The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.

Purpose: To investigate the effect of culturing human corneal endothelial cells (HCEnCs) from older donors on extracellular matrix (ECM) derived from human corneal endothelial cell line (HCEC‐12).Methods: HCEC‐12 cells were cultured on lab‐tek chamber slides for 9 days. Upon confluence, the cells were ruptured using ammonium hydroxide leaving the released ECM on the slide surface which was visualized using scanning electron microscope (SEM). HCEnCs from old aged donor tissues (n = 40) were isolated and cultured on either fibronectin‐collagen (FNC) or HCEC‐12 ECM at passage (P) 0. At subsequent passages (P1 and P2), cells were sub‐cultured on FNC and ECM separately. Live/dead analysis and tight junction using ZO‐1 staining were used to record percentage viability and morphological changes. The protein composition of HCEC‐12 ECM was then analysed using liquid chromatography–mass spectrometry.Results: SEM images showed long fibrillar‐like structures and a fully laid ECM upon confluence. HCEnCs cultured from older donor tissues on this ECM showed significantly better proliferation and morphometric characteristics at subsequent passages. Out of 1307 proteins found from the HCEC‐12 derived ECM, 93 proteins were evaluated to be matrix oriented out of which 20 proteins were exclusively found to be corneal endothelial specific.Conclusions: ECM derived from HCEC‐12 retains protein and growth factors that stimulate the growth of HCEnCs. As the current clinical trials are from younger donors that are not available routinely for cell culture, HCEnCs from older donors can be cultured on whole ECM and passaged successfully.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine

Background/Objectives: Zinc, an important trace metal, requires daily intake but dietary antagonists including phytic acid reduce its absorption. It is unclear if phytic acid affects zinc absorption at the level of bioaccessibility (how much soluble zinc is available from digestion) or bioavailability (how much zinc is absorbed by the intestine). This study investigates at which level this occurs at and if the zinc source alters the response. Methods: Following a standardised in vitro digestion (INFOGEST), the yield of soluble zinc was measured as the bioaccessible fraction from inorganic and chelated zinc sources, with and without phytic acid. Bioavailability was assessed by measuring cellular zinc uptake in intestinal cell lines (Caco-2 and IPEC-J2). Results: Phytic acid affected the bioaccessibility of zinc, with varying impacts depending on the zinc source. Zinc proteinate had the highest bioaccessibility (42%) without phytic acid, while inorganic zinc sulphate (24%) and zinc bisglycinate (27%) were lower. ZnSO₄ was more susceptible to phytic acid antagonism than chelated zinc sources (from 2:100 molar ratio of phytic acid: zinc), while the chelated zinc sources were only affected at a molar ratio of 4:100, with zinc bisglycinate being more susceptible than zinc proteinate. Cellular zinc uptake (bioavailability) and toxicity at equimolar concentrations were unaffected by phytic acid. Conclusions: This study found that phytic acid affected bioaccessibility, not bioavailability. The zinc source impacts the response. Zinc proteinate was consistently more bioaccessible while both chelated zinc sources were less susceptible to phytic acid than inorganic zinc.

It is well established that oxaliplatin, one of the three Pt(II) anticancer drugs approved worldwide, and phenanthriplatin, an important preclinical monofunctional Pt(II) anticancer drug, possess a different mode of action from that of cisplatin and carboplatin, namely, the induction of nucleolar stress. The exact mechanisms that lead to Pt-induced nucleolar stress are, however, still poorly understood. As such, studies aimed at better understanding the biological targets of both oxaliplatin and phenanthriplatin are urgently needed to expand our understanding of Pt-induced nucleolar stress and guide the future design of Pt chemotherapeutics. One approach that has seen great success in the past is the use of Pt-click complexes to study the biological targets of Pt drugs. Herein, we report the synthesis and characterization of the first examples of click-capable phenanthriplatin complexes. Furthermore, through monitoring the relocalization of nucleolar proteins, RNA transcription levels, and DNA damage repair biomarker γH2AX, and by investigating their in vitro cytotoxicity, we show that these complexes successfully mimic the cellular responses observed for phenanthriplatin treatment in the same experiments. The click-capable phenanthriplatin derivatives described here expand the existing library of Pt-click complexes. Significantly they are suitable for studying nucleolar stress mechanisms and further elucidating the biological targets of Pt complexes.

Drug-mediated correction of abnormal biological zinc homeostasis could provide new routes to treating neurodegeneration, cancer, and viral infections. Designing therapeutics to facilitate zinc transport intracellularly is hampered by inadequate concentrations of endogenous zinc, which is often protein-bound in vivo. We found strong evidence that hydroxychloroquine, a drug used to treat malaria and employed as a potential treatment for COVID-19, does not bind and transport zinc across biological membranes through ionophoric mechanisms, contrary to recent claims. In vitro complexation studies and liposomal transport assays are correlated with cellular zinc assays in A549 lung epithelial cells to confirm the indirect mechanism of hydroxychloroquine-mediated elevation in intracellular zinc without ionophorism. Molecular simulations show hydroxychloroquine-triggered helix perturbation in zinc-finger protein without zinc chelation, a potential alternative non-ionophoric mechanism.