, 2008, Teramitsu et al., 2010 and Teramitsu and White, 2006). During song development, FoxP2 knockdown in Area Palbociclib ic50 X by lentivirus-mediated RNA interference causes inaccurate song imitation and a reduction in neural spine density ( Haesler et al., 2007). Thus, the thalamocortical–basal ganglia circuit is thought to contribute during development to song learning and vocal control in songbirds, with FoxP2 intimately involved, similar to the situation in humans ( Haesler et al., 2007, Schulz et al., 2010 and Teramitsu et al., 2010). Overall, these results suggest that the FoxP2 expression pattern in the thalamocortical–basal

ganglia circuit is conserved between marmoset and other species. The IO is important for learning and timing of motor control ( De Zeeuw et al., 1998), and is closely associated with the cerebellum. Jurgens and Richter (1986) reported that vocalizations can be induced by electrically stimulating the IO ( Jurgens & Richter, 1986). Therefore, although the relationship between the IO and

speech is unclear, it may be associated with vocalization in humans. Future studies are necessary to investigate the role of the IO and speech disorder-related genes in vocalization. Almost all speech impairments and reading disabilities are find more learning disorders, prevalent in childhood. Most of the genes associated with these disorders play important roles in neural development, yet show different expression Thiamet G patterns in different brain areas. Furthermore, expression levels or patterns of these genes also changed during development in the marmoset brain (Table 2). Non-human primates do not have language or acquire

vocalization in the way that humans do, because of differing neuroanatomical connectivity of the auditory–vocal regions between humans and non-human primates. The arcuate fasciculus is a white-matter fiber tract that links the lateral temporal cortex with the frontal cortex, via a dorsal projection that arches around the Sylvian fissure (Rilling et al., 2008). The arcuate fasciculus shows significant differences between human and monkey brain, with projections to the middle and inferior temporal gyrus absent in monkey (Thiebaut de Schotten, Dell’Acqua, Valabregue, & Catani, 2012). In addition, from the point of view of vocal learning, direct connections between the telencephalon and medullary vocal motor nucleus have been reported in a limited number of vertebrates. In mammals, direct connections between the primary motor cortex and nucleus ambiguus that controls the vocal organ, are present in humans (Iwatsubo et al., 1990, Kuypers, 1958a and Schoen, 1969), but not observed in monkeys (Jurgens, 1976, Jurgens, 2002, Kuypers, 1958b and Simonyan and Jurgens, 2002). In contrast, neural activation in the homologue of Broca’s area is observed in vocalizing marmosets using gene expression analysis of immediate early genes (Simoes et al., 2010).

Amino acid metabolism pathways appear to be more downregulated in testes, but central genes such as the GOT1 (Glutamic-oxaloacetic transaminase 1) gene are downregulated both in ovary and testis. Compared to ovaries and testes, much fewer genes were found to AZD6244 in vitro be significantly

regulated in the frontal tissue. This is, at least in part, caused by the nature of this tissue section, which contains a number of different tissue types. Based on morphology, this selection of tissue contains not only neuronal, (endo and exocrine) glandular tissues but also muscle, subcuticular tissue and the anterior part of the gut. This observation is confirmed by the transcription of gene hallmark to subcuticular tissues: vitellogenins and muscle: actin, tropomyosin and titin. However, upregulated genes in the frontal tissue also included genes expected to be found in neuronal tissue such as GABA receptor (subunit: alpha), glycine and glutamate receptors, rhodopsin found in the eye and a chloride channel (bestrophin). Transcripts from the frontal tissue are Selleck MLN0128 good candidates for products that could be excreted by the salmon louse and act as potential modulators of the host fish. Candidates for such genes

are upregulated genes annotated as angiotensin-converting enzyme and calmodilin. When identifying genes regulated in the intestine the transcription patterns for the subcuticular tissue was considered relative to all tissues except the frontal tissue, as this may contain intestine tissue contaminants (see Material and methods). Among the upregulated Carnitine palmitoyltransferase II genes we found several proteases (e.g. carboxypeptidase A, cathepsins, elastase, neprilysin and trypsins) and other genes (e.g. Lipase, CD63, fadD and oligotransporters) associated with pancreatic secretion, protein and lipid digestion and lysosomal activity.

However, genes encoding protein components in the apical complex of the lysosomes were downregulated. The previously characterized trypsins, LsTryp1–5 (Kvamme et al., 2004) were among the genes with high relative expression in the gut along with a high affinity copper uptake protein. In addition 2 MFS solute transporters (gradient driven) and an aquaporin were upregulated relative to the other tissues. Genes involved in both glycogen synthesis (KO0500, e.g. glycogen synthetase) and metabolism (e.g. glycogen debranching enzyme and glycogen phosphorylase) and genes involved in synthesis of Triacylglycerol (TAG) are not significantly differentially expressed compared to other tissues. 28 cytochrome P450 (CYP) genes, commonly involved in oxidation of metabolic intermediates including lipids and xenobiotic substances, are upregulated in the gut, whereas no CYP genes are upregulated in other tissues.

For short-term treatment, cells were treated for 6 h at 37 °C in the presence or absence of 5% S9, after which, the medium was discarded, and the cells were rinsed with PBS(−) and cultured for another 18 h at 37 °C in fresh medium. For continuous treatment, cells were treated for 24 h in the absence of metabolic activation. At the start and end of the treatment period, and at the end of culturing, whether

or not the test sample had precipitated was checked macroscopically. At the end of culturing, the number of cells was counted by using a Microcell counter (CDA-500; Sysmex, Hyogo, Japan) after trypsinization, and the cell growth find more rate at each dose was calculated. Finally, the prepared specimens were stained with Giemsa solution (Merck, Darmstadt, Germany) and 200 metaphase cells per dose were evaluated for structural aberrations and numerical aberrations, respectively. 2) Evaluation of results The in vitro chromosomal aberration test was considered positive when the frequency of cells with structural aberrations or numerically aberrant cells was 10% or more and the increase was dose-related. All other cases were considered negative. No statistical analyses were used. A total of four SDs and five STs were detected in the test sample (Table 3 and Fig. 1). The total SD and ST content of the test sample was approximately 88%; approximately 12% of the styrene oligomers

in the test sample were of tetrameric or greater size (data not check details shown). Prior to conducting the Ames test, a dose range-finding study was conducted using six doses: 156, 313, 625, 1250, 2500, and 5000 μg/plate. No increase in the number of revertant colonies and no bacterial growth inhibition were observed at any of the doses examined (data not shown). Therefore, the same six doses were used for the first Ames test. To confirm the reproducibility of the results, a second Ames

test was conducted, but this time in consideration of the results of the first test, only five doses were used: 313, 625, 1250, 2500, and 5000 μg/plate. Because the dose–response curves obtained from the two Ames tests were comparable, only Terminal deoxynucleotidyl transferase the dose–response curves from the first test are presented (Fig. 2). The number of revertant colonies in each dose was similar to that in the negative control for all tester strains. No inhibition of bacterial growth and no precipitation of the test substance were observed for any of the test conditions in either the presence or absence of S9 mix. Therefore, because the number of revertant colonies in all treatment groups for all tester strains was less than twice that in each negative control in the presence or absence of S9 mix in both the first and second Ames test, the genotoxicity of the test solution was considered negative. The numbers of revertant colonies in the negative control and positive control were within the historical range for our laboratory (data not shown).

The Airn and Kcnq1ot1 genes are up to several hundred kilobases away from the EXEL genes they regulate ( Figure 1a and b), and in both cases correlative evidence suggests that the ncRNA product is causing repression at a distance, as described for Kcnq1ot1 above. In the placenta, the Airn macro ncRNA product is located in close proximity to the silent paternal promoter of the EXEL gene Slc22a3 that also carries a repressive H3K9me3 histone mark [ 35••]. Silencing of Slc22a3 ZD1839 ic50 depends on the lysine methyltransferase EHMT2 [ 35••] whose main activity is to catalyse H3K9me2, but which can also catalyse H3K9me3 at some loci [ 30, 36 and 37].

As Airn also associates with EHMT2 in placenta, it is possible that the Airn ncRNA product is responsible for the recruitment of EHMT2 to the 17-AAG nmr Slc22a3 promoter and therefore for its silencing. The Tagging and Recovery of Associated Proteins (TRAP) method that is dependent on detecting the ncRNA by in situ hybridization was used to detect the close proximity of the Airn ncRNA and the Slc22a3 promoter [ 35••]. Interestingly

this technique was initially used to discover a chromosome loop connecting enhancers in the β-globin locus control region with the β-globin promoter [ 38]. Applying the U0126 in vitro same concept to the Airn TRAP data implies that the Slc22a3 promoter is close to the Airn transcription unit in three-dimensional space. With this in mind we propose a model consistent with the published data, where the mature ncRNA product is not responsible for silencing genes at a distance, but rather Airn transcription blocks the binding of transcriptional activators that are required to facilitate chromosomal looping and activation of Slc22a2 and Slc22a3 expression. In this model, early development is defined by a ground state chromatin conformation that allows low-level biallelic expression of protein-coding

genes on both parental alleles (Figure 2a and b, top). This ground state is well established for Igf2r in pre-implantation embryos [ 39 and 40], and for Slc22a2 and Slc22a3, which are not upregulated until post-implantation [ 11••]. In this ground state Airn is not made, because DNA methylation of the ICE prevents Airn expression on the maternal chromosome [ 11••] and most probably essential transcription factors are not yet expressed to activate the paternal allele [ 41]. In the post-implantation embryo, following the binding of transcriptional activators, activating loops form on the maternal chromosome between enhancers and the promoters of Slc22a2 and Slc22a3, causing their upregulation ( Figure 2a, middle and bottom).

For MCPA the valid results increased clearly when applying the higher limit value and the range of valid data even included the range of invalid in full. This

effect is mainly due to the inclusion of absorption results obtained with six reconstructed human skin samples which were obviously higher, but based on TEWL cut-off limit 13 g m−2 h−1 classified NU7441 price as valid. The very slow penetrating test compound 14C-MCPA-2EHE showed no clear difference of absorption values in valid and invalid skin samples. This was observed with all integrity tests (Table 4, Table 5 and Table 6). Mean, min and max values did not differ significantly for the two different limit values of TWF (Table 6). However, the stricter limit value for TEER (2 kΩ) led to a different distribution (Table 4). Only 2 of 90 skin samples were classified as invalid with 1 kΩ as the limit, but 28 with 2 kΩ. Applying the limit value 2 kΩ, the majority of the reconstructed human skin samples with higher

absorption results for the test compounds were classified as invalid (23 out of 30). In contrast, five excised human skin samples were classified as invalid despite of absorption data in reasonable ranges. Analog to TEWL, differentiation with TEER (limit: 2 kΩ) and TWF resulted in obvious higher absorption means for invalid find more skin samples than for valid skin samples as well as in significant Progesterone overlapping of results. In a second step linear regression analyses for the absorption values (AD, maxKp, dependent variable y) and integrity test results (independent variable x) were used to check whether integrity tests TEER, TEWL, TWF, ISTD and BLUE are able to display minor barrier

differences between skin samples continuously. Besides human skin, rat skin was included in these analyses. Table 7 shows mean, min and max values of slopes and R2 derived from analysis for the different experimental groups. One group covers experiments using one defined combination of test compound (testosterone, caffeine, MCPA or MCPA-2EHE) and skin preparation (excised human skin, reconstructed human skin or excised rat skin). The correlations varied over a wide range for all five methods, four test compounds and three skin preparation types. The best correlations in average (R2: 0.484) and maximal (R2: 0.911) were achieved with the ISTD. Partially good correlations were observed for TEWL: the maximal R2 of 0.790 was achieved with test compound 14C-testosterone applied to reconstructed human skin. Even inverse correlations were occasionally obtained with TEWL, TEER, TWF and BLUE but not for ISTD. The dataset of the special investigation comprising all experiments with 14C-MCPA applied to undamaged and gradually damaged rat skin covers a wide range of absorption (AD 6–100%) and absorption rates (Marzulli-Class: slow to very fast) ( Marzulli and Brown, 1969).

Food and water were provided ad libitum. The experimental protocol was approved by the Ethics Committee on the Use of Animals, Health Sciences Center, Federal University Omipalisib supplier of Rio de Janeiro (Protocol IBCCF 012). Two separate experiments, with equal procedures, were necessary for this study. The first one used thirty-four mice, randomly

divided into 6 groups (5–6 animals per group) for pulmonary mechanics and histological analyses. The second experiment had 30 animals sacrificed for all biochemical analyses. We had 4 control animals at all time points in the first set of experiments. After running a one-way ANOVA followed by Bonferroni’s multiple comparisons test using the mechanics data, all control groups were statistically similar. Thus, one animal was randomly picked up from each group and, thus, SAL group was formed (n = 5). In the second batch of animals 5 mice were used as controls. SAL animals received a single intratracheal instillation (i.t.) of 50 μL of saline solution (NaCl 0.9%). Cylindrospermopsin groups (CYN) received a single sublethal dose of semi-purified extract of cylindrospermopsin (70 μg/kg body weight, i.e., 45–55 μL, i.t.). This dose

was chosen based on the cylindrospermopsin LD50 in mice (i.p.), namely, 200 μg/kg BW ( Terao et al., 1994). All animals (25–30 g) were selleck screening library analyzed 2, 8, 24, 48 and 96 h after instillation. For intratracheal instillation mice were anesthetized with sevoflurane, and saline or cylindrospermopsin was gently 4��8C instilled into their tracheas with the aid of an ultra-fine U-100 insulin syringe. The animals rapidly recovered after instillation. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical

Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Academy of Sciences, USA. The animals were exposed to a semi-purified extract of C. raciborskii. The cylindrospermopsin producer strain CYP 011K, kindly provided by Dr. Andrew Humpage and Dr. Peter Baker (Australian Water Quality Centre, Adelaide, Australia) was cultured in ASM-1 medium, the lyophilized biomass was extracted in ultrapure water, centrifuged and passed through a C18 cartridge to remove part of the matrix interference. The process ensured the removal of any cyanobacterial LPS in the extract. The extraction step and HPLC analysis of toxin content were done according to Welker et al. (2002). At 2, 8, 24, 48 and 96 h after instillation of saline or cylindrospermopsin the animals were sedated with diazepam (1 mg, i.p.), anesthetized with pentobarbital sodium (20 mg/kg BW, i.p.), tracheotomized, and a snugly fitting cannula (0.8 mm i.d.) was introduced into the trachea. The animals were then paralyzed with pancuronium bromide (0.1 mg/kg, i.v.

1C). Rarely, parasite-positive areas were seen during the chronic phase PI3K inhibitor (data not shown). Histopathological analyses revealed that in T. cruzi-infected C3H/He mice, brain inflammation was restricted to the acute phase of infection, when inflammatory cells were seen in the parenchyma and perivascular cuffs with one or more layers of infiltrating cells ( Fig. 1D). In the acutely infected C3H/He mice, several CNS areas were affected including hippocampus ( Fig. 1D), a brain region involved in depression in mouse models ( Bahi and Dreyer, in press). In contrast, no inflammatory infiltrates were detected in the brain of acutely and chronically T. cruzi-infected C57BL/6

mice ( Fig. 1D), resembling the CNS of NI controls. These data are summarized in Table S1. Therefore, these models allowed us to test whether behavioral alterations were induced during chronic T. cruzi infection and whether they were a long-term consequence of acute CNS inflammation. To test whether behavioral alterations are present in T. cruzi infection, we initially subjected infected

mice to the open-field test and analyzed the numbers buy GSK2118436 of peripheral and central crossed lines and rearing episodes. Acutely infected C57BL/6 mice exhibited a significant (p < 0.001; t (11) > 5.124) decrease in locomotor/exploratory activity compared with the NI controls in five-, ten- and thirty-minute sessions ( Fig. S1A). Chronically T. cruzi-infected C57BL/6 mice also presented a significant decrease in locomotor/exploratory activity expressed as the reductions in the number of crossed peripheral (p < 0.0001; t (9) = 11.89) and central (p < 0.01; t Farnesyltransferase (9) = 4.107) lines and rearing episodes (p < 0.0001; t (9) = 8.888) in five-minute sessions ( Fig. S1B). This finding confirms our previous data ( Silva et al., 2010). Conversely, when T. cruzi-infected C3H/He mice were compared with sex- and age-matched NI controls, there were no significant differences (p > 0.05; t (6) < 1.500)

in the numbers of crossed peripheral and central lines or rearing episodes during the acute (30 dpi; Fig. 2A) or chronic (90 dpi; Fig. 2B) phases of infection in five-minute sessions of the open-field test. Furthermore, no significant (p > 0.05; t (11) < 1.000) behavioral alterations were detected in acutely ( Fig. S2A) or chronically ( Fig. S2B) T. cruzi-infected C3H/He mice when their performances in ten- and thirty-minute sessions of the open-field test were analyzed. Considering that sickness features may contribute to behavioral alterations such as decreases in spontaneous locomotor/exploratory activity ( Rogers et al., 2001), we further assessed sickness behavior by checking body weight loss (which reveals loss of appetite), apathy and increase in temperature (indicative of fever). During the recorded interval (from 7 to 150 dpi), apathy, characterized as prostration, was not detected in C3H/He and C57BL/6 mice infected with a low-level inoculum of the Colombian T. cruzi strain.

(2006). The midgut of P. nigrispinus, like other Heteroptera Pentatomorpha (see for example D. peruvianus, Silva et al., 1995), is divided into three major chambers, from which the first (AM, anterior midgut) and the last (PM, posterior midgut) are dilated and the middle chamber (MM, middle midgut) is cylindrical ( Fig. 1B). Salivary and midgut luminal contents are mildly acidic. The mean Bleomycin and standard errors of pH values (n = 10) are 6.0 ± 0.1 in salivary gland and 5.6 ± 0.1 in AM, 5.7 ± 0.1 in MM, and 5.8 ± 0.1 in PM. The fine structure of P. nigrispinus

midgut cells do not differ much from other Heteroptera Pentatomomorpha, like D. peruvianus ( Silva et al., 1995) and Brontocoris tabidus (Heteroptera: Pentatomidae) ( Guedes et al., 2007 and Fialho

et al., 2009). Hence, only the apical features that are interesting in the context of this work will be described. The apex of midgut cells display microvilli that are ensheathed with glove-like finger membranes, the perimicrovillar membranes (Fig. 2A and B). What are remarkable are the muscles fibers visible in the midgut lumen (Fig. 2C and D). These ingested muscles fibers are discernible only in the anterior and middle midgut, suggesting that they are digested as the food moves toward the hindgut. Activities measured in extracts of midguts and salivary glands are described in table 1. The substrate Suc-AAPF-MCA was not hydrolyzed by homogenates of salivary glands and midguts, thus ruling out the occurrence of chymotrypsin as a digestive enzyme. Z-FR-MCA (like benzoyl-Arg-p-nitroanilide, BApNA) may be hydrolyzed by both cathepsin L-like enzymes, which are cysteine proteinases, and trypsin, which is a serine proteinase. They may be Selleck HDAC inhibitor distinguished, however, by their pH optima and their response in the presence of sulfhydryl reagents (usually Cys) and E-64 (Terra and Ferreira, 1994). By these criteria, the major proteinase in both salivary glands and midgut is cathepsin L, although a small amount of trypsin activity is present in salivary glands. These conclusions are based in the following

observations: (1) assays with Z-FR-MCA at pH 6.0 in the presence of DNA ligase Cys were strongly inhibited by E-64 (100 ± 10% in midguts and 92 ± 7% in salivary glands), revealing the presence of a cathepsin L activity; (2) assays with Z-FR-MCA at pH 7.5 with salivary gland homogenates were inhibited by only 31 ± 8% in the presence of E-64, indicating the occurrence of a trypsin activity. No activity was found at those conditions in midgut samples, discounting the presence of significant trypsin activity in midguts. Activity on Z-RR-MCA is only 2% of the activity determined with Z-FR-MCA, confirming that the enzyme is really a cathepsin L, discounting the possibility of being a cathepsin B (also a cysteine proteinase), which would be more activity on Z-RR-MCA (Barrett et al., 2004). Enzyme activities determined with native collagen (thus presenting a triple helix) correspond to the metalloproteinase collagenase.

6). Snakes venoms contain peptides with structural and functional equivalents of mammalian NPs (ANP, BNP and CNP), which present dose-dependent hypotensive effects [10], [34] and [40]. In addition to natriuretic peptide studies, a 38-amino acid residues peptide (DNP) was isolated from the venom of Dendroaspis angusticeps (the green mamba snake), demonstrating properties that are similar to both

Ion Channel Ligand Library chemical structure human ANP and BNP [33]. Other NPs from snake venoms were identified from Lachesis muta (Lm-CNP), Bothrops jararacusu (Bj-CNP) and other snakes presenting a homologous structure for the human CNP [28] and [35]. The hypotensive effect of Coa_NP2, presented herein, occurred in association with a significant increase in plasma nitrite levels, corroborating with previously data suggesting that NPs are able to stimulate nitric oxide (NO) production [4]. Together, a NO-dependent hypotensive effect was identified with a peptide isolated

from the snake venom of Agkistrodon acutus [34], and it was shown that infusion of NP isolated from Crotalus durissus cascavella venom was responsible for the increased nitrite levels [10]. Thus, these findings support the notion that Coa_NP2 exerts its hypotensive action, at least in part, through stimulation selleck screening library of NO production. As such, there are three different receptor isoforms for the NPs, namely, natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C), in which the human NP family have been shown natriuretic, diuretic, hypotensive and vasodilator actions [20] and [22]. It has recently been suggested that BNP exerts its vascular effects through the same pathway as ANP, i.e. the NPR-A. This guanylate cyclase-coupled receptor

is located both on endothelial and vascular smooth muscle cells [37]. Activation of NPR-A generates the second messenger cyclic guanosine monophosphate (cGMP) which, in turns, activates Ca2+ channels and ATP-sensitive K+-channels leading to vasorelaxation [21] and [29]. However, CNP binds to the NPR-B, a specific tetracosactide guanylate cyclase-coupled receptor, and it is located on the vascular smooth muscle cell, also leading to vasodilatation through hyperpolarization [19]. To evaluate the possible mechanisms responsible for these dose-dependent hypotensive effects, we used endothelium-denuded rings preparations (Coa_NP2-e−). It was observed that vasorelaxation produced by the Coa_NP2 in thoracic aortic rings precontracted with phenylephrine was endothelium-dependent, as evidenced by its abolition when it was used Coa_NP2-e−. (Coa_NP2-e+ or Coa_NP2-e− group, respectively, Fig. 5). Similar findings were revealed by other NPs originated from different snake venoms [10] and [38]. The vasorelaxant effect caused by Coa_NP2 seems not to be involved in the NP receptor type A (NPR-A).

A river has physical integrity when river process and form are actively connected under the current hydrologic and sediment regime. One component of ecological or physical integrity is sustainability. Sustainability

is most effectively defined within a specified time interval, but implies the ability to maintain existing conditions during that time interval. Another component of integrity is resilience, which refers to the ability Epacadostat clinical trial of a system to recover following disturbance. A resilient ecosystem recovers the abundance and diversity of organisms and species following a drought or a tropical cyclone, for example, and a resilient river recovers channel geometry and sediment fluxes following a large flood. Drawing on concepts of ecological and physical integrity, a composite definition for critical

zone integrity and sustainability might be a region in which critical zone processes respond to fluxes of matter and energy in a manner that sustains a landscape and an ecosystem with at least minimum levels of diversity. R428 price The core concept of this definition is that biotic and non-biotic processes can respond to fluctuations in matter and energy through time and space, rather than being rigidly confined to a static condition. In other words, hillslopes have the ability to fail in landslides during intense precipitation, rather than being shored up by rock bolts and retaining walls, and fish populations

have the ability to migrate to different portions of a river network in response to flooding or Demeclocycline drought, rather than being partitioned into sub-populations by impassable barriers such as dams or culverts. Layers of vagueness are built into this definition, however. Over what time span must the landscape and ecosystem be sustained? What constitutes an acceptable minimum level of physical or biological diversity? These are not simple questions to answer, but in addressing these questions for specific situations, geomorphologists can make vital and needed contributions to ongoing dialogs about how to preserve vitally important ecosystem services and biodiversity. Focusing on these questions can also force geomorphologists to explicitly include biota in understanding surface processes and landforms. The stabilization of hillslopes or the partitioning of rivers does not really matter in a purely physical context. Although geomorphologists may be interested to know that hillslopes cannot adjust because of stabilization or rivers cannot continue to move sediment downstream because of dams, these issues become critically important only in the context of increased hazards for humans in the hillslope example, or loss of ecosystem services for biotic communities in the dam example. The issues raised above are complex and difficult to address.