Physiological and pharmacological stimulation for in vitro maturation of substrate metabolism in human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.

Adjunct use of optical coherence tomography increases the detection of recurrent basal cell carcinoma over clinical and dermoscopic examination alone.

BACKGROUND: Basal cell carcinoma (BCC) is a locally destructive form of skin cancer, mainly affecting Caucasians. In the last few years non-surgical treatments of BCC have become widely used and non-invasive methods for treatment monitoring and follow-up are therefore becoming increasingly warranted. The objective of this study was to investigate the utility of adjunct use of non-invasive optical coherence tomography (OCT) imaging for the detection of recurrent BCC over clinical and dermoscopic examination alone, in a real-world setting. METHODS: A total of 58 patients, previously treated with curettage and/or MAL-PDT for BCC, were included in the study. The patients were examined clinically and dermoscopically for recurrence by a dermatologist before joining the study. The included patients were then OCT scanned and if the OCT images raised suspicion of recurrent BCC the area was biopsied. RESULTS: In 6 cases the clinical examination revealed suspicions recurrent lesions and OCT correctly identified all of these (6/6). In 49 cases the follow-up examinations showed no clinical or dermoscopic signs of recurrence, but in 12.2% (6/49) of these cases the subsequent OCT examination revealed a subclinical recurrent BCC lesion. These were all confirmed by histology. In 3 cases both the clinical and the OCT diagnosis was unclear and recurrent BCC could not be ruled out, but histology showed no sign of malignancy. CONCLUSIONS: These results suggest that the adjunct use of OCT increases the detection rate of recurrent BCC over clinical/dermoscopy examination alone.

A case report of differentiating staphylococcal scalded skin syndrome and toxic epidermal necrolysis by optical coherence tomography.

BACKGROUND: Staphylococcal scalded skin syndrome (SSSS) and toxic epidermal necrolysis (TEN) both present with acute onset, high morbidity and significant mortality. Rapid diagnosis is therefore of importance. The aim of this study was to investigate and compare the presentation of these diseases using optical coherence tomography (OCT). METHODS: Two male patients with bullous diseases, SSSS and TEN, respectively, were photographed digitally, examined using dermoscopy, OCT scanned and subsequently biopsied in the said order. RESULTS: The bullous skin was visualized by OCT showing two distinct images: the SSSS-patient displayed superficial hyporefletive flaccid structures with a split high in the thickened (0.51 mm vs. 0.12 mm) epidermis while the TEN-patient demonstrated a larger hyporeflective ovoid structure with a split right below the thickened epidermis (0.18 mm vs. 0.06 mm). CONCLUSION: These findings suggest that there is a potential for the application of OCT scanning in the acute phase of SSSS and TEN in order to distinguish them for a faster diagnosis and better management and treatment.

Imaging of collagen deposition disorders using optical coherence tomography.

BACKGROUND: Collagen deposition disorders such as hypertrophic scars, keloids and scleroderma can be associated with significant stigma and embarrassment. These disorders often constitute considerable impairment to quality of life, with treatment posing to be a substantial challenge. Optical coherence tomography (OCT) provides a non-invasive, easily applicable bedside optical imaging method for assessment of the skin. It is hypothesized that OCT imaging may be useful in assessing fibrosis to avoid additional biopsies that could potentially worsen the scarring. METHOD: Thirty-three patients with ordinary scars, hypertrophic scars, keloid scarring, lichen sclerosus et atrophicus and localized or systemic scleroderma were recruited for this pilot study. Affected tissue and adjacent healthy skin were scanned using OCT and digitally photographed. Density measurements were performed in ImageJ on OCT images from scleroderma patients, both systemic and morphea (10 patients), keloid patients (10 patients) and healthy skin adjacent to keloids (10 patients). RESULTS: OCT images of scarring diseases showed varying degrees of disruption to the skin architecture. OCT characteristics were identified for each lesion type. Hypertrophic scars displayed an increased vascularity and signal-rich bands correlating to excessive collagen deposition. Keloids depicted a disarray of hyper-reflective areas primarily located in the upper dermis. Additionally, the dermis displayed a heterogeneous morphology without indications of any vascular supply or lymphatic network. In contrast to keloids, scleroderma displayed a more cohesive backscattering indicating a difference in density of collagen or other dermal structures. OCT images demonstrated no significant differences between mean density measurements in OCT images of scleroderma, keloid and healthy skin (P = 0.07). CONCLUSION: The OCT imaging appears to identify different scarring mechanisms, and therefore be of potential use in the assessment of outcomes following non-invasive therapy of e.g. early or progressive lesions.

Fluorescent prey traps in carnivorous plants.

Carnivorous plants acquire most of their nutrients by capturing ants, insects and other arthropods through their leaf-evolved biological traps. So far, the best-known attractants in carnivorous prey traps are nectar, colour and olfactory cues. Here, fresh prey traps of 14 Nepenthes, five Sarracenia, five Drosera, two Pinguicula species/hybrids, Dionaea muscipula and Utricularia stellaris were scanned at UV 366 nm. Fluorescence emissions of major isolates of fresh Nepenthes khasiana pitcher peristomes were recorded at an excitation wavelength of 366 nm. N. khasiana field pitcher peristomes were masked by its slippery zone extract, and prey capture rates were compared with control pitchers. We found the existence of distinct blue fluorescence emissions at the capture spots of Nepenthes, Sarracenia and Dionaea prey traps at UV 366 nm. These alluring blue emissions gradually developed with the growth of the prey traps and diminished towards their death. On excitation at 366 nm, N. khasiana peristome 3:1 CHCl3­MeOH extract and its two major blue bands showed strong fluorescence emissions at 430­480 nm. Masking of blue emissions on peristomes drastically reduced prey capture in N. khasiana pitchers. We propose these molecular emissions as a critical factor attracting arthropods and other visitors to these carnivorous traps. Drosera, Pinguicula and Utricularia prey traps showed only red chlorophyll emissions at 366 nm.

Vectorial release of matrix metalloproteinases (MMPs) from porcine RPE-choroid explants following selective retina therapy (SRT): towards slowing the macular ageing process.

The purpose of this study was to investigate release of matrix metalloproteinases (MMP) 2 and 9 during retinal pigment epithelium (RPE) wound healing after Selective Retina Therapy (SRT) with laser energy levels below and above the threshold of RPE cell death. Following exposure to SRT using a prototype pulsed Nd:YLF laser with energies of 80-180 mJ/cm(2) fresh porcine RPE-monolayers with Bruch's membrane and choroid were cultured in modified Ussing chambers which separate the apical (RPE-facing) and basal (choroid facing) sides of the RPE monolayer. Threshold energy for RPE cell death and wound healing were determined with calcein-AM viability test. Inactive and active forms of MMP 2 and 9 were quantified within tissue samples and in the culture medium of the apical and basal compartments of the Ussing chamber using gelatine zymography. Laser energies of 160-180 mJ/cm(2) resulted in cell death within 1 h while 120-140 mJ/cm(2) resulted in delayed death of exposed RPE cells. All cells survived 80 and 100 mJ/cm(2). Laser spots healed within 6 days after SRT accompanied by a transient vectorial increase of MMPs. SRT with 180 mJ/cm(2) increased active MMP 2 by 1.9 (p < 0.05) and 1.6 (p < 0.05) fold in tissue and basal compartments, respectively, without alterations in the apical compartment. Pro-MMP 2 levels were also significantly increased in all compartments (p < 0.05). Release of MMP 9 was not altered. Laser energy below the threshold of RPE cell death did not alter the release of MMP 2 or 9. The findings suggest that the release of active MMP 2 on the basal side of the RPE during wound healing following SRT may address age-related pathological changes of Bruch's membrane with a potential to slow degenerative macular ageing processes before irreversible functional loss has occurred.

Macromolecular diffusion characteristics of ageing human Bruch's membrane: implications for age-related macular degeneration (AMD).

Macromolecular species such as retinal binding protein, transferrin, ceruloplasmin, etc., released by the fenestrated choroidal capillaries must diffuse across Bruch's membrane for interaction with the basal membranes of the retinal pigment epithelium (RPE) for delivery of essential metabolites to the neural retina. The patency of this pathway through ageing Bruch's was examined by quantifying the diffusional flux of a 21.2 kDa fluorescein-isothiocyanate labelled dextran. Dextran flux measurements across Bruch's membrane from the macular region of the human fundus showed a highly significant decrease (p < 0.001) with ageing of donor such that diffusional transport in the ninth decade was about 6.5% of that in the first decade of life. Peripheral regions also showed a highly significant decline (p < 0.001) but ageing changes were considerably slowed in comparison to the macula with diffusional rates in the ninth decade being about 44% of that in the first decade. Peripheral samples from AMD donors displayed diffusional rates that were lower than the control population. The age-related decline in macromolecular diffusion across Bruch's membrane suggests that in the elderly, the patency of the conducting pathways may be compromised and in the more advanced ageing of Bruch's associated with AMD, the metabolic trafficking of carrier proteins may be severely impaired.

An experimental study of the elastic properties of the human Bruch's membrane-choroid complex: relevance to ageing.

AIM: To investigate the mechanical properties (stress-strain relation, elasticity, hysteresis, response to stress spikes and drops) of isolated human Bruch's membrane-choroid, as well as the effect of ageing and aged related macular degeneration (AMD). METHODS: 13 Bruch's membrane-choroid complexes were obtained from human donors (21-97 years). Two samples (aged 85 and 95) showed signs of AMD including large, soft drusen, choroidal neovascularisation, and/or disciform scars. Various hydrostatic pressures (stress) were applied to the choroidal surface of mid-peripheral samples mounted in a modified open Ussing chamber. Linear scans of the tissue were recorded by optical coherence tomography (OCT) and the pressure induced deformation (strain), elasticity, hysteresis, and response to pressure spikes and drops measured. RESULTS: The elasticity of human Bruch's membrane-choroid complex decreased linearly with ageing (p<0.001) after the age of 21 with an approximate reduction of 1% per year. The decrease was not exaggerated in AMD. The recoil capacity of Bruch's membrane-choroid was not affected by ageing. The response to pressure spikes/drops was similar in age matched normal and AMD eyes. The results suggest that although the aged induced decrease in Bruch's membrane elasticity may contribute to breaks in this membrane in AMD leading to neovascularisation this is not sufficient. The presence of other factors is required for its development. CONCLUSION: The elasticity of Bruch's membrane-choroid complex decreases with age while recoil capacity does not. The decrease was not exaggerated in AMD.

Baseline cardiac troponin I in patients on maintenance dialysis.

INTRODUCTION AND OBJECTIVES: There have been conflicting reports in the medical literature regarding baseline levels of cardiac troponins and their usefulness as specific markers of acute myocardial injury in patients on maintenance dialysis. Hence we undertook a study of the normal range of baseline cardiac troponin I (cTnI) in patients on maintenance dialysis in our center. METHODOLOGY: Seventy-five patients aged 45 years or more on maintenance dialysis for at least 1 month were included in the study. None had had an acute cardiac event requiring admission to the hospital during the preceding month. Samples of blood were collected and cTnI levels were estimated using an immunoenzymatic assay (Access Immunoassay system, Beckmann). Correlation of cTnI with gender, age, body mass index (BMI), and adequacy of dialysis (Kt/v) were also done. RESULTS: Forty-six (61.3%) patients were on maintenance hemodialysis and 29 (38.6%) were on peritoneal dialysis. Twenty-four (32%) were over the age of 65 years and 37 (49.3%) were men. Levels of cTnI ranged from 0.000 to 0.568 ng/mL, with mean of 0.040 +/- 0.080. Only six (8%) had levels above 0.1 ng/mL, which was the cut off level for significance in our laboratory. There was no statistically significant correlation of cTnI levels with gender, age, BMI, or Kt/v. CONCLUSIONS: Baseline cTnI levels were below significant levels in the majority of maintenance dialysis patients aged at least 45 years or more. Hence cTnI can be used as a valid marker of acute myocardial injury in this group of patients.

Hydraulic conductivity of fixed retinal tissue after sequential excimer laser ablation: barriers limiting fluid distribution and implications for cystoid macular edema.

OBJECTIVES: To measure the hydraulic conductivity (HC) of human retina and to determine the presence and location of high-resistance barriers to fluid movement through the retina. METHODS: Forty-one pairs of human eyes were investigated using an HC chamber. Once baseline HC had been determined, the effect of ablating through varying thickness of retina from the vitreous or photoreceptor surface using an excimer laser (193 nm) was investigated. Tissue samples were then processed for histological investigation. RESULTS: The HC of fixed intact human retina was 2.54 x 10(-10) m/s per pascal at 539 Pa (range, 0.6 x 10(-10) to 3.3 x 10(-10) m/s per pascal; SD, 0.6 x 10(-10) m/s per pascal [1 mm Hg equals 133 Pa]). Ablation from either surface resulted in little change in HC until a critical depth was reached, at which point there was an order of magnitude increase. The critical depth was approximately 170 microm from the inner limiting membrane when ablating from the vitreous surface and 70 microm from the inner limiting membrane when ablating from the photoreceptor surface. Histological specimens showed that these barriers were the synaptic portion of the outer plexiform layer, and the inner plexiform layer, respectively. CONCLUSIONS: The 2 high-resistance barriers to fluid flow through the retina are the synaptic portion of the outer plexiform layer, and the inner plexiform layer. CLINICAL RELEVANCE: These observations help to explain the distribution of cystoid macular edema seen in histological studies and with optical coherence tomography.

Chloramine-T in radiolabeling techniques. IV. Penta-O-acetyl-N-chloro-N-methylglucamine as an oxidizing agent in radiolabelling techniques.

Chloramine-T (CAT) is commonly used in radiolabeling of bioactive molecules by halogenation. CAT is used to release radioactive elemental iodine by oxidation of its salts. Unfortunately, CAT is a strong oxidizing agent and can cause significant damage to peptides and proteins. This may lower the yield of the iodination reaction and may produce undesirable side products. Recently, it was found that the in situ formation of N-chlorosecondary amines, by the addition of secondary amines to CAT prior to exposure to the substrate, can reduce the oxidative damage caused by CAT. To simplify the method, we prepared penta-O-acetyl-N-chloro-N-methylglucamine (NCMGE) as a solid N-chlorosecondary amine. The chemical reactivity of NCMGE toward a model amino acid, 1-aminocyclohexane carboxylic acid, was compared with that of chloramine-T. In the presence of the model amino acid, CAT lost all its chlorine titer within 60 min while NCMGE retained 99% of its chlorine titer. NCMGE was compared to CAT for the iodination of l-tyrosine and leucine enkephalin. For both substrates, the NCMGE method produced larger or equal yields of the monoiodo and diiodo products and less decomposition. It is proposed that the method employing NCMGE to release diatomic iodine is more convenient and efficient for radiolabeling peptides and proteins than currently used methods.

Nasal absorption of ondansetron in rats: an alternative route of drug delivery.

PURPOSE: Ondansetron (OND) is a 5-HT3 receptor antagonist that is used therapeutically for the prevention of nausea and vomiting associated with emetogenic cancer therapy. There is a need for nasal drug delivery in specific patient populations where the use of commercially available intravenous and oral dosage forms may be inconvenient and/or unfeasible. METHODS: OND (Zofran Injection, 2 mg/ml) was administered at a dose of 1 mg/kg to male Sprague-Dawley rats intravenously or intranasally (n = 3 in each group). A special surgical procedure was performed to ensure that the drug solution was held in the nasal cavity. OND was injected into the femoral vein for the intravenous group. Blood samples were collected at appropriate times for 60 min. An HPLC method was employed to determine OND in the plasma. RESULTS: The results clearly showed that OND was readily and rapidly absorbed through the nasal mucosa of the rat. The peak plasma level was attained within 10 min. OND was also completely absorbed as the plasma concentration-time profiles for the two routes were comparable. The terminal elimination half-lives were also similar. CONCLUSIONS: The nasal administration route for OND is apparently as effective as the intravenous route. If one considers the limitations of delivering OND orally or intravenously to patients undergoing emetogenic cancer therapy, it becomes obvious that the intranasal route is a potential alternative modality to prevent nausea and vomiting associated with such therapy.

Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups.

A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage.

Age-dependent variation in metalloproteinase activity of isolated human Bruch's membrane and choroid.

PURPOSE: To characterize and determine the effect of aging on the matrix metalloproteinase (MMP) component of the extracellular matrix-remodeling mechanism of isolated human Bruch's-choroid. METHODS: Immunohistochemical techniques and western blot analysis were used to detect and localize various members of the MMP family of proteolytic enzymes in the Bruch's-choroid complex. Gelatin substrate zymography was used to detect and quantify the levels of MMP-2 and -9 in homogenates of Bruch's-choroid from both macular and peripheral regions of the human fundus. Aging alterations in these enzymes were quantified by densitometric analysis of photographic negatives of the zymography gels. RESULTS: Intact preparations of Bruch's-choroid showed the presence of inactive forms of two gelatinases (MMP-2, 65 kDa, and MMP-9, 92 kDa), interstitial collagenase (MMP-1, 52 kDa) and stromelysin (MMP-3, 57 kDa). MMP-1 and -3 were localized primarily to Bruch's membrane. MMP-9 was distributed evenly in Bruch's membrane with some patchy presence in the choroidal mass. Distribution of MMP-2 was similar to that of MMP-9, but the staining in Bruch's was much fainter. On gelatin zymography, an active form of MMP-2 (58-kDa species) was frequently observed in peripheral samples but only occasionally in macular regions. The levels of MMP-2 and -9 increased with aging in both the macular and the peripheral regions of the fundus (P < 0.05). MMP-2 levels were lower in macular regions than in the periphery but no such variation was observed with MMP-9. Both these inactive gelatinases could be activated in vitro. CONCLUSIONS: A matrix-degrading mechanism essential for extracellular remodeling was shown to be present in Bruch's membrane. In macular regions, increasing levels of inactive forms of metalloproteinase and scarcity of active forms of MMP-2 suggests possible involvement of impaired extracellular degradation in both aging and macular degeneration.

Target organ involvement in hypertensive patients in Eastern Sudan.

Hypertension has become a major cause of cardiovascular morbidity and mortality in both the developed and the developing nations. In a cross-sectional study we assessed the state of blood pressure (BP) control and the pattern of target organ complications in 198 Sudanese patients treated in a teaching hospital in Kassala town in Eastern Sudan, (mean age 53 years, 76% women). Excellent BP control (BP <140/90 mm Hg) was achieved in 46% of the patients. Stage 2 or Stage 3 target organ involvement, particularly albuminuria and ischaemic heart disease, were detected in one-third of the patients. These complications were found to be related to both the severity and the duration of hypertension as well as to the frequency of cigarette smoking. Factors such as poor compliance, adverse socioeconomic status, as well as obesity and cigarette smoking may account for poor BP control and hence the development of hypertensive complications. We conclude that optimal BP control is not yet achieved in the majority of hypertensives in the Sudan. Reasons for this failure should be identified and corrected in order to avoid hypertensive target organ damage.

Localization of the site of major resistance to fluid transport in Bruch's membrane.

PURPOSE: To determine the site of highest resistance to the movement of water across Bruch's membrane in humans. METHODS: A hydraulic conductivity chamber was designed that enabled us to measure flow across Bruch's membrane while ablating its subepithelial aspect using an excimer laser (193 nm). When resistance was lost, samples were fixed and processed for electron microscopy. RESULTS: Changes in the hydraulic conductivity of Bruch's membrane in response to the excimer-mediated sequential removal of tissue layers was studied in four control eyes of donors 26, 46, 61, and 76 years of age and in one eye of an 83-year-old donor with age-related macular degeneration. The number of laser pulses required to abolish the resistance in Bruch's membrane was found to be dependent on the age of the donor. The ablation rate was approximately 0.11 micron per pulse. Loss of resistance correlated with removal of layers internal to the layer of elastin. CONCLUSIONS: This study indicates that the inner collage-nous layer imparts the major resistance to fluid movement between the retinal pigment epithelium and the choroid. Aging changes in the ultrastructure of these compartments could be responsible for the decrease in hydraulic conductivity observed in early life in previous studies.

Non-retinal abnormalities associated with progressive retinal atrophy (PRA) in the miniature poodle.

Osmotic fragility was examined in red blood cells taken from miniature poodles with progressive retinal atrophy (PRA). In this study, the median osmotic fragility for erythrocytes (concentration of NaCl required for 50% haemolysis) was found to be significantly lower for clinically affected, compared to unaffected animals. These differences were maintained regardless of changes in erythrocyte incubation pH (7.0, 7.5). There was no difference in the slope parameter of erythrocyte osmotic fragility profiles for both sets of animals, indicating a similar variability of erythrocyte age. In groups of animals maintained with the same diet and fasted for 12 hr, and in groups of animals with no dietary constraints, total plasma cholesterol was significantly lower for affected, compared to unaffected animals. Red blood cell indices were also assessed; mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), red blood cell count (RBC) and blood haemoglobin concentration (Hb) were normal, while erythrocyte packed cell volume (PCV) was significantly reduced for affected, compared to unaffected. Data from this study suggest that the abnormal erythrocyte osmotic fragility of affected animals is unlikely to be a consequence of a higher erythrocyte surface area/volume ratio, higher plasma cholesterol concentration, or greater proportion of new to old erythrocytes, and that a general membrane defect might be associated with prcd in the miniature poodle.