International Counseling Values: Recognizing Valued Approaches Identified by International Counseling Professionals Through Qualitative Inquiry.

People may assume that the counseling profession functions with a shared set of values that promote well-being and mental health to individuals, families, and communities across the globe. Common values, such as described in training programs, ethical codes, and other areas, reflect the approach and direction for providing professional counseling services among counseling professionals throughout the world. The researchers designed this qualitative study using a phenomenological approach to explore how counseling values are experienced and implemented across various cultures. The 16 participants of the study include counseling professionals from different countries to increase representation from eight regions of the world. The researchers recognize valued approaches commonly identified among the participants implementing counseling services, including marital and family counseling, child and school counseling, faith integration, indigenous practices, and person-centered safe spaces. While each of these valued approaches is described in detail, final applications of the data offer proposed steps to improve the advancement of counseling on a global scale, including strategies for transcultural counseling training, resource adaptability, and bilateral development in the profession.

Newly Identified Chemicals Preserve Mitochondrial Capacity and Decelerate Loss of Photoreceptor Cells in Murine Retinal Degeneration Models.

Purpose: Metabolic stress and associated mitochondrial dysfunction are implicated in retinal degeneration irrespective of the underlying cause. We identified seven unique chemicals from a Chembridge DiverSET screen and tested their protection against photoreceptor cell death in cell- and animal-based approaches. Methods: Calcium overload (A23187) was triggered in 661W murine photoreceptor-derived cells, and changes in redox potential and real-time changes in cellular metabolism were assessed using the MTT and Seahorse Biosciences XF assay, respectively. Cheminformatics to compare structures, and biodistribution in the living pig eye aided in selection of the lead compound. In-situ, retinal organ cultures of rd1 mouse and S334ter-line-3 rat were tested, in-vivo the light-induced retinal degeneration in albino Balb/c mice was used, assessing photoreceptor cell numbers histologically. Results: Of the seven chemicals, six were protective against A23187- and IBMX-induced loss of mitochondrial capacity, as measured by viability and respirometry in 661W cells. Cheminformatic analyses identified a unique pharmacophore with 6 physico-chemical features based on two compounds (CB11 and CB12). The protective efficacy of CB11 was further shown by reducing photoreceptor cell loss in retinal explants from two retinitis pigmentosa rodent models. Using eye drops, CB11 targeting to the pig retina was confirmed. The same eye drops decreased photoreceptor cell loss in light-stressed Balb/c mice. Conclusions: New chemicals were identified that protect from mitochondrial damage and lead to improved mitochondrial function. Using ex-vivo and in-vivo models, CB11 decreased the loss of photoreceptor cells in murine models of retinal degeneration and may be effective as treatment for different retinal dystrophies.

SAHA is neuroprotective in in vitro and in situ models of retinitis pigmentosa.

PURPOSE: Recent reports linking HDAC6 to mitochondrial turnover and neurodegeneration led us to hypothesize that an inhibitor such as Vorinostat (suberoylanilide hydroxamic acid, SAHA) may reduce mitochondrial damage found in retinitis pigmentosa (RP), a progressive neurodegenerative disease of the eye. Here we tested the efficacy of SAHA for its ability to protect photoreceptors in in-vitro and in-situ models of RP. As the stressor, we focused on calcium overload. Calcium is one of the main drivers of cell death, and is associated with rod loss in the rd1 mouse retina, which harbors a mutation in the Pde6b gene similar to that found in human patients suffering from autosomal recessive RP. METHOD: Murine photoreceptor cell line (661W) were exposed to agents that led to calcium stress. Cell survival and redox capacity were measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time changes in cellular metabolism were assessed using the Seahorse Biosciences XF24 analyzer, and mitochondrial fission-fusion using imaging. In-situ, neuroprotection was assessed in RPE/retina organ cultures of the rd1 mouse. SAHA effects on cell survival were compared in 661W cells with those of the specific HDAC6 inhibitor tubastatin A, and those on protein acetylation by Western blotting. RESULTS: In stressed 661W cells, SAHA was found to increase cell survival that was associated with improved mitochondrial respiration and reduced mitochondrial fission. The protective effects of SAHA were also observed on photoreceptor cell survival in whole retinal organ explants of the rd1 mouse. Even though tubastatin A was ineffective in increasing cell survival in 661W cells, HDAC6 activity was confirmed in 661W cells after SAHA treatment with protein acetylation specific for HDAC6, defined by an increase in tubulin, but not histone acetylation. CONCLUSIONS: SAHA was found to protect mitochondria from damage, and concomitantly reduced photoreceptor cell death in cell and organ cultures. The lack of activity of tubastatin A suggests that there must be an additional mechanism of action involved in the protective mechanism of SAHA that is responsible for its neuroprotection. Overall, SAHA may be a useful treatment for the prevention of photoreceptor degeneration associated with human RP. The results are discussed in the context of the effects of inhibitors that target different classes and members of the HDAC family and their effects on rod versus cone survival.

Small Molecules that Protect Mitochondrial Function from Metabolic Stress Decelerate Loss of Photoreceptor Cells in Murine Retinal Degeneration Models.

One feature common to many of the pathways implicated in retinal degeneration is increased metabolic stress leading to impaired mitochondrial function. We found that exposure of cells to calcium ionophores or oxidants as metabolic stressors diminish maximal mitochondrial capacity. A library of 50,000 structurally diverse "drug-like" molecules was screened for protection against loss of calcium-induced loss of mitochondrial capacity in 661W rod-derived cells and C6 glioblastomas. Initial protective hits were then tested for protection against IBMX-induced loss of mitochondrial capacity as measured via respirometry. Molecules that protected mitochondria were then evaluated for protection of rod photoreceptor cells in retinal explants from rd1 mice. Two of the molecules attenuated loss of photoreceptor cells in the rd1 model. In the 661W cells, exposure to calcium ionophore or tert-butylhydroperoxide caused mitochondrial fragmentation that was blocked with the both compounds. Our studies have identified molecules that protect mitochondria and attenuate loss of photoreceptors in models of retinal degeneration suggesting that they could be good leads for development of therapeutic drugs for treatment of a wide variety of retinal dystrophies.

Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death.

Although genetic and environmental factors contribute to neurodegenerative disease, the underlying etiology common to many diseases might be based on metabolic demand. Mitochondria are the main producer of ATP, but are also the major source of reactive oxygen species. Under normal conditions, these oxidants are neutralized; however, under environmental insult or genetic susceptibility conditions, oxidative stress may exceed cellular antioxidant capacities, leading to degeneration. We tested the hypothesis that loss in mitochondrial reserve capacity plays a causative role in neuronal degeneration and chose a cone photoreceptor cell line as our model. 661W cells were exposed to agents that mimic oxidant stress or calcium overload. Real-time changes in cellular metabolism were assessed using the multi-well Seahorse Biosciences XF24 analyzer that measures oxygen consumption (OCR) and extracellular acidification rates (ECAR). Cellular stress resulted in an early loss of mitochondrial reserve capacity, without affecting basal respiration; and ECAR was increased, representing a compensatory shift of ATP productions toward glycolysis. The degree of change in energy metabolism was correlated with the amount of subsequent cell death 24-hours post-treatment, the concentration-dependent loss in mitochondrial reserve capacity correlated with the number of live cells. Our data suggested first, that loss in mitochondrial reserve capacity is a major contributor in disease pathogenesis; and second, that the XF24 assay might represent a useful surrogate assay amenable to the screening of agents that protect against loss of mitochondrial reserve capacity. In future experiments, we will explore these concepts for the development of neuroprotective agents.

Assessment of mitochondrial damage in retinal cells and tissues using quantitative polymerase chain reaction for mitochondrial DNA damage and extracellular flux assay for mitochondrial respiration activity.

Mitochondrial dysfunction and genomic instability are associated with a number of retinal pathologies including age-related macular degeneration, diabetic retinopathy, and glaucoma. Consequences of mitochondrial dysfunction within cells include elevation of the rate of ROS production due to damage of electron transport chain proteins, mitochondrial DNA (mtDNA) damage, and loss of metabolic capacity. Here we introduce the quantitative polymerase chain reaction assay (QPCR) and extracellular flux assay (XF) as powerful techniques to study mitochondrial behavior. The QPCR technique is a gene-specific assay developed to analyze the DNA damage repair response in mitochondrial and nuclear genomes. QPCR has proved particularly valuable for the measurement of oxidative-induced mtDNA damage and kinetics of mtDNA repair. To assess the functional consequence of mitochondrial oxidative damage, real-time changes in cellular bioenergetics of cell monolayers can be measured with a Seahorse Biosciences XF24 analyzer. The advantages and limitations of these procedures will be discussed and detailed methodologies provided with particular emphasis on retinal oxidative stress.

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage.

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC(50)) for most of the tested polyphenols are between 8 and 480 µM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu(I)-mediated DNA damage less effectively than Fe(II)-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu(I), and H(2)O(2), it is likely that weak polyphenol-Cu(I) interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu(I), H(2)O(2), and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy.

Kinetics of iron oxidation upon polyphenol binding.

Polyphenol prevention of iron-mediated DNA damage occurs primarily through iron binding. Once bound, iron in the Fe(2+)-polyphenol complex autooxidizes to Fe(3+) in the presence of O(2). To determine the correlation between the rate of Fe(2+)-polyphenol autooxidation and polyphenol antioxidant ability, kinetic studies at pH = 6.0 in the presence of oxygen were performed using UV-vis spectrophotometry. Initial rates of iron-polyphenol complex oxidation for epigallocatechin gallate (EGCG), methyl-3,4,5-trihydroxybenzoate (MEGA), gallic acid (GA), epicatechin (EC), and methyl-3,4-dihydroxybenzoate (MEPCA) were in the range of 0.14-6.7 min(-1). Polyphenols with gallol groups have faster rates of iron oxidation than their catechol analogs, suggesting that stronger iron binding results in faster iron oxidation. Concentrations of polyphenol, Fe(2+), and O(2) were varied to investigate the dependence of the Fe(2+)-polyphenol autooxidation on these reactants for MEGA and MEPCA. For these analogous gallate and catecholate complexes of Fe(2+), iron oxidation reactions were first order in Fe(2+), polyphenol, and O(2), but gallate complexes show saturation behavior at much lower Fe(2+) concentrations. Thus, gallol-containing polyphenols promote iron oxidation at a significantly faster rate than analogous catechol-containing compounds, and iron oxidation rate also correlates strongly with polyphenol inhibition of DNA damage for polyphenol compounds with a single iron-binding moiety.

A review of the antioxidant mechanisms of polyphenol compounds related to iron binding.

In this review, primary attention is given to the antioxidant (and prooxidant) activity of polyphenols arising from their interactions with iron both in vitro and in vivo. In addition, an overview of oxidative stress and the Fenton reaction is provided, as well as a discussion of the chemistry of iron binding by catecholate, gallate, and semiquinone ligands along with their stability constants, UV-vis spectra, stoichiometries in solution as a function of pH, rates of iron oxidation by O(2) upon polyphenol binding, and the published crystal structures for iron-polyphenol complexes. Radical scavenging mechanisms of polyphenols unrelated to iron binding, their interactions with copper, and the prooxidant activity of iron-polyphenol complexes are briefly discussed.

Predicting how polyphenol antioxidants prevent DNA damage by binding to iron.

Prevention of oxidative DNA damage due to hydroxyl radical is important for the prevention and treatment of disease. Because of their widely recognized antioxidant ability, 12 polyphenolic compounds were assayed by gel electrophoresis to directly quantify the inhibition of DNA damage by polyphenols with Fe(2+) and H2O2. All of the polyphenol compounds have IC50 values ranging from 1-59 microM and inhibit 100% of DNA damage at 50-500 microM concentrations. Gel electrophoresis results with iron(II)EDTA and UV-vis spectroscopy experiments confirm that binding of the polyphenol to iron is essential for antioxidant activity. Furthermore, antioxidant potency of polyphenol compounds correlates to the pKa of the first phenolic hydrogen, representing the first predictive model of antioxidant potency based on metal-binding. Understanding this iron-coordination mechanism for polyphenol antioxidant activity will aid in the design of more-potent antioxidants to treat and prevent diseases caused by oxidative stress, and help develop structure-activity relationships for these compounds.

The central role of metal coordination in selenium antioxidant activity.

Oxidative DNA damage occurs in vivo by hydroxyl radical generated in metal-mediated Fenton-type reactions. Cell death and mutation caused by this DNA damage are implicated in neurodegenerative and cardiovascular diseases, cancer, and aging. Treating these conditions with antioxidants, including highly potent selenium antioxidants, is of growing interest. Gel electrophoresis was used to directly quantify DNA damage inhibition by selenium compounds with copper and H(2)O(2). Selenocystine inhibited all DNA damage at low micromolar concentrations, whereas selenomethionine showed similar inhibition at 40 times these concentrations, and 2-aminophenyl diselenide showed no effect. DNA damage inhibition by these selenium compounds does not correspond to their glutathione peroxidase activities, and UV-vis and gel electrophoresis results indicate that selenium-copper coordination is essential for DNA damage inhibition. Understanding this novel metal-coordination mechanism for selenium antioxidant activity will aid in the design of more potent antioxidants to treat and prevent diseases caused by oxidative stress.