Among these preparations, S-allyl cysteine (SAC), one of the major organic garlic compounds that had been known to possess a powerful anti-oxidant property, can be candidate for potential formula compared with other organosulfur compounds, including diallyl bisulfide, diallyl trisulfide (DAT3S), or diallyl-tetrasulfide (DAT4S).[9, 10] Studies in mouse and rodent models have shown that SAC can be rapidly absorbed in the GI tract and has very low acute/subacute toxicity (LD50 value > 54.7 mM/kg oral; > 20 mM/kg intraperitoneal), which is 30-fold less toxic than other typical garlic compounds, such as allicin Selleck Ruxolitinib and diallyl disulfide.[11, 12] Moreover, SAC is two hydrophilic cysteine-containing compounds

naturally formed in CDK inhibitor garlic. It has been shown to exert anti-inflammatory and anti-oxidative effects in various models, but never been tried against GI inflammation.[13] Recently, phytoceuticals or phytochemicals were proven to possess the ability to orchestrate gastric inflammation more globally through histone deacetylator (HDAC) inhibitory activity, as well as potentiate the host defensive phase enzyme induction like heme oxygenase-1 (HO-1) through nuclear factor erythroid-derived 2-related factor (Nrf2) transcriptional activation, a transcription factor that in humans

is encoded by the NFE2L2 gene, of which Nrf2 anti-oxidant response pathway is “the primary cellular defense” against the cytotoxic effects of oxidative stress, in our study provoked by indomethacin. The object of our study was either to document the preventive efficacy of selleckchem SAC against indomethacin-induced gastric damage or to identify the underlying molecular mechanisms on how SAC could impose protection. An animal model of indomethacin-induced gastric damage model was established, and tumor necrosis

factor-α (TNF-α)-stimulated cell models were used for current experiment. All chemical reagents were obtained from Sigma (St. Louis, MO, USA). Synthetic SAC was provided by Pharmaking Co., Ltd (Seongnam, Korea). Rebamipide was provided by Otsuka Pharmaceutical Co., Ltd (Tokushima, Japan). PD98095 and SB202190 were provided from Calbiochem (Billerica, MA, USA). Western blotting detection reagents were obtained from Amersham Biotechnology (Bucks, UK). Antibodies for COX-2, β-actin, inducible nitric oxide synthase (iNOS), α-tubulin, superoxide dismutase-1 (SOD-1), glutathione peroxidase-2 (GPX-2), glutathione-S-transferase-π (GST-π), Nrf2, γ-Glutamylcysteine Synthetase (γ-GCS), NAD(P)H:quinone oxidoreductase-1 (NQO-1), phospho-extracellular-signal-regulated kinase (pERK), and ERK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for calcium-dependent phospholipase A2 (cPLA2), phosphor-IκBα, IκBα, p65, p50, phospho-p38, and p38 were purchased from Cell Signaling Technology (Denver, MA, USA). Antibody for heme oxygenase 1 (HO-1) was from R&D Systems, Inc. (Minneapolis, MN, USA).

Among these preparations, S-allyl cysteine (SAC), one of the major organic garlic compounds that had been known to possess a powerful anti-oxidant property, can be candidate for potential formula compared with other organosulfur compounds, including diallyl bisulfide, diallyl trisulfide (DAT3S), or diallyl-tetrasulfide (DAT4S).[9, 10] Studies in mouse and rodent models have shown that SAC can be rapidly absorbed in the GI tract and has very low acute/subacute toxicity (LD50 value > 54.7 mM/kg oral; > 20 mM/kg intraperitoneal), which is 30-fold less toxic than other typical garlic compounds, such as allicin click here and diallyl disulfide.[11, 12] Moreover, SAC is two hydrophilic cysteine-containing compounds

naturally formed in MAPK Inhibitor Library garlic. It has been shown to exert anti-inflammatory and anti-oxidative effects in various models, but never been tried against GI inflammation.[13] Recently, phytoceuticals or phytochemicals were proven to possess the ability to orchestrate gastric inflammation more globally through histone deacetylator (HDAC) inhibitory activity, as well as potentiate the host defensive phase enzyme induction like heme oxygenase-1 (HO-1) through nuclear factor erythroid-derived 2-related factor (Nrf2) transcriptional activation, a transcription factor that in humans

is encoded by the NFE2L2 gene, of which Nrf2 anti-oxidant response pathway is “the primary cellular defense” against the cytotoxic effects of oxidative stress, in our study provoked by indomethacin. The object of our study was either to document the preventive efficacy of selleck chemicals SAC against indomethacin-induced gastric damage or to identify the underlying molecular mechanisms on how SAC could impose protection. An animal model of indomethacin-induced gastric damage model was established, and tumor necrosis

factor-α (TNF-α)-stimulated cell models were used for current experiment. All chemical reagents were obtained from Sigma (St. Louis, MO, USA). Synthetic SAC was provided by Pharmaking Co., Ltd (Seongnam, Korea). Rebamipide was provided by Otsuka Pharmaceutical Co., Ltd (Tokushima, Japan). PD98095 and SB202190 were provided from Calbiochem (Billerica, MA, USA). Western blotting detection reagents were obtained from Amersham Biotechnology (Bucks, UK). Antibodies for COX-2, β-actin, inducible nitric oxide synthase (iNOS), α-tubulin, superoxide dismutase-1 (SOD-1), glutathione peroxidase-2 (GPX-2), glutathione-S-transferase-π (GST-π), Nrf2, γ-Glutamylcysteine Synthetase (γ-GCS), NAD(P)H:quinone oxidoreductase-1 (NQO-1), phospho-extracellular-signal-regulated kinase (pERK), and ERK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for calcium-dependent phospholipase A2 (cPLA2), phosphor-IκBα, IκBα, p65, p50, phospho-p38, and p38 were purchased from Cell Signaling Technology (Denver, MA, USA). Antibody for heme oxygenase 1 (HO-1) was from R&D Systems, Inc. (Minneapolis, MN, USA).

The characteristics of auditory functions and vestibular BMN 673 mw symptoms and signs were assessed and reviewed by a blinded physician. Results.— The whole sample was found audiologically normal. In group A, 6 subjects had normal vestibular test results, whereas vestibular testing disclosed either peripheral

or central sufferance or both, in the remaining 16 patients (73%). Twelve subjects from group B had normal vestibular test results whereas positive vestibular test results were reported in the remaining 6 subjects (33%). Conclusions.— This single-blind work outlines the brain stem abnormalities in children with migraine in the form of direct involvement of peripheral or central vestibular pathways or both. Interestingly, some children with migraine but without vestibular symptoms also had abnormal results at vestibular testing. This could demonstrate a subclinical

involvement of vestibular pathways without clinical presentation. The subjects are still being followed up to evaluate the evolution of symptomatology. “
“Background.— Headache Crenolanib purchase is one of the most common medical complaints reported by individuals suffering from human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), but limited and conflicting data exist regarding their prevalence, prototypical characteristics, and relationship to HIV disease variables in the current era of highly active antiretroviral therapy (HAART). Objectives.— The aims of see more the present cross-sectional study were to characterize headache symptoms among patients with HIV/AIDS and to assess relations between headache and HIV/AIDS disease variables. Methods.— Two hundred HIV/AIDS patients (49% female; mean age = 43.22 ± 12.30 years; 74% African American) from an internal medicine clinic and an AIDS outreach clinic were administered a structured headache diagnostic interview to assess headache characteristics and features consistent with International Classification of Headache Disorders (ICHD)-II diagnostic semiologies. They also

completed 2 measures of headache-related disability. Prescribed medications, most recent cluster of differentiation (CD4) cell count, date of HIV diagnosis, possible causes of secondary headache, and other relevant medical history were obtained via review of patient medical records. Results.— One hundred seven patients (53.5%) reported headache symptoms, the large majority of which were consistent with characteristics of primary headache disorders after excluding 4 cases attributable to secondary causes. Among those who met criteria for a primary headache disorder, 88 (85.44%) met criteria for migraine, most of which fulfilled ICHD-II appendix diagnostic criteria for chronic migraine. Fifteen patients (14.56%) met criteria for episodic or chronic tension-type headache.

The characteristics of auditory functions and vestibular selleck kinase inhibitor symptoms and signs were assessed and reviewed by a blinded physician. Results.— The whole sample was found audiologically normal. In group A, 6 subjects had normal vestibular test results, whereas vestibular testing disclosed either peripheral

or central sufferance or both, in the remaining 16 patients (73%). Twelve subjects from group B had normal vestibular test results whereas positive vestibular test results were reported in the remaining 6 subjects (33%). Conclusions.— This single-blind work outlines the brain stem abnormalities in children with migraine in the form of direct involvement of peripheral or central vestibular pathways or both. Interestingly, some children with migraine but without vestibular symptoms also had abnormal results at vestibular testing. This could demonstrate a subclinical

involvement of vestibular pathways without clinical presentation. The subjects are still being followed up to evaluate the evolution of symptomatology. “
“Background.— Headache see more is one of the most common medical complaints reported by individuals suffering from human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), but limited and conflicting data exist regarding their prevalence, prototypical characteristics, and relationship to HIV disease variables in the current era of highly active antiretroviral therapy (HAART). Objectives.— The aims of selleck the present cross-sectional study were to characterize headache symptoms among patients with HIV/AIDS and to assess relations between headache and HIV/AIDS disease variables. Methods.— Two hundred HIV/AIDS patients (49% female; mean age = 43.22 ± 12.30 years; 74% African American) from an internal medicine clinic and an AIDS outreach clinic were administered a structured headache diagnostic interview to assess headache characteristics and features consistent with International Classification of Headache Disorders (ICHD)-II diagnostic semiologies. They also

completed 2 measures of headache-related disability. Prescribed medications, most recent cluster of differentiation (CD4) cell count, date of HIV diagnosis, possible causes of secondary headache, and other relevant medical history were obtained via review of patient medical records. Results.— One hundred seven patients (53.5%) reported headache symptoms, the large majority of which were consistent with characteristics of primary headache disorders after excluding 4 cases attributable to secondary causes. Among those who met criteria for a primary headache disorder, 88 (85.44%) met criteria for migraine, most of which fulfilled ICHD-II appendix diagnostic criteria for chronic migraine. Fifteen patients (14.56%) met criteria for episodic or chronic tension-type headache.

This selleck compound has led to a rise in H. pylori-negative PUD [21]. Meta-analysis of currently available studies shows an overall decline of both incidence and prevalence of PUD [21]. Current population-based incidences of PUD diagnoses range between 100/100.000 and 190/100.000 per year in Western countries, and current population-based prevalences range between 120/100.000 and 4700/100.000.

The prevalence of PUD in endoscopic series was reported in three large series, ranging from 5 to 16% of patients undergoing upper gastrointestinal endoscopy [21]. Remarkably, data on epidemiology of PUD in Asian countries are lacking. In contrast to the clear declining incidence of PUD, available data on the incidence of PUD complications are conflicting. Some studies have shown a stable rate of hospitalizations for complicated PUD [22–24], whereas others showed a decreasing incidence of complications, probably because Selleck Carfilzomib of widespread use of proton-pump

inhibitors [25,26]. The most common complication of PUD is bleeding. This occurs in about 10–20% of patients with H. pylori-associated PUD and is the most common cause of nonvariceal upper gastrointestinal bleeding. Bleeding of peptic ulcer is a complication with major morbidity and mortality. As the risk for recurrent PUD bleeding is high, adequate management should include identification of the etiology of the ulcer, followed by proper targeted treatment. A recent international consensus therefore strongly recommended to always test patients with peptic ulcer bleeding for H. pylori infection [27]. H. pylori-positive patients need eradication therapy followed by testing to confirm eradication [27]. A negative H. pylori test in the acute setting should be repeated during follow-up,

as the negative predictive value of H. pylori tests is low in this situation [27]. Testing for H. pylori should also be performed in patients with PUD bleeding while taking NSAIDs or aspirin. Guidelines advise to manage all etiologic factors contributing to PUD bleeding, and give adequate selleck inhibitor gastroprotection to those cases who require continuation of the NSAID or aspirin [27]. A recent randomized trial showed that H. pylori eradication in NSAID users was associated with healing of gastritis despite continued NSAID use [28]. A recent randomized trial in 156 patients at high risk for secondary cardiovascular complications comparing early versus late continuation of aspirin after PUD bleeding showed that early aspirin continuation during PPI gastroprotection was associated with a higher risk of rebleeding, but a lower risk of mortality because of the reduction in cardiovascular events [29]. Finally, recent research in this area has led to the identification of larger numbers of patients with idiopathic, H. pylori-negative, aspirin and NSAID-negative PUD. A cohort study of 333 patients with PUD bleeding showed that H.

This selleck has led to a rise in H. pylori-negative PUD [21]. Meta-analysis of currently available studies shows an overall decline of both incidence and prevalence of PUD [21]. Current population-based incidences of PUD diagnoses range between 100/100.000 and 190/100.000 per year in Western countries, and current population-based prevalences range between 120/100.000 and 4700/100.000.

The prevalence of PUD in endoscopic series was reported in three large series, ranging from 5 to 16% of patients undergoing upper gastrointestinal endoscopy [21]. Remarkably, data on epidemiology of PUD in Asian countries are lacking. In contrast to the clear declining incidence of PUD, available data on the incidence of PUD complications are conflicting. Some studies have shown a stable rate of hospitalizations for complicated PUD [22–24], whereas others showed a decreasing incidence of complications, probably because click here of widespread use of proton-pump

inhibitors [25,26]. The most common complication of PUD is bleeding. This occurs in about 10–20% of patients with H. pylori-associated PUD and is the most common cause of nonvariceal upper gastrointestinal bleeding. Bleeding of peptic ulcer is a complication with major morbidity and mortality. As the risk for recurrent PUD bleeding is high, adequate management should include identification of the etiology of the ulcer, followed by proper targeted treatment. A recent international consensus therefore strongly recommended to always test patients with peptic ulcer bleeding for H. pylori infection [27]. H. pylori-positive patients need eradication therapy followed by testing to confirm eradication [27]. A negative H. pylori test in the acute setting should be repeated during follow-up,

as the negative predictive value of H. pylori tests is low in this situation [27]. Testing for H. pylori should also be performed in patients with PUD bleeding while taking NSAIDs or aspirin. Guidelines advise to manage all etiologic factors contributing to PUD bleeding, and give adequate this website gastroprotection to those cases who require continuation of the NSAID or aspirin [27]. A recent randomized trial showed that H. pylori eradication in NSAID users was associated with healing of gastritis despite continued NSAID use [28]. A recent randomized trial in 156 patients at high risk for secondary cardiovascular complications comparing early versus late continuation of aspirin after PUD bleeding showed that early aspirin continuation during PPI gastroprotection was associated with a higher risk of rebleeding, but a lower risk of mortality because of the reduction in cardiovascular events [29]. Finally, recent research in this area has led to the identification of larger numbers of patients with idiopathic, H. pylori-negative, aspirin and NSAID-negative PUD. A cohort study of 333 patients with PUD bleeding showed that H.

Here, we undertook

a broad study of the phosphorus (P)–related behavior of marine Synechococcus isolates from all previously described ribotypes (sensu Fuller et al. 2003). A wide variability in P-related physiology was noted among members of this genus, particularly in the CP 868596 utilization of organic P sources. However, some characteristics (e.g., cell size change during P limitation and the ability to accumulate polyphosphate) were largely consistent with their phylogenetic lineage and inferred ecology, with clear distinctions between oligotrophic, mesotrophic, and opportunistic lineages. Similarly, the ability to induce protein expression in response to P limitation was consistent with the presence/absence of phoB/R regulatory capacity of the corresponding strain. Taxonomic differences in P uptake, storage, and utilization strategies could explain the ubiquitous distribution of marine Synechococcus throughout the world’s oceans and explain the coexistence and/or ecological partitioning

of multiple learn more phototrophic taxa in the photic zone of tropical and subtropical oligotrophic oceans. “
“Asexual reproduction by cloning may affect the genetic structure of populations, their potential to evolve, and, among foundation species, contributions to ecosystem functions. Macroalgae of the genus Fucus are known to produce attached click here plants only by sexual recruitment. Recently, however, clones of attached plants recruited by asexual reproduction were observed in a few populations of Fucus radicans Bergström

et L. Kautsky and F. vesiculosus L. inside the Baltic Sea. Herein we assess the distribution and prevalence of clonality in Baltic fucoids using nine polymorphic microsatellite loci and samples of F. radicans and F. vesiculosus from 13 Baltic sites. Clonality was more common in F. radicans than in F. vesiculosus, and in both species it tended to be most common in northern Baltic sites, although variation among close populations was sometimes extensive. Individual clonal lineages were mostly restricted to single or nearby locations, but one clonal lineage of F. radicans dominated five of 10 populations and was widely distributed over 550 × 100 km of coast. Populations dominated by a few clonal lineages were common in F. radicans, and these were less genetically variable than in other populations. As thalli recruited by cloning produced gametes, a possible explanation for this reduced genetic variation is that dominance of one or a few clonal lineages biases the gamete pool resulting in a decreased effective population size and thereby loss of genetic variation by genetic drift. Baltic fucoids are important habitat-forming species, and genetic structure and presence of clonality have implications for conservation strategies.

Here, we undertook

a broad study of the phosphorus (P)–related behavior of marine Synechococcus isolates from all previously described ribotypes (sensu Fuller et al. 2003). A wide variability in P-related physiology was noted among members of this genus, particularly in the check details utilization of organic P sources. However, some characteristics (e.g., cell size change during P limitation and the ability to accumulate polyphosphate) were largely consistent with their phylogenetic lineage and inferred ecology, with clear distinctions between oligotrophic, mesotrophic, and opportunistic lineages. Similarly, the ability to induce protein expression in response to P limitation was consistent with the presence/absence of phoB/R regulatory capacity of the corresponding strain. Taxonomic differences in P uptake, storage, and utilization strategies could explain the ubiquitous distribution of marine Synechococcus throughout the world’s oceans and explain the coexistence and/or ecological partitioning

of multiple selleck phototrophic taxa in the photic zone of tropical and subtropical oligotrophic oceans. “
“Asexual reproduction by cloning may affect the genetic structure of populations, their potential to evolve, and, among foundation species, contributions to ecosystem functions. Macroalgae of the genus Fucus are known to produce attached selleck chemicals llc plants only by sexual recruitment. Recently, however, clones of attached plants recruited by asexual reproduction were observed in a few populations of Fucus radicans Bergström

et L. Kautsky and F. vesiculosus L. inside the Baltic Sea. Herein we assess the distribution and prevalence of clonality in Baltic fucoids using nine polymorphic microsatellite loci and samples of F. radicans and F. vesiculosus from 13 Baltic sites. Clonality was more common in F. radicans than in F. vesiculosus, and in both species it tended to be most common in northern Baltic sites, although variation among close populations was sometimes extensive. Individual clonal lineages were mostly restricted to single or nearby locations, but one clonal lineage of F. radicans dominated five of 10 populations and was widely distributed over 550 × 100 km of coast. Populations dominated by a few clonal lineages were common in F. radicans, and these were less genetically variable than in other populations. As thalli recruited by cloning produced gametes, a possible explanation for this reduced genetic variation is that dominance of one or a few clonal lineages biases the gamete pool resulting in a decreased effective population size and thereby loss of genetic variation by genetic drift. Baltic fucoids are important habitat-forming species, and genetic structure and presence of clonality have implications for conservation strategies.

13 If present in high titer, anti-LKM1 strongly support the diagnosis of AIH, even if liver biopsy is precluded by other clinical

considerations. The mainstay technique for autoantibody screening is indirect immunofluorescence on composite sections of freshly frozen rodent stomach, kidney and liver.14 This technique not only permits the detection of ANA, SMA, anti-LKM1, and AMA but also suggests the presence of other autoantibodies of an evolving clinical importance, such as antibody learn more to liver cytosol type 1 (anti-LC1)111,121 and antibody to liver kidney microsome type 3 (anti LKM-3).122,123 Confirmation of the presence of the latter autoantibody is obtained with assays detecting antibodies to their molecular targets, formiminotransferase Ferrostatin-1 research buy cyclo-deaminase (FTCD) and family 1 UDP-glucuronosyl-transferases (UGT1A), respectively (Table 4). Other autoantibodies that may be useful in classifying patients who lack the conventional serological findings are anti-SLA124-128 and

atypical pANCA.119,129,130,131-139 Atypical pANCA, originally considered specific for PSC and inflammatory bowel disease (IBD),124,125 are frequently present in patients with AIH,126,127 and occasionally can be the only autoantibodies detected (Table 4).128 ANCA typically do not coexist with anti-LKM1.127 Recent evidence indicates that the target of atypical pANCA is located within the nuclear membrane. For this reason, a more suitable designation may be peripheral anti-neutrophil nuclear antibody (pANNA) (Table 4).102,103 Anti-SLA129 and anti–liver-pancreas (anti-LP),130 originally described as separate autoantibodies in AIH, were later found to target the same antigen and to

represent a single serological entity. These antibodies are now referred to as anti-SLA or anti-SLA/LP. Their molecular target is a transfer ribonucleoprotein (Table 4).119,131,132 SLA has recently been renamed SEPSECS (Sep [O-phosphoserine] see more tRNA synthase) Selenocysteine Synthase. Anti-SLA are occasionally found in patients with AIH who are negative for ANA, SMA, and anti-LKM1,133 but are more commonly found in association with the conventional autoantibodies, especially if sensitive immunoassays are used.133-136 Anti-SLA are highly specific for the diagnosis of autoimmune liver disease,133 and their detection may identify patients with more severe disease and worse outcome.137-140 Commercial ELISAs are available for their detection. The conventional and nonstandard autoantibodies described in AIH are shown in Table 4. Figure 4 provides an algorithm for the use of autoantibodies in the diagnosis of AIH. Multiple genetic associations with AIH have been described in different ethnic groups.29,141-154 The primary genetic association is with the major histocompatibility complex locus, and associations of HLA alleles with disease predisposition, clinical phenotype, response to therapy, and outcome have been studied.

13 If present in high titer, anti-LKM1 strongly support the diagnosis of AIH, even if liver biopsy is precluded by other clinical

considerations. The mainstay technique for autoantibody screening is indirect immunofluorescence on composite sections of freshly frozen rodent stomach, kidney and liver.14 This technique not only permits the detection of ANA, SMA, anti-LKM1, and AMA but also suggests the presence of other autoantibodies of an evolving clinical importance, such as antibody CB-839 in vivo to liver cytosol type 1 (anti-LC1)111,121 and antibody to liver kidney microsome type 3 (anti LKM-3).122,123 Confirmation of the presence of the latter autoantibody is obtained with assays detecting antibodies to their molecular targets, formiminotransferase see more cyclo-deaminase (FTCD) and family 1 UDP-glucuronosyl-transferases (UGT1A), respectively (Table 4). Other autoantibodies that may be useful in classifying patients who lack the conventional serological findings are anti-SLA124-128 and

atypical pANCA.119,129,130,131-139 Atypical pANCA, originally considered specific for PSC and inflammatory bowel disease (IBD),124,125 are frequently present in patients with AIH,126,127 and occasionally can be the only autoantibodies detected (Table 4).128 ANCA typically do not coexist with anti-LKM1.127 Recent evidence indicates that the target of atypical pANCA is located within the nuclear membrane. For this reason, a more suitable designation may be peripheral anti-neutrophil nuclear antibody (pANNA) (Table 4).102,103 Anti-SLA129 and anti–liver-pancreas (anti-LP),130 originally described as separate autoantibodies in AIH, were later found to target the same antigen and to

represent a single serological entity. These antibodies are now referred to as anti-SLA or anti-SLA/LP. Their molecular target is a transfer ribonucleoprotein (Table 4).119,131,132 SLA has recently been renamed SEPSECS (Sep [O-phosphoserine] check details tRNA synthase) Selenocysteine Synthase. Anti-SLA are occasionally found in patients with AIH who are negative for ANA, SMA, and anti-LKM1,133 but are more commonly found in association with the conventional autoantibodies, especially if sensitive immunoassays are used.133-136 Anti-SLA are highly specific for the diagnosis of autoimmune liver disease,133 and their detection may identify patients with more severe disease and worse outcome.137-140 Commercial ELISAs are available for their detection. The conventional and nonstandard autoantibodies described in AIH are shown in Table 4. Figure 4 provides an algorithm for the use of autoantibodies in the diagnosis of AIH. Multiple genetic associations with AIH have been described in different ethnic groups.29,141-154 The primary genetic association is with the major histocompatibility complex locus, and associations of HLA alleles with disease predisposition, clinical phenotype, response to therapy, and outcome have been studied.