD. This derivatised CD was used to prepare precipitated polymers (0.3-7.2 μm) in a 40% aqueous acetonitrile porogen. A polymerisable hydrocortisone methacrylate monomer was successfully synthesised for use in semi-covalent imprinting which produced discrete microspherical beads (1.2-5.1 μm) using divinylbenzene as the crosslinker

Recent Advances in Ophthalmic Drug Delivery

Due to population aging and to the increasing prevalence of diseases such as diabetes, chronic eye disorders such as glaucoma, age-related macular degeneration, and diabetic retinopathy have increased significantly, becoming responsible for a high percentage of blindness and vision impairment cases at a global level [...

Polystyrene-co-Divinylbenzene PolyHIPE Monoliths in 1.0 mm Column Formats for Liquid Chromatography

The reversed phase liquid chromatographic (RP-HPLC) separation of small molecules using a polystyrene-co-divinylbenzene (PS-co-DVB) polyHIPE stationary phases housed within 1.0 mm i.d. silcosteel columns is presented within this study. A 90% PS-co-DVB polyHIPE was covalently attached to the walls of the column housing by prior wall modification with 3-(trimethoxysilyl) propyl methacrylate and could withstand operating backpressures in excess of 200 bar at a flow rate of 1.2 mL/min. Permeability studies revealed that the monolith swelled slightly in 100% acetonitrile relative to 100% water but could nevertheless be used to separate five alkylbenzenes using a flow rate of 40 µL/min (linear velocity: 0.57 mm/s). Remarkable column-to-column reproducibility is shown with retention factor variation between 2.6% and 6.1% for two separately prepared columns

Long-term clinical manifestations of retained bullet fragments within the intervertebral disk space

A retrospective review of 12 patients who were victims of penetrating trauma with a bullet or bullet fragments lodged within the intervertebral disk space was conducted. The objective of the review was to evaluate the potential systemic effects of lead resorption at long-term follow-up. Literature regarding the potential for lead toxicity due to retained bullet fragments within the intervertebral disk space is lacking. Between January 1969 and June 1993, a total of 238 patients with a gunshot wound to the spine were identified. Twelve of the 238 were found to have a bullet or bullet fragments within the intervertebral disk space. All patients were fully screened for evidence of plumbism. The average age at time of gunshot injury was 35.8 years; the average time for follow-up was 7.8 years. One of the 12 patients showed clinical evidence of plumbism. The patient subsequently underwent a partial laminectomy and diskectomy with excision of the bullet fragments. The patient's complaints, specific for plumbism, resolved 2 months postoperatively. We conclude that patients with retained lead-based bullet fragments in the intervertebral disk should be educated about the rare potential for plumbism due to partial bullet fragment resorption and that long-term observation for this disorder is recommended