Key considerations and challenges in the application of social-network research for environmental decision making.

Attempts to better understand the social context in which conservation and environmental decisions are made has led to increased interest in human social networks. To improve the use of social-network analysis in conservation, we reviewed recent studies in the literature in which such methods were applied. In our review, we looked for problems in research design and analysis that limit the utility of network analysis. Nineteen of 55 articles published from January 2016 to June 2019 exhibited at least 1 of the following problems: application of analytical methods inadequate or sensitive to incomplete network data; application of statistical approaches that ignore dependency in the network; or lack of connection between the theoretical base, research question, and choice of analytical techniques. By drawing attention to these specific areas of concern and highlighting research frontiers and challenges, including causality, network dynamics, and new approaches, we responded to calls for increasing the rigorous application of social science in conservation.

Consideraciones y Retos Importantes en la Aplicación de la Investigación por medio de Redes Sociales para la Toma de Decisiones Ambientales Resumen Los intentos por tener un mejor entendimiento del contexto social en el que se toman las decisiones ambientales y de conservación han derivado en un incremento en el interés por las redes sociales humanas. Para mejorar el uso del análisis de redes sociales en la conservación, buscamos en la literatura los estudios recientes que hayan aplicado dichos métodos y los sometimos a una revisión. En esta revisión, examinamos los problemas en el diseño de la investigación y del análisis que limitan la utilidad del análisis de redes. Diecinueve de los 55 artículos publicados entre enero 2016 y junio 2019 exhibieron al menos uno de los siguientes problemas: aplicación de métodos analíticos inadecuados o sensibles a la información incompleta sobre las redes; aplicación de estrategias estadísticas que ignoran la dependencia en la red; o falta de conexión entre la base teórica, la pregunta de investigación y la selección de técnica analítica. Al llamar la atención hacia estas áreas específicas de interés y resaltar las fronteras y retos de la investigación, incluyendo la causalidad, las dinámicas de redes y las estrategias nuevas, respondimos a la necesidad de incrementar la aplicación rigurosa de las ciencias sociales en la conservación.

Fragile X Mental Retardation Protein expression in the retina is regulated by light.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick.

'We had to change to single beds because I itch in the night': a qualitative study of the experiences, attitudes and approaches to coping of patients with cutaneous T-cell lymphoma.

BACKGROUND: Primary cutaneous T-cell lymphoma (CTCL) is a rare but prevalent condition which can have a significant impact on many aspects of quality of life. However, there is little evidence of patients' lived experience of CTCL. OBJECTIVES: To understand in greater depth patients' experiences of living and coping with CTCL, and to inform the development of models of care for this population in line with U.K. METHODS: Semi-structured interviews were conducted with a purposive sample of patients with CTCL who attended an inner-city teaching hospital. Participants were purposively selected according to their disease stage, age, sex and ethnicity. RESULTS: Nineteen patients with CTCL (stages IB-IVB), aged between 41 and 83 years, participated in a single interview. This included 10 men; 15 people identified themselves as white British. Interviews lasted a median of 55 (range 28-170) min. Two main themes emerged: issues regarding diagnosis, particularly a perceived delay in diagnosis, and the impact of CTCL (subthemes related to physical well-being, practical concerns and psychological and social well-being and coping). CONCLUSIONS: Findings from this study illuminate the diverse effects of CTCL on patients' lives. The universal experience of delays in diagnosis was striking and a concern to patients. The disease, particularly its physical symptoms, had a significant impact on patients' lives, including employment, leisure and relationships. Despite the symptom burden and its impact, participants described effective coping strategies such as drawing on social support, maintaining normal lives and becoming well informed about CTCL. Proactive holistic assessment and management of the range of patient concerns is needed in providing care for patients with CTCL and their family and friends.

Comparison of in vivo and in vitro effects of continuous exposure of L1210 cells to 6-thioguanine.

In this study the cytokinetic and antitumor effects of 12-h continuous treatment with 6-thioguanine (TG) were studied in L1210 cells in vivo and in vitro. Loss of clonogenicity in vitro was maximized at a drug concentration of 0.2 microM. Higher drug concentrations produced less cell kill, and the surviving fraction observed after exposure to 25 microM TG was 1 log higher than at 0.2 microM (2% versus 0.2% of control cloning efficiency, respectively). Delayed G2 arrest in vitro was also found to be most pronounced at 0.2 microM, with G1 arrest more predominant at higher concentrations. Studies in vivo were conducted using C57BL X DBA/2 F1 mice, with or without advanced L1210 ascites tumor. In initial experiments performed on animals without tumor, the 50% lethal dose for 12-h s.c. infusions of TG was approximately 0.8 mumol/kg/min. Correlation of steady-state TG plasma levels with infusion rate revealed a linear relationship up to 0.62 mumol/kg/min, above which the TG plasma concentration increased disproportionately to input rate. Total body clearance of TG, calculated from the linear portion of this curve, was 123 ml/kg/min. The antitumor effects of TG infusions were correlated with steady state plasma concentrations achieved in each individual animal, and it was found that dose rates yielding levels from 1 to 10 microM increased survival time by about 40%, with no apparent optimum plasma level in this range. Examination of the cytokinetic effects caused by TG infusions at the low and high ends of this maximally therapeutic range showed that, as was the case in vitro, lower concentrations of TG caused delayed G2 arrest, while higher concentrations induced more rapid G1 arrest. On the basis of these, as well as previous findings, we propose that the operative mechanism of cell kill by TG in vivo may be dose dependent and may be reflected by the relative degree of G2 versus G1 arrest. We also suggest that the appropriate strategy for the clinical use of TG is to determine the drug concentration which produces maximum G2 arrest of tumor cells, and to infuse continuously at a rate to achieve that level for the maximum time tolerated by the patient, rather than to select an arbitrary length of infusion followed by escalation to maximum tolerated drug concentration.

Clusterin: a role in cell survival in the face of apoptosis?

Clusterin is a multifunctional glycoprotein complex found in virtually all body fluids and on the surface of cells lining body cavities. Demonstrated and proposed functions include the transport of lipoproteins, the inhibition of complement-mediated cell lysis and the modulation of cell-cell interactions. On the basis of its elevated expression in apoptotic tissues, it was originally proposed that the protein might be casually involved in apoptosis. Here, we discuss the recent data that, in contrast to the earlier notion, suggest that clusterin expression is not enhanced, but rather is down-regulated in the cells undergoing apoptosis and that its expression in the apoptotic tissue is restricted to the vital neighboring cells. These results led to the proposal that rather than being a cell death gene, clusterin is a cell survival gene, exerting a protective function on the surviving bystander cells.

Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, in the mammalian retina.

Synaptophysin and synaptoporin are homologous proteins that are among the most abundant synaptic vesicle proteins. Despite their high degree of sequence similarity, they are differentially distributed in the brain. The distribution of synaptophysin and synaptoporin was examined in the adult rat and rabbit retina by using single- and double- labeling immunocytochemistry with conventional light microscopy and confocal laser scanning microscopy. In the rat retina, synaptophysin immunoreactivity was found in the outer plexiform layer in terminals of photoreceptors and was homogeneously distributed throughout the inner plexiform layer. Synaptoporin immunoreactivity, however, was restricted to the inner plexiform layer. Labeling was most prominent in three distinct bands of the inner plexiform layer separated by two bands of very low synaptoporin immunoreactivity. In the rabbit retina, synaptophysin and synaptoporin immunoreactivity were found in the inner and outer plexiform layers. In the inner plexiform layer, labeling for both vesicle proteins was homogeneous, with no detectable stratification. In the outer plexiform layer, synaptophysin was present in photoreceptor terminals, and synaptoporin was present in horizontal cells. Staining of isolated rabbit retinal cells confirmed that both the axonless A type and the axon-bearing B type horizontal cells are immunoreactive for synaptoporin. In addition, electron microscopy of synaptoporin-immunostained rabbit retinas revealed no labeling of photoreceptor terminals but of putative synaptic sites in horizontal cells in the outer plexiform layer. No functional correlation was found in the expression of either synaptic vesicle protein with the type of neuron or synapse (ribbon or conventional).

Localization of the alpha(1F) calcium channel subunit in the rat retina.

PURPOSE: The molecular identity of the calcium channels that mediate glutamate release from photoreceptors is unknown. Mutations in the recently identified, retina-specific alpha(1F) calcium channel subunit cause incomplete X-linked congenital stationary night blindness (CSNB2), the phenotype of which is consistent with a defect in neurotransmission within the retina. The purpose of this study was to determine the cellular distribution of the alpha(1F) subunit in the retina. METHODS: Antibodies were raised against a unique peptide from the human alpha(1F) sequence. Rat retina sections were labeled with affinity-purified alpha(1F) antibodies and the immunofluorescence analyzed by confocal microscopy. The alpha(1F) staining was compared with that obtained with a pan-alpha(1) antibody, used to reveal the distribution of known voltage-gated calcium channels in the retina. Some sections were double labeled for alpha(1F) and the photoreceptor synaptic ribbon marker, bassoon. RESULTS: Staining of retina sections with anti-alpha(1F) resulted in strong punctate labeling in the outer plexiform layer (OPL) and weak punctate labeling in the inner plexiform layer (IPL), consistent with a synaptic localization. Staining was also observed in the outer nuclear layer. Within the OPL, alpha(1F) immunoreactivity was clustered in discrete, horseshoe-shaped patches, the shape and dimensions of which are characteristic of rod active zones. Similar structures were labeled with the pan-alpha(1) antibody. Localization of alpha(1F) immunoreactivity to rod active zones was confirmed by double labeling for bassoon, a component of photoreceptor synaptic ribbons. CONCLUSIONS: The distribution of alpha(1F) immunoreactivity in the OPL suggests that calcium influx through alpha(1F) or alpha(1F)-like channels mediates glutamate release from rod photoreceptors.

Expression of the alpha1F calcium channel subunit by photoreceptors in the rat retina.

PURPOSE: The CACNA1F gene encodes a voltage-gated calcium channel alpha1 subunit, alpha1F, which is expressed in the human retina. Mutations in this gene cause incomplete X-linked congenital stationary night blindness (CSNB2). The aim of this study was to obtain the sequence of the rat alpha1F cDNA and localize the encoded polypeptide in the rat retina. METHODS: The full-length rat alpha1F sequence was compiled from sequencing of overlapping alpha1F PCR fragments amplified from rat retinal cDNA. Antiserum was raised against a human alpha1F peptide. It was found that the human alpha1F peptide used to generate the antiserum was conserved at only 11 out of 19 residues in the cloned rat sequence. Therefore, antibodies were affinity purified against either the human alpha1F peptide or the equivalent rat peptide and used for immunofluorescent staining of rat retina sections. RESULTS: The rat alpha1F amino acid sequence was found to be 91% and 95% identical to the human and mouse alpha1F sequences, respectively. Antibodies affinity purified against the human alpha1F peptide stained both the outer nuclear layer (ONL) and outer plexiform layer of rat retina sections. In contrast, staining with antibodies affinity purified against the corresponding rat alpha1F peptide was restricted to the ONL. CONCLUSIONS: The rat alpha1F amino acid sequence is highly homologous to the human and mouse sequences. The immunohistochemical results indicate the existence of distinct alpha1F isoforms or alpha1F-like channels, which are differentially distributed in the cell bodies and synaptic terminals of photoreceptors in the rat retina.

Presynaptic proteins of ribbon synapses in the retina.

The synapses of photoreceptors and bipolar cells in the retina are characterized ultrastructurally by the presence of an electron-dense bar, the synaptic ribbon, lying perpendicular to the plasma membrane at the active zone and extending about 0.5 microm into the cytoplasm. Hence, these synapses are known as ribbon synapses. All neurons that make ribbon synapses release neurotransmitter tonically. That is, neurotransmitter is released continuously from these neurons and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven, phasic release from other neurons. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but several differences that may be important determinants of tonic transmitter release have been identified in the retina by immunohistochemistry. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.

SNAP-25 is present on the Golgi apparatus of retinal neurons.

SNAP-25 is a neuronal SNARE protein required for synaptic vesicle exocytosis and neurite outgrowth. Here we show that in addition to synaptic staining, SNAP-25 immunoreactivity is also localized to an intracellular, perinuclear compartment of retinal neurons. Double-labeling with an antibody against the 58 kD resident protein of the trans-golgi network indicates that the intracellular SNAP-25 is localized to the Golgi complex. Immuno-electron microscopic localization of SNAP-25 confirmed its presence on the Golgi apparatus of photoreceptors, bipolar cells, amacrine cells and ganglion cells in the retina. These data implicate SNAP-25 in the trafficking of Golgi-derived vesicles in neurons in addition to the synaptic vesicle cycle.

The distribution of neuronal calcium sensor-1 protein in the developing and adult rat retina.

We have examined the distribution of the neuronal calcium-binding protein, neuronal calcium sensor 1 (NCS-1) in the developing and adult rat retina using subcellular fractionation of the rat retina and immunohistochemistry. NCS-1 immunoreactivity was situated primarily in the ganglion cells, a class of amacrine cells, and in the inner plexiform layer (IPL). During development, NCS-1 protein expression closely followed that of the synaptic vesicle protein, synaptophysin, increasing dramatically in the IPL at postnatal day 3, the time when conventional synapses are formed in the retina. These findings suggest that NCS-1 plays a role in synaptogenesis in the retina and in synaptic transmission at conventional synapses but not ribbon synapses in the adult rat retina.

Modulation of the cytotoxic mechanism of 6-thioguanine by 4-amino-5-imidazolecarboxamide.

Previous evidence has indicated that either purine starvation or incorporation into DNA may be the dominant biochemical effect of the antileukemic agent 6-thioguanine (TG), depending on exposure conditions. Furthermore, it has been suggested that the paradoxical decrease in TG-induced cytotoxicity at high drug concentrations may be due to an antagonistic interaction between these two mechanisms, in which purine starvation inhibits DNA synthesis and, therefore, incorporation of TG into DNA. In this report we test the hypothesis that by concurrent treatment of L1210 cells with TG and the purine precursor 4-amino-5-imidazolecarboxamide (AIC) it is possible to alleviate DNA synthesis inhibition caused by high concentrations of TG, thus enhancing TG incorporation into DNA and TG-induced cell kill. Both the cytotoxic and cytokinetic results presented support this hypothesis. However, gross incorporation of TG into DNA was not increased by AIC under conditions in which a significant enhancement of cytotoxicity (i.e., 1 log) was observed. These findings suggest that the potentiating effect of AIC may be most prominent on the subpopulation of cells that are resistant to treatment with TG alone, and they demonstrate that the cytotoxic effects of TG treatments are more accurately reflected by observing specific cytokinetic changes (delayed late S/G2 arrest) than by measuring the average extent of TG incorporation into DNA within a given population. Finally, we propose that it may be possible to select conditions for administration of TG that favor one or the other cytotoxic mechanism, depending on whether the clinical objective is induction of remission (where rapid cell lysis due to purine starvation would be desired) or eradication of subclinical disease during remission (where proliferation-dependent cytotoxicity due to DNA incorporation should be more effective.

Calcium-induced calcium release contributes to synaptic release from mouse rod photoreceptors.

We tested whether calcium-induced calcium release (CICR) contributes to synaptic release from rods in mammalian retina. Electron micrographs and immunofluorescent double labeling for the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2) and synaptic ribbon protein, ribeye, showed a close association between ER and synaptic ribbons in mouse rod terminals. Stimulating CICR with 10 microM ryanodine evoked Ca(2+) increases in rod terminals from mouse retinal slices visualized using confocal microscopy with the Ca(2+)-sensitive dye, Fluo-4. Ryanodine also stimulated membrane depolarization of individual mouse rods. Inhibiting CICR with a high concentration of ryanodine (100 microM) reduced the electroretinogram (ERG) b-wave but not a-wave consistent with inhibition of synaptic transmission from rods. Ryanodine (100 microM) also inhibited light-evoked voltage responses of individual rod bipolar cells (RBCs) and presumptive horizontal cells recorded with perforated patch recording techniques. A presynaptic site of action for ryanodine's effects is further indicated by the finding that ryanodine (100 microM) did not alter currents evoked in voltage-clamped RBCs by puffing the mGluR6 antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), onto bipolar cell dendrites in the presence of the mGluR6 agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4). Ryanodine (100 microM) also inhibited glutamatergic outward currents in RBCs evoked by electrical stimulation of rods using electrodes placed in the outer segment layer. Together, these results indicate that, like amphibian retina, CICR contributes to synaptic release from mammalian (mouse) rods. By boosting synaptic release in darkness, CICR may improve the detection of small luminance changes by post-synaptic neurons.

Calcium channel heterogeneity among cone photoreceptors in the tree shrew retina.

Retinal photoreceptors are depolarized in darkness and release neurotransmitter tonically. They respond to light by hyperpolarization and a concomitant reduction in transmitter release. The calcium-dependent release of transmitter is coupled to graded changes in membrane potential by L-type calcium channels in the photoreceptor terminals. This paper reports the immuno-localization of an L-type channel alpha1D subunit to most, but not all, synaptic terminals of cones in the tree shrew retina. Double labelling for the alpha1D subunit and the plasma membrane Ca2+-ATPase, which has been shown to be present in all tree shrew cones, revealed a subpopulation of cone terminals that did not react with the alpha1D antibody. The nonimmunoreactive synaptic terminals represented approximately 5.8% of the total and formed a highly regular array across the retina reminiscent of the blue cones. Double-staining for the alpha1D subunit and blue cone opsin confirmed that these are the blue cones. The observed differences in calcium channel immunoreactivity between long and short wavelength cones points to previously unsuspected heterogeneity in the molecular machinery governing transmitter release from spectrally different cone types.

Neurotransmitter release at ribbon synapses in the retina.

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.

Interaction between band 3 and ankyrin begins in early compartments of the secretory pathway and is essential for band 3 processing.

In many cell types, membrane proteins are specifically segregated to particular areas of the cell surface. It is known that this segregation is stabilized by anchorage proteins which interact with the cytoskeleton. However, the mechanism by which the interactions with anchorage proteins occur, as well as whether they may also play a role in the process of sorting, is not known. Using differentiated murine erythroleukemic cells, we have investigated the association between band 3 (a major transmembrane anion exchanger), and ankyrin (a cytoplasmic protein that links band 3 to the cytoskeleton). Our data demonstrate that the association between band 3 and ankyrin occurs in the endoplasmic reticulum or the first Golgi compartment. These data support a model in which the band 3-ankyrin complex is inserted as a "cassette" at the plasma membrane into the cytoskeletal network. Biosynthetic studies on cotransfected 293 cells with cDNAs encoding band 3 and the band 3 binding fragment of ankyrin (AnK-90), suggest that ankyrin is not only responsible for the anchorage of band 3 to the cytoskeleton but is also involved in the exit of band 3 out of the endoplasmic reticulum.

Generation of truncated brain AE3 isoforms by alternate mRNA processing.

AE3 gene is a member of the AE anion exchanger gene family that is expressed primarily in brain and heart. The principal product of the AE3 gene in rodent brain, FL-AE3p, when expressed in heterologous cell lines, gives rise to chloride-dependent changes in intracellular pH consistent with its operation as an anion exchanger. We have identified two novel isoforms of mouse AE3 that are generated by tissue-specific alternate RNA processing. One of these isoforms encodes a polypeptide, 14-AE3p, that corresponds to a portion of the NH2-terminal cytoplasmic domain of AE3. 14-AE3p lacks the entire transmembrane domain that-in FL-AE3p forms the anion exchange channel. Immunoblots with antibodies to the NH2- and COOH-termini confirm that FL-AE3 and 14-AE3 are expressed in rat brain as 160 kDa and 74 kDa polypeptides, respectively. Unlike FL-AE3p, however, 14-AE3p is insoluble in non-ionic detergent, suggesting a possible association with the cytoskeleton.

Localization and properties of voltage-gated calcium channels in cone photoreceptors of Tupaia belangeri.

In spiking neurons, phasic, calcium-dependent transmitter release is initiated when a presynaptic action potential activates voltage-dependent calcium channels. Vertebrate photoreceptors are nonspiking neurons that continuously release transmitter. This study uses patch-clamp recording to examine the electrophysiological properties of mammalian cones in intact retina. The cell capacitance was 10+/-1 pF and the input resistance was 0.52+/-0.46 G omega at -65 mV (31 cells). A specific membrane capacitance of 1.2 pF/cm2 was calculated. The cones did not appear to be chemically or electrically coupled. The calcium conductance averaged 3+/-1 nS (five cells). Fifty percent of the calcium channels were active at -40 mV, and at this voltage the number of active channels changed e-fold for a 6-mV voltage change. At 25 degrees C, the current reached a peak within about 1 ms after onset of a step to -35 mV. The calcium influx produced by depolarization activated a chloride conductance with a delay of a few milliseconds. The channels did not completely inactivate during maintained depolarization. The calcium channels were partially blocked by high concentrations of nifedipine, an L-type specific antagonist, and were recognized by an antibody raised against the L-type subunit alpha-1D. The immunohistochemical staining shows that the calcium channels are localized to the synaptic terminals. The immunohistochemical, physiological, and pharmacological properties indicate that the calcium channels in mammalian photoreceptors may represent a novel isoform, possibly with some homologies to the L-type class. The activation range of the channels matches the physiological operating range of photoreceptors.