Current understanding of subclinical diabetic retinopathy informed by histology and high-resolution in vivo imaging.

The escalating incidence of diabetes mellitus has amplified the global impact of diabetic retinopathy. There are known structural and functional changes in the diabetic retina that precede the fundus photography abnormalities which currently are used to diagnose clinical diabetic retinopathy. Understanding these subclinical alterations is important for effective disease management. Histology and high-resolution clinical imaging reveal that the entire neurovascular unit, comprised of retinal vasculature, neurons and glial cells, is affected in subclinical disease. Early functional manifestations are seen in the form of blood flow and electroretinography disturbances. Structurally, there are alterations in the cellular components of vasculature, glia and the neuronal network. On clinical imaging, changes to vessel density and thickness of neuronal layers are observed. How these subclinical disturbances interact and ultimately manifest as clinical disease remains elusive. However, this knowledge reveals potential early therapeutic targets and the need for imaging modalities that can detect subclinical changes in a clinical setting.

Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo.

INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Variability in Capillary Perfusion Is Increased in Regions of Retinal Ischemia Due to Branch Retinal Vein Occlusion.

Purpose: To investigate alterations in macular perfusion variability due to branch retinal vein occlusion (BRVO) using a novel approach based on optical coherence tomography angiography (OCTA) coefficient of variation (CoV) analysis. Methods: Thirteen eyes of 13 patients with macular ischemia due to BRVO were studied. Multiple consecutive en face OCTA images were acquired. Bias field correction, spatial alignment, and normalization of intensities across the images were performed followed by pixelwise computation of standard deviation divided by the mean to generate a CoV map. Region of interest-based CoV values, derived from this map, for arterioles, venules, and the microvasculature were compared between regions with macular ischemia and control areas of the same eye. Control areas were regions of the same macula that were not affected by the BRVO and had normal retinal vascular structure as seen on multimodal imaging and normal retinal vascular density measurements as quantified using OCTA. Results: CoV increased by a mean value of 17.6% within the microvasculature of ischemic regions compared to the control microvasculature (P < 0.0001). CoV measurements of microvasculature were consistently greater in the ischemic area of all 13 eyes compared to control. There were no differences in CoV measurements between ischemic and control areas for arterioles (P = 0.13) and venules (P = 1.0). Conclusions: Greater variability in microvasculature perfusion occurs at sites of macular ischemia due to BRVO. We report a novel way for quantifying macular perfusion variability using OCTA. This technique may have applicability for studying the pathophysiology of other retinal vascular diseases.

Studies of the retinal microcirculation using human donor eyes and high-resolution clinical imaging: Insights gained to guide future research in diabetic retinopathy.

The microcirculation plays a key role in delivering oxygen to and removing metabolic wastes from energy-intensive retinal neurons. Microvascular changes are a hallmark feature of diabetic retinopathy (DR), a major cause of irreversible vision loss globally. Early investigators have performed landmark studies characterising the pathologic manifestations of DR. Previous works have collectively informed us of the clinical stages of DR and the retinal manifestations associated with devastating vision loss. Since these reports, major advancements in histologic techniques coupled with three-dimensional image processing has facilitated a deeper understanding of the structural characteristics in the healthy and diseased retinal circulation. Furthermore, breakthroughs in high-resolution retinal imaging have facilitated clinical translation of histologic knowledge to detect and monitor progression of microcirculatory disturbances with greater precision. Isolated perfusion techniques have been applied to human donor eyes to further our understanding of the cytoarchitectural characteristics of the normal human retinal circulation as well as provide novel insights into the pathophysiology of DR. Histology has been used to validate emerging in vivo retinal imaging techniques such as optical coherence tomography angiography. This report provides an overview of our research on the human retinal microcirculation in the context of the current ophthalmic literature. We commence by proposing a standardised histologic lexicon for characterising the human retinal microcirculation and subsequently discuss the pathophysiologic mechanisms underlying key manifestations of DR, with a focus on microaneurysms and retinal ischaemia. The advantages and limitations of current retinal imaging modalities as determined using histologic validation are also presented. We conclude with an overview of the implications of our research and provide a perspective on future directions in DR research.

Angiographic biomarkers are significant predictors of treatment response to intravitreal aflibercept in diabetic macular edema.

This prospective single-center study aims to identify biomarkers that predict improvement in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) at 6 months, in 76 eyes with diabetic macular edema (DME) treated monthly with intravitreal aflibercept. At baseline, all patients underwent standardized imaging with color photography, optical coherence tomography (OCT), fluorescein angiography (FA) and OCT angiography (OCTA). Glycosylated hemoglobin, renal function, dyslipidemia, hypertension, cardiovascular disease and smoking were recorded. Retinal images were graded in a masked fashion. Baseline imaging, systemic and demographic variables were investigated to detect associations to BCVA and CRT change post aflibercept. Predictors of BCVA improvement included greater macular vessel density quantified using OCTA (p = 0.001) and low-density lipoprotein (LDL) ≥ 2.6 mmol/L (p = 0.017). Lower macular vessel density eyes showed a significant reduction in CRT but no BCVA improvement. Predictors of CRT reduction included peripheral non-perfusion seen on ultrawide-field FA (p = 0.005) and LDL ≥ 2.6 mmol/L (p < 0.001). Retinal angiographic biomarkers derived from OCTA and ultrawide-field FA may help predict functional and anatomic response to anti-vascular endothelial growth factor (VEGF) therapy in patients with DME. Elevated LDL is associated with treatment response in DME. These results may be used to better-select patients who will benefit from intravitreal aflibercept for treatment of DME.

An assessment of microvascular hemodynamics in human macula.

An adequate blood supply to meet the energy demands is essential for any tissue, particularly for high energy demand tissues such as the retina. A critical question is: How is the dynamic match between neuronal demands and blood supply achieved? We present a quantitative assessment of temporal and spatial variations in perfusion in the macular capillary network in 10 healthy human subjects using a non-invasive and label-free imaging technique. The assessment is based on the calculation of the coefficient of variation (CoV) of the perfusion signal from arterioles, venules and capillaries from a sequence of optical coherence tomography angiography images centred on the fovea. Significant heterogeneity of the spatial and temporal variation was found within arterioles, venules and capillary networks. The CoV values of the capillaries and smallest vessels were significantly higher than that in the larger vessels. Our results demonstrate the presence of significant heterogeneity of spatial and temporal variation within each element of the macular microvasculature, particularly in the capillaries and finer vessels. Our findings suggest that the dynamic match between neuronal demands and blood supply is achieved by frequent alteration of local blood flow evidenced by capillary perfusion variations both spatially and temporally in the macular region.

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved ß-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

Frail patients having vascular surgery during the early COVID-19 pandemic experienced high rates of adverse perioperative events and amputation.

BACKGROUND: Frailty predicts adverse perioperative outcomes and increased mortality in patients having vascular surgery. Frailty assessment is a potential tool to inform resource allocation, and shared decision-making about vascular surgery in the resource constrained COVID-19 pandemic environment. This cohort study describes the prevalence of frailty in patients having vascular surgery and the association between frailty, mortality and perioperative outcomes. METHODS: The COVID-19 Vascular Service in Australia (COVER-AU) prospective cohort study evaluates 30-day and six-month outcomes for consecutive patients having vascular surgery in 11 Australian vascular units, March-July 2020. The primary outcome was mortality, with secondary outcomes procedure-related outcomes and hospital utilization. Frailty was assessed using the nine-point visual Clinical Frailty Score, scores of 5 or more considered frail. RESULTS: Of the 917 patients enrolled, 203 were frail (22.1%). The 30 day and 6 month mortality was 2.0% (n = 20) and 5.9% (n = 35) respectively with no significant difference between frail and non-frail patients (OR 1.68, 95%CI 0.79-3.54). However, frail patients stayed longer in hospital, had more perioperative complications, and were more likely to be readmitted or have a reoperation when compared to non-frail patients. At 6 months, frail patients had twice the odds of major amputation compared to non-frail patients, after adjustment (OR 2.01; 95% CI 1.17-3.78), driven by a high rate of amputation during the period of reduced surgical activity. CONCLUSION: Our findings highlight that older, frail patients, experience potentially preventable adverse outcomes and there is a need for targeted interventions to optimize care, especially in times of healthcare stress.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Synergistic effect of plasma-activated medium and novel indirubin derivatives on human skin cancer cells by activation of the AhR pathway.

Due to the increasing number of human skin cancers and the limited effectiveness of therapies, research into innovative therapeutic approaches is of enormous clinical interest. In recent years, the use of cold atmospheric pressure plasma has become increasingly important as anti-cancer therapy. The combination of plasma with small molecules offers the potential of an effective, tumour-specific, targeted therapy. The synthesised glycosylated and non glycosylated thia-analogous indirubin derivatives KD87 and KD88, respectively, were first to be investigated for their pharmaceutical efficacy in comparison with Indirubin-3'-monoxime (I3M) on human melanoma (A375) and squamous cell carcinoma (A431) cells. In combinatorial studies with plasma-activated medium (PAM) and KD87 we determined significantly decreased cell viability and cell adhesion. Cell cycle analyses revealed a marked G2/M arrest by PAM and a clear apoptotic effect by the glycosylated indirubin derivative KD87 in both cell lines and thus a synergistic anti-cancer effect. I3M had a pro-apoptotic effect only in A431 cells, so we hypothesize a different mode of action of the indirubin derivatives in the two skin cancer cells, possibly due to a different level of the aryl hydrocarbon receptor and an activation of this pathway by nuclear translocation of this receptor and subsequent activation of gene expression.

Small Molecules in the Treatment of Squamous Cell Carcinomas: Focus on Indirubins.

Skin cancers are the most common malignancies in the world. Among the most frequent skin cancer entities, squamous cell carcinoma (SCC) ranks second (~20%) after basal cell carcinoma (~77%). In early stages, a complete surgical removal of the affected tissue is carried out as standard therapy. To treat advanced and metastatic cancers, targeted therapies with small molecule inhibitors are gaining increasing attention. Small molecules are a heterogeneous group of protein regulators, which are produced by chemical synthesis or fermentation. The majority of them belong to the group of receptor tyrosine kinase inhibitors (RTKIs), which specifically bind to certain RTKs and directly influence the respective signaling pathway. Knowledge of characteristic molecular alterations in certain cancer entities, such as SCC, can help identify tumor-specific substances for targeted therapies. Most frequently, altered genes in SCC include TP53, NOTCH, EGFR, and CCND1. For example, the gene CCND1, which codes for cyclin D1 protein, is upregulated in nearly half of SCC cases and promotes proliferation of affected cells. A treatment with the small molecule 5'-nitroindirubin-monoxime (INO) leads to inhibition of cyclin D1 and thus inhibition of proliferation. As a component of Danggui Longhui Wan, a traditional Chinese medicine, indirubins are used to treat chronic diseases and have been shown to inhibit inflammatory reactions. Indirubins are pharmacologically relevant small molecules with proapoptotic and antiproliferative activity. In this review, we discuss the current literature on indirubin-based small molecules in cancer treatment. A special focus is on the molecular biology of squamous cell carcinomas, their alterations, and how these are rendered susceptible to indirubin-based small molecule inhibitors. The potential molecular mechanisms of the efficacy of indirubins in killing SCC cells will be discussed as well.

Colour-Value Based Method for Polydopamine Coating-Stability Characterization on Polyethersulfone Membranes.

Porous polyethersulfone membranes as used in oenology were investigated in order to evaluate temperature-dependent permeances in a temperature range from 10 to 35 °C. A temperature correction factor was determined for this type of membrane to get accurate and comparable results for further developments. Moreover, the membranes were modified with a bio-inspired polydopamine coating in order to reduce fouling. The performance of the membranes could be increased with respect to permeance and flux recovery under cross-flow conditions. In order to test the applicability and stability of the coating layer, they were treated with basic and acidic cleaning agents as used in industry for fouled membranes. The chemical stability of the coating layer was studied under basic and acidic conditions, by systematic observation of the colour change of the coated membranes over treatment time.

Synthesis and antiproliferative activity of selenoindirubins and selenoindirubin-N-glycosides.

Selenoindirubins and selenoindirubin-N-glycosides were prepared by the reaction of isatins and isatin-N-glycosides with 3-acetoxy-benzo[b]selenophene, respectively. While selenoindirubin-N-glycosides have not been reported before, three non-glycosylated selenoindirubins were previously reported, but without quantities, yields, scales, experimental details and spectroscopic data. In addition, the work could, in our hands, not be reproduced to prepare pure products. The present paper includes an optimized procedure for the synthesis of selenoindirubins and their complete characterization. Both selenoindirubins and selenoindirubin-N-glycosides showed antiproliferative activity in lung cancer cell lines. In melanoma cells, antiproliferative effects were further accompanied by induced apoptosis in combination with the death ligand TRAIL.

Synthesis of N,N'-diglycosylated isoindigos.

The first N,N'-diglycosylated isoindigos were prepared by P(NEt(2))(3)-mediated dimerization of acetyl protected N-glycosyl-5-alkylisatins. Subsequent deacetylation yielded the corresponding deprotected N,N'-diglycosylated isoindigos. Isatine-N-glycosides containing an unsubstituted or alkyl-substituted heterocyclic moiety and ß-D-manno- and ß-L-rhamno configured carbohydrate substituents were successfully transformed into the corresponding isoindigos. In contrast, isatins with ß-D-gluco- or ß-D-xylo configured substituents failed to give the desired products.

Methyl 4',5-dichloro-2-hy-droxy-4,6-dimethyl-biphenyl-3-carboxyl-ate.

In the title compound, C(16)H(14)Cl(2)O(3), the dihedral angle between the mean planes of the two benzene rings is 55.30 (5)°. The methyl ester group lies within the ring plane due to an intra-molecular O-H⋯O hydrogen bond [maximum deviation from the C(8)O(2) mean plane is 0.0383 (13) Å]. In the crystal, mol-ecules are held together by rather weak C-H⋯O hydrogen bonds.

Synthesis and characterization of bis-cyclopropanated 1,3,5-tricarbonyl compounds. A combined synthetic, spectroscopic and theoretical study.

Bis-cyclopropanated 1,3,5-tricarbonyl compounds were prepared by a sequence of Claisen condensations and cyclopropanations. The optimization of the conditions proved to be very important to suppress retro-Claisen reactions. The conformation of these molecules was studied by experimental and computational methods. The syn/syn;syn/syn conformation is present for all derivatives. It is exclusively present in the case of the derivative containing a phenyl group located at the terminal carbon atom. In most cases, equilibria with other conformers are found.

Sensitization of melanoma cells for death ligand-induced apoptosis by an indirubin derivative--Enhancement of both extrinsic and intrinsic apoptosis pathways.

Until today effective therapies are lacking for metastatic melanoma. The death ligand TRAIL appears as promising in cancer treatment; however, melanoma cells reveal both preexisting and inducible TRAIL resistance. Here, we present evidence that the recently described indirubin derivative 8-Rha-ß enhances melanoma cell sensitivity for death ligands and overcomes resistance to TRAIL and CD95 agonists. Indirubin is known from traditional Chinese medicine and is a potent kinase inhibitor. Unraveling of apoptotic signaling pathways revealed that TRAIL resulted in a quick (within 8h) downregulation of both agonistic TRAIL receptors DR4 and DR5, in a kind of negative feed-back loop. Treatment with indirubin, however, mediated upregulation of both receptors, thus compensating this negative feed-back loop by TRAIL. Furthermore, indirubin activated intrinsic apoptosis pathways, seen in loss of mitochondrial membrane potential and release of cytochrome c. The mitochondrial response appeared as related to upregulation of Bax and Bad and to downregulation of Mcl-1. Remarkably, indirubin in combination with TRAIL was also able to overcome apoptosis resistance due to ectopic Bcl-2 overexpression. The tumor suppressor p53 appeared as master regulator of these propapoptotic changes and is the transactivator of proapoptotic proteins which was upregulated by indirubin. Taking into account the physiological role of death ligands in immune surveillance, sensitization of melanoma cells for death ligands may be supportive for an anti-tumor immune response. Furthermore, combinations with kinase inhibitors, such as indirubin 8-Rha-ß may help for a breakthrough of TRAIL-mediated strategies in melanoma.