Eicosanoids as diagnostics for preterm labor

Despite continuing significant research efforts and an increasing prevalence of preterm labor (PTL) worldwide, a comprehensive understanding of the mechanisms involved remains to be established; such is the complexity of the interactions involved. Closing the knowledge gaps in this field would afford an opportunity to improve mortality and morbidity rates on a global scale. Early identification of pregnancies at risk of PTL would increase the possibility of delaying birth (where appropriate) prior to initiation of labor. This requires identification of appropriate biomarkers that are readily detectable in easily obtained clinical samples. A family of arachidonic acid metabolites, called eicosanoids. Eicosanoids have shown promise as possible diagnostic biomarkers for PTL. The inherent problems of antibody cross-reactivity in immunoassay methods has, however, resulted in possible misinterpretation of past results. The following mini-review outlines the necessity of such diagnostic targets and the problem with previous research methods and also summarizes recent findings in the field

Cytoprotective silk biomaterials to repair retinal degeneration

A curative treatment for age-related macular degeneration (AMD) still eludes medical science, despite being the leading cause of blindness in developed countries and third highest cause of blindness worldwide. AMD can be categorized as either “wet” or “dry”, with the former causing the most acute devastation and the latter being refractive to currently available treatments. The leaky choroidal neovascular growth in the wet form can be combatted with anti-angiogenic drugs. Although repeated intraocular injections of drugs (e.g. anti-vascular endothelial growth factor) are the current best practice for AMD, the oxidative stress-mediated-death of key retinal cells remains unaddressed in both forms. Furthermore, the progression of AMD disease ruins the normal architecture of Bruch’s membrane, which underpins the retinal pigment epithelium (RPE). The ideal therapy option for both types of AMD would reinstate the RPE population and replace the damaged Bruch’s membrane, while providing antioxidative protection against cell death. Thus, a transplantable scaffold is sought to deliver a monolayer of RPE grown upon an inherently antioxidative biomaterial.Silk proteins have a long-established history of medical use, with research predominately focussed on the inner fibrous protein, Bombyx mori silk fibroin (BMSF). Although BMSF has impressive mechanical properties when manufactured into a thin film, it does not support cell attachment when used without mammalian-derived extracellular matrix (ECM) coatings. These ECM coatings can carry an increased risk of contamination with known and unknown pathogens, and elevated risk of immunogenicity. Bombyx mori silk sericin (BMSS), the globular polypeptide complex that surrounds the fibroin strands, possesses a host of advantageous properties including enhanced cell attachment, proliferation, and antioxidation. Hence, in the present study, BMSS was investigated as a biomaterial for restoration of the RPE population and Bruch’s membrane in a single transplant.This thesis presents the optimized method for BMSS isolation and manufacture into thin coatings with impressive cytocompatibility for both the RPE cell line, ARPE-19, and human embryonic stem cell-derived RPE (hESC-RPE). Freestanding membranes were created by employing Transwell® and BMSF membranes as structural supports for BMSS coatings. The inherent antioxidative capacity of BMSS preparations was quantified by a radical ion scavenging assay. Accordingly, the most antioxidative formulation was investigated as both a film and gel substrate for RPE cell culture in a model of oxidative stress and was found to protect ARPE-19 populations against significant losses. Thus, the project has provided proof-of-concept for an all-silk, BMSS-coated BMSF biomaterial with innate antioxidative activity. With further development, this inherently cytoprotective BMSS biomaterial may be employed for RPE transplantation as it meets the criteria of an ideal treatment for both forms of AMD

Further development of silk sericin as a biomaterial: comparative investigation of the procedures for its isolation from Bombyx mori silk cocoons

There is significant research dedicated to fibroin and sericin, the two major proteinaceous components of the silk threads produced by the domesticated silkworm, Bombyx mori. While fibroin is accepted as an established biomaterial, sericin (BMSS) has been largely neglected in this respect on the account of a hypothetical allergenic activity. Research over the past decade, including our previous study (Prog Biomater 2:14, 2013), demonstrated the biocompatibility of sericin and feasibility of its use as a biomaterial. However, the current procedures for isolating BMSS from the raw silk cocoons can only provide degraded proteins, where the size and distribution of their molecular masses are significantly altered. Based on the plausible assumption that such effects can have a negative impact on the properties of sericin as a biomaterial, in this study we investigated comparatively four different extraction procedures in order to find the method that would cause the least hydrothermal degradation of BMSS. The products resulting from commonly used procedures (extraction in boiling water, alkaline extraction, and extraction in autoclave) were compared to those resulting from aqueous extraction in mild conditions as described a long time ago by Anderlini. The molecular mass distribution in BMSS resulting from each procedure was examined by electrophoretic analysis performed on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE), while the conformational changes pertaining to secondary structure of BMSS were evaluated by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectrometry. The electrophoretograms indicated that the aqueous extraction in mild conditions conducted at 50 degrees C for durations up to 4 weeks, with/ without stirring, afforded the least degraded BMSS. The infrared spectrometric analysis showed that BMSS resulting from the mild extraction method contained predominantly beta-sheet conformations, while the more degradative methods (alkaline, autoclave) led to BMSS where the random-coil conformations were preferential. The long-duration aqueous extraction at 50 degrees C (but not at 60 degrees C) appeared as a valid option for obtaining BMSS products where the hydrothermally induced fragmentation of the polypeptidic components is minimized

Autoclave technique for the isolation of Bombyx mori sericin hydrolysate for potential neuroprotective applications

Silk and its componentsSilk from Bombyx mori silkworms has been recommended for use in medical applications for millennia, and is commonly known for its use as sutures. Each strand of silk is made of two fibrous threads of insoluble protein called fibroin, surrounded by a soluble glue-like coating collectively referred to as “sericin”. Evidence of “sericin” bioactivityStudies have revealed a broad range of activities of “sericin”: anti-tyrosine activity, improved adhesion and proliferation of a corneal epithelial cell line, as well as the inhibition of lipid peroxidation in rat brain cells and colon cancer cell proliferation. More recently, it was found that “sericin” had a protective effect on rat neural cells which may be due to inherent antioxidative properties.Lack of standard procedureCalling the outer coating “sericin” is a misnomer as it is really a complex mixture containing a composite of at least 6 polypeptides with molecular weights ranging from 10kDa to 400kDa, now collectively known as sericin, along with other components including waxes, lipids, carbohydrates, pigments, and flavonoids. The outer coating can be easily separated from the fibroin strands by boiling and filtering, but a variety of methods have been devised to improve extraction efficiency and alter the final product in terms of sericin molecular weight and retention of small molecules.Flaws in previous isolation methods There are inherent flaws in the previous methods for isolation of silk’s outer coating (see table, below): lack of a standardised procedure; lengthy, labour intensive protocols; unmonitored loss of potentially bioactive small molecules through dialysis steps; low yields; inconsistency in protein degradation; and no method which produced a soluble powder for stable room temperature storage which retained all of the native outer coating components. Our aimWe aim to exploit the neuroprotective effect of sericin by using it as a thin film for transplantation of protected replacement retinal cells and as an antioxidant to prevent retinal cell damage from occurring in retinal degenerative diseases like age-related macular degeneration (AMD) and glaucoma. These conditions are two of the leading causes of blindness worldwide and both involve decades of reactive oxygen species accumulation; therefore the incidence will rise with the aging population. Oxidative pathophysiology lies at the heart of many diseases; hence this research may have applications not only in ophthalmology but also for the broader research community

Optimized Bombyx mori silk sericin biomaterial for retinal pigment epithelium regeneration.

Purpose: A transplantable cell carrier is sought to restore the retinal pigment epithelium (RPE) in age-related macular degeneration. Preparations of Bombyx mori silk sericin (BMSS) have shown in vivo and in vitro protective, wound healing, and antioxidative properties; however, current isolation techniques lead to inconsistent structure and bioactivity. We present an optimized method for BMSS isolation and manufacture into a biomaterial capable ofsupporting RPE.Methods: Gravimetric yield analysis, SDS-PAGE, solubility, transparency, and the ability to support viable human adult RPE cells (ARPE-19) were determined for the BMSS films created from four increasing durations of high temperature high pressure (HTHP) extraction, without dialysis. The new preparations were compared to both the original HTHPmethod (30-min HTHP with 4-h dialysis) and to uncoated tissue culture plastic (TCP), as a positive control. The effect of water annealing the films was also investigated. The optimized method was tested as a biomaterial for humanembryonic stem cell derived-RPE (hESC-RPE) cells against Geltrex coated and uncoated transwell inserts (Fig. 1).Results: The yield was not adversely affected by increased duration of HTHP (n=5) and surpassed previous methods. The polypeptide component of BMSS degraded in a time-dependent manner, increasing solubility of the lyophilised product. Films were suitable for biomaterial applications if cast from freshly prepared solutions but not if reconstituted from lyophilised BMSS. Films prepared from both the new and original 30-min HTHP methods (and not annealed) performed comparably to TCP in terms of ARPE19 cell viability at 4 h and 7 days (2-way ANOVA, n=3, p>0.05). As dialysis proved unnecessary, the new 30 min method was selected for improved speed, yield, and theoretical retention of potentially bioactive components. Long term hESC-RPE established for 8 weeks upon optimized BMSS filmsdisplayed a monolayer culture (1-2 cells deep) with correct cobblestone morphology (Fig. 2) and pigmentation.Conclusions: Films prepared by the new optimized method of 30-min HTHP without dialysis or water annealing supported ARPE19 cells with viability equivalent to TCP controls, and outperformed Geltrex and uncoated transwells in developing correct hESC-RPE morphology and pigmentation

Comparison of penetrating and endothelial keratoplasty in patients with iridocorneal endothelial syndrome: A registry study

Background: To compare graft survival of endothelial keratoplasty (EK) versus penetrating keratoplasty (PK) in patients with iridocorneal endothelial (ICE) syndrome and identify ocular features associated with graft survival. Methods: Observational, prospective, cohort study. A total of 30 806 first grafts performed between 1985 and 2020 were identified through the Australian Corneal Graft Registry and included in this observational, prospective cohort study. A total of 196 eyes underwent a primary corneal graft for ICE syndrome. Kaplan–Meier graft survival plots and Chi-squared tests were performed to identify graft survival rates for EK and PK. A history of raised intraocular pressure (IOP) was also recorded and analysed. Graft survival of eyes with ICE syndrome were compared to that of other indications. Results: Grafts performed for ICE syndrome increased to 0.8% of all cases during the 2005 to 2020 period compared with 0.5% between 1985 to 2004 (χ2=9.35, p = 0.002). From 2010, EK surpassed PK as the preferred graft type. Survival of primary grafts in eyes with ICE syndrome was lower than for other indications (log-rank = 56.62, p < 0.001). Graft survival was higher following PK than Descemet stripping (automated) endothelial keratoplasty (DS(A)EK) (log-rank = 10.56, p = 0.001). Graft survival was higher in eyes without a history of raised IOP compared to those with a reported history of raised IOP (log-rank = 13.06, p < 0.001). Conclusions: ICE syndrome carries a poor prognosis for graft survival. DS(A)EK had a poorer prognosis than PK. A history of raised IOP is associated with higher risk of graft failure.</p

The role of endocannabinoids in pregnancy

Endocannabinoids are a family of lipid signalling molecules. As with prostaglandins (PGs), endocannabinoids are derived from polyunsaturated fatty acids and affect cell function via receptor-mediated mechanisms. They also bind to PG receptors, although at a lower affinity. The endocannabinoid network is regulated in pregnancy from embryo development to labour onset. Even small changes in endocannabinoid exposure can retard embryo development and affect implantation success. There is now compelling evidence that aberrant expression of factors involved in the endocannabinoid pathway in the placenta and circulating lymphocytes results in spontaneous miscarriage and poor pregnancy outcomes. It is likely that competition between endocannabinoids, PGs and other similar lipids ultimately determines how phospholipid/fatty acid substrates are metabolised and, thus, the balance between the uterotonic and tocolytic activities. We, therefore, hypothesise that endocannabinoid profiles may be used as a biomarker to predict and/or identify spontaneous labour onset