Biocorrosion and Cytotoxicity Studies on Biodegradable Mg-Based Multicomponent Alloys.

Magnesium-based multicomponent alloys with different compositions, namely Mg60Al20Zn5Cu10Mn5 (Mg60 alloy), Mg70Al15Zn5Cu5Mn5 (Mg70 alloy), and Mg80Al5Cu5Mn5Zn5 (Mg 80) alloys, were prepared using the disintegrated melt deposition technique. The DMD technique is a distinctive method that merges the benefits from gravity die casting and spray forming. This approach facilitates high solidification rates, process yields, and reduced metal wastage, resulting in materials with a fine microstructure and minimal porosity. Their potential as biodegradable materials was assessed through corrosion in different simulated body fluids (SBFs), microstructure, and cytotoxicity tests. It was observed that the Mg60 alloy exhibited low corrosion rates (~× 10-5 mm/year) in all SBF solutions, with a minor amount of corrosive products, and cracks were observed. This can be attributed to the formation of the Mg32(AlZn)49 phase and to its stability due to Mg(OH)2 film, leading to excellent corrosion resistance when compared to the Mg70 and M80 alloys. Conversely, the Mg80 alloy exhibited high corrosion rates, along with more surface degradation and cracks, due to active intermetallic phases, such as Al6Mn, Al2CuMg, and Al2Cu phases. The order of corrosion resistance for the Mg alloy was found to be ASS > HBSS > ABP > PBS. Further, in vitro cytotoxicity studies were carried out using MDA-MB-231 tumor cells. By comparing all three alloys, in terms of proliferation and vitality, the Mg80 alloy emerged as a promising material for implants, with potential antitumor activity.

Human equivalent doses of L-DOPA rescues retinal morphology and visual function in a murine model of albinism.

L-DOPA is deficient in the developing albino eye, resulting in abnormalities of retinal development and visual impairment. Ongoing retinal development after birth has also been demonstrated in the developing albino eye offering a potential therapeutic window in humans. To study whether human equivalent doses of L-DOPA/Carbidopa administered during the crucial postnatal period of neuroplasticity can rescue visual function, OCA C57BL/6 J-c2J OCA1 mice were treated with a 28-day course of oral L-DOPA/Carbidopa at 3 different doses from 15 to 43 days postnatal age (PNA) and for 3 different lengths of treatment, to identify optimum dosage and treatment length. Visual electrophysiology, acuity, and retinal morphology were measured at 4, 5, 6, 12 and 16 weeks PNA and compared to untreated C57BL/6 J (WT) and OCA1 mice. Quantification of PEDF, ßIII-tubulin and syntaxin-3 expression was also performed. Our data showed impaired retinal morphology, decreased retinal function and lower visual acuity in untreated OCA1 mice compared to WT mice. These changes were diminished or eliminated when treated with higher doses of L-DOPA/Carbidopa. Our results demonstrate that oral L-DOPA/Carbidopa supplementation at human equivalent doses during the postnatal critical period of retinal neuroplasticity can rescue visual retinal morphology and retinal function, via PEDF upregulation and modulation of retinal synaptogenesis, providing a further step towards developing an effective treatment for albinism patients.

A laser-induced mouse model of progressive retinal degeneration with central sparing displays features of parafoveal geographic atrophy.

There are no disease-modifying treatments available for geographic atrophy (GA), the advanced form of dry age-related macular degeneration. Current murine models fail to fully recapitulate the features of GA and thus hinder drug discovery. Here we describe a novel mouse model of retinal degeneration with hallmark features of GA. We used an 810 nm laser to create a retinal lesion with central sparing (RLCS), simulating parafoveal atrophy observed in patients with progressive GA. Laser-induced RLCS resulted in progressive GA-like pathology with the development of a confluent atrophic lesion. We demonstrate significant changes to the retinal structure and thickness in the central unaffected retina over a 24-week post-laser period, confirmed by longitudinal optical coherence tomography scans. We further show characteristic features of progressive GA, including a gradual reduction in the thickness of the central, unaffected retina and of total retinal thickness. Histological changes observed in the RLCS correspond to GA pathology, which includes the collapse of the outer nuclear layer, increased numbers of GFAP + , CD11b + and FcγRI + cells, and damage to cone and rod photoreceptors. We demonstrate a laser-induced mouse model of parafoveal GA progression, starting at 2 weeks post-laser and reaching confluence at 24 weeks post-laser. This 24-week time-frame in which GA pathology develops, provides an extended window of opportunity for proof-of-concept evaluation of drugs targeting GA. This time period is an added advantage compared to several existing models of geographic atrophy.

Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1.

BACKGROUND: T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases.  METHODS: In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-ß1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177. RESULTS: We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-ß1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death. CONCLUSION: This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.

Characterization of age-related macular degeneration in Indian donor eyes.

Purpose: The purpose of this study was to test the reliability of fundus stereomicroscopy in postmortem eyes to assign severity of age-related macular degeneration (AMD) using the Minnesota grading and confirmation by histology using Alabama and Sarks grading scales and to assess the incidence of AMD pathology in donor eyes from a South Indian population. Methods: Eyes (199) from 153 donors (55-95 years) after obtaining fundus images were processed for histology. Fundus images were graded according to the Minnesota grading system based on drusen size, area of depigmentation, and atrophy. At least one eye from each donor displaying the AMD phenotypes were subjected to histological examination. The fundus grading was correlated with histology and the stages of AMD assigned for early AMD by the Alabama AMD grading system and for both early and advanced AMD by the Sarks classification. Results: Stereoscopic examination of the fundus found that 10 of the 153 donors had features of early AMD and 3 advanced AMD. Following histological examination, one of the early AMD eyes was reclassified as advanced AMD. Early AMD features that were observed on histology included soft drusen (>63 µm), basal laminar deposits, photoreceptor outer segment degeneration, disorganization of retinal pigment epithelium (RPE), Bruch's membrane thickening. Advanced AMD features observed in histology are extensive atrophy of RPE, choroidal neovascularization and disciform scar formation. . Conclusion: Identification of either early or advanced AMD using stereomicroscopic assessment (SMA) showed high sensitivity and specificity. However, misclassification between AMD stages can occur when only SMA is used.

Progress in developing rodent models of age-related macular degeneration (AMD).

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss, typically affecting individuals from mid-life onwards. Its multifactorial aetiology and the lack of any effective treatments has spurred the development of animal models as research and drug discovery tools. Several rodent models have been developed which recapitulate key features of AMD and provide insights into its underlying pathology. These have contributed to making significant progress in understanding the disease and the identification of novel therapeutic targets. However, a major caveat with existing models is that they do not demonstrate the full disease spectrum. In this review, we outline advances in rodent AMD models from the last decade. These models feature various hallmarks associated with AMD, including oxidative stress, hypoxia, immune dysregulation, genetic mutations and environmental risk factors. The review summarises the methods by which each model was created, its pathological characteristics as well as its relation to the disease in humans.

Clinical application of non-coding RNAs in sepsis.

PURPOSE OF REVIEW: Studies indicating that non-coding RNAs (ncRNAs) play a regulatory role in sepsis are increasing rapidly. This present review summarizes recent publications on the role of microRNAs and long non-coding RNAs (lncRNAs) in sepsis. RECENT FINDINGS: MicroRNAs (miRNAs) and lncRNAs are being identified as potential sepsis biomarkers and therapeutic targets. Experimental studies have examined the biological mechanisms that might underpin the regulatory role of these ncRNAs in sepsis. SUMMARY: Clinical applications of miRNAs and lncRNAs in sepsis are on the horizon. These data could lead to the identification of novel treatments or indeed support the repurposing of existing drugs for sepsis. Validation of the findings from these preliminary studies and crucially integration of multiomics datasets will undoubtedly revolutionize the clinical management of sepsis.

Cystic fibrosis epithelial cells are primed for apoptosis as a result of increased Fas (CD95).

BACKGROUND: Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells. METHODS: Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines. RESULTS: Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. CONCLUSIONS: Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR.

Extremely complex populations of small RNAs in the mouse retina and RPE/choroid.

PURPOSE: MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 22 nucleotides in length that regulate gene expression. They are widely expressed in the retina, being both required for its normal development and perturbed in disease. The aim of this study was to apply new high-throughput sequencing techniques to more fully characterize the miRNAs and other small RNAs expressed in the retina and retinal pigment epithelium (RPE)/choroid of the mouse. METHODS: Retina and RPE/choroid were dissected from eyes of 3-month-old C57BL/6J mice. Small RNA libraries were prepared and deep sequencing performed on a genome analyzer. Reads were annotated by alignment to miRBase, other noncoding RNA databases, and the mouse genome. RESULTS: Annotation of 9 million reads to 320 miRNAs in retina and 340 in RPE/choroid provides the most comprehensive profiling of miRNAs to date. Two novel miRNAs were identified in retina. Members of the sensory organ-specific miR-183, -182, -96 cluster were among the most highly expressed, retina-enriched miRNAs. Remarkably, miRNA "isomiRs," which vary slightly in length and are differentially detected by Taqman RT-qPCR assays, existed for all the microRNAs identified in both tissues. More variation occurred at the 3' ends, including nontemplated additions of T and A. Drosha-independent mirtron miRNAs and other small RNAs derived from snoRNAs were also detected. CONCLUSIONS: Deep sequencing has revealed the complexity of small RNA expression in the mouse retina and RPE/choroid. This knowledge will improve the design and interpretation of future functional studies of the role of miRNAs and other small RNAs in retinal disease.

Para-inflammation-mediated retinal recruitment of bone marrow-derived myeloid cells following whole-body irradiation is CCL2 dependent.

Previous studies have shown that following whole-body irradiation bone marrow (BM)-derived cells can migrate into the central nervous system, including the retina, to give rise to microglia-like cells. The detailed mechanism, however, remains elusive. We show in this study that a single-dose whole-body γ-ray irradiation (8 Gy) induced subclinical damage (i.e., DNA damage) in the neuronal retina, which is accompanied by a low-grade chronic inflammation, para-inflammation, characterized by upregulated expression of chemokines (CCL2, CXCL12, and CX3CL1) and complement components (C4 and CFH), and microglial activation. The upregulation of chemokines CCL2 and CXCL12 and complement C4 lasted for more than 160 days, whereas the expression of CX3CL1 and CFH was upregulated for 2 weeks. Both resident microglia and BM-derived phagocytes displayed mild activation in the neuronal retina following irradiation. When BM cells from CX3CR1(gfp/+) mice or CX3CR1(gfp/gfp) mice were transplanted to wild-type C57BL/6 mice, more than 90% of resident CD11b(+) cells were replaced by donor-derived GFP(+) cells after 6 months. However, when transplanting CX3CR1(gfp/+) BM cells into CCL2-deficient mice, only 20% of retinal CD11b(+) cells were replaced by donor-derived cells at 6 month. Our results suggest that the neuronal retina suffers from a chronic stress following whole-body irradiation, and a para-inflammatory response is initiated, presumably to rectify the insults and maintain homeostasis. The recruitment of BM-derived myeloid cells is a part of the para-inflammatory response and is CCL2 but not CX3CL1 dependent.