Ocular delivery of Pigment Epithelium-Derived Factor (PEDF) as a neuroprotectant for Geographic Atrophy.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), that starts with atrophic lesions in the outer retina that expand to cover the macula and fovea, leading to severe vision loss over time. Pigment Epithelium-Derived Factor (PEDF) has a diverse-range of properties, including its ability to promote cell survival, reduce inflammation, inhibit angiogenesis, combat oxidative stress, regulate autophagy, and stimulate anti-apoptotic pathways, making it a promising therapeutic candidate for GA. However, the relatively short half-life of PEDF protein has precluded its potential as a clinical therapy for GA since it would require frequent injections. Therefore, we describe administration of a PEDF gene, comparing and contrasting delivery routes, viral and non-viral vectors, and consider the critical challenges for PEDF as a neuroprotectant for GA.

Inverted repeats in the monkeypox virus genome are hot spots for mutation.

The current monkeypox virus (MPXV) strain differs from the strain arising in 2018 by 50+ single nucleotide polymorphisms (SNPs) and is mutating much faster than expected. The cytidine deaminase apolipoprotein B messenger RNA editing enzyme, catalytic subunit B (APOBEC3) was hypothesized to be driving this increased mutation. APOBEC has recently been identified to preferentially mutate cruciform DNA secondary structures formed by inverted repeats (IRs). IRs were recently identified as hot spots for mutation in severe acute respiratory syndrome coronavirus 2, and we aimed to identify whether IRs were also hot spots for mutation within MPXV genomes. We found that MPXV genomes were replete with IR sequences. Of the 50+ SNPs identified in the 2022 outbreak strain, 63.9% of these were found to have arisen within IR regions in the 2018 reference strain (MT903344.1). Notably, IR sequences found in the 2018 reference strain were significantly lost over time, with an average of 32.5% of these sequences being conserved in the 2022 MPXV genomes. This evidence was highly indicative that mutations were arising within IRs. This data provides further support to the hypothesis that APOBEC may be driving MPXV mutation and highlights the necessity for greater surveillance of IRs of MPXV genomes to detect new mutations.

Targeting Microtubules for the Treatment of Heart Disease.

Heart disease remains the leading cause of morbidity and mortality worldwide. With the advancement of modern technology, the role(s) of microtubules in the pathogenesis of heart disease has become increasingly apparent, though currently there are limited treatments targeting microtubule-relevant mechanisms. Here, we review the functions of microtubules in the cardiovascular system and their specific adaptive and pathological phenotypes in cardiac disorders. We further explore the use of microtubule-targeting drugs and highlight promising druggable therapeutic targets for the future treatment of heart diseases.

Analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets?

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.

Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress.

BACKGROUND: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. MATERIALS AND METHODS: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. RESULTS: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. CONCLUSION: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation.

Cardiovascular Mechanisms of Action of Anthocyanins May Be Associated with the Impact of Microbial Metabolites on Heme Oxygenase-1 in Vascular Smooth Muscle Cells.

Anthocyanins are reported to have cardio-protective effects, although their mechanisms of action remain elusive. We aimed to explore the effects of microbial metabolites common to anthocyanins and other flavonoids on vascular smooth muscle heme oxygenase-1 (HO-1) expression. Thirteen phenolic metabolites identified by previous anthocyanin human feeding studies, as well as 28 unique mixtures of metabolites and their known precursor structures were explored for their activity on HO-1 protein expression in rat aortic smooth muscle cells (RASMCs). No phenolic metabolites were active when treated in isolation; however, five mixtures of phenolic metabolites significantly increased HO-1 protein expression (127.4-116.6%, p ≤ 0.03). The present study demonstrates that phenolic metabolites of anthocyanins differentially affect HO-1 activity, often having additive, synergistic or nullifying effects.

Signatures of anthocyanin metabolites identified in humans inhibit biomarkers of vascular inflammation in human endothelial cells.

SCOPE: The physiological relevance of contemporary cell culture studies is often perplexing, given the use of unmetabolized phytochemicals at supraphysiological concentrations. We investigated the activity of physiologically relevant anthocyanin metabolite signatures, derived from a previous pharmacokinetics study of 500 mg 13 C5 -cyanidin-3-glucoside in eight healthy participants, on soluble vascular adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6) in human endothelial cells. METHODS AND RESULTS: Signatures of peak metabolites (previously identified at 1, 6, and 24 h post-bolus) were reproduced using pure standards and effects were investigated across concentrations ten-fold lower and higher than observed mean (<5 µM) serum levels. Tumor necrosis factor-α (TNF-α)-stimulated VCAM-1 was reduced in response to all treatments, with maximal effects observed for the 6 and 24 h profiles. Profiles tested at ten-fold below mean serum concentrations (0.19-0.44 µM) remained active. IL-6 was reduced in response to 1, 6, and 24 h profiles, with maximal effects observed for 6 h and 24 h profiles at concentrations above 2 µM. Protein responses were reflected by reductions in VCAM-1 and IL-6 mRNA, however there was no effect on phosphorylated NFκB-p65 expression. CONCLUSION: Signatures of anthocyanin metabolites following dietary consumption reduce VCAM-1 and IL-6 production, providing evidence of physiologically relevant biological activity.

Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells.

BACKGROUND: Flavonoids have been implicated in the prevention of cardiovascular disease; however, their mechanisms of action have yet to be elucidated, possibly because most previous in vitro studies have used supraphysiological concentrations of unmetabolized flavonoids, overlooking their more bioavailable phenolic metabolites. OBJECTIVE: We aimed to explore the effects of phenolic metabolites and their precursor flavonoids at physiologically achievable concentrations, in isolation and combination, on soluble vascular cellular adhesion molecule-1 (sVCAM-1). METHOD: Fourteen phenolic acid metabolites and 6 flavonoids were screened at 1 µM for their relative effects on sVCAM-1 secretion by human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha (TNF-α). The active metabolites were further studied for their response at different concentrations (0.01 µM-100 µM), structure-activity relationships, and effect on vascular cellular adhesion molecule (VCAM)-1 mRNA expression. In addition, the additive activity of the metabolites and flavonoids was investigated by screening 25 unique mixtures at cumulative equimolar concentrations of 1 µM. RESULTS: Of the 20 compounds screened at 1 µM, inhibition of sVCAM-1 secretion was elicited by 4 phenolic metabolites, of which protocatechuic acid (PCA) was the most active (-17.2%, P = 0.05). Investigations into their responses at different concentrations showed that PCA significantly reduced sVCAM-1 15.2-36.5% between 1 and 100 µM, protocatechuic acid-3-sulfate and isovanillic acid reduced sVCAM-1 levels 12.2-54.7% between 10 and 100 µM, and protocatechuic acid-4-sulfate and isovanillic acid-3-glucuronide reduced sVCAM-1 secretion 27.6% and 42.8%, respectively, only at 100 µM. PCA demonstrated the strongest protein response and was therefore explored for its effect on VCAM-1 mRNA, where 78.4% inhibition was observed only after treatment with 100 µM PCA. Mixtures of the metabolites showed no activity toward sVCAM-1, suggesting no additive activity at 1 µM. CONCLUSIONS: The present findings suggest that metabolism of flavonoids increases their vascular efficacy, resulting in a diversity of structures of varying bioactivity in human endothelial cells.