Ascophyllum nodosum as a nutrient supporting oral health in dogs and cats: a review.

Home dental care is a key element of periodontal therapy in veterinary patients. Among many strategies of passive home dental care there is a supplementation of animal diet with seaweed Ascophyllum nodosum which have been shown to reduce both calculus and plaque accumulation after oral administration in both dogs and cats. Ascophyllum nodosum contains numerous biologically active ingredients, including micro-elements, vitamins, and several other compounds, however the exact mechanism of its beneficial action remains unclear. The very first metabolomic data suggest that it could change the composition of dog saliva. Several products containing Ascophyllum nodosum had been assessed clinically according to standards and requirements provided by the Veterinary Oral Health Council. The conducted clinical trials in dogs and cats revealed that Ascophyllum nodosum exerts the strongest preventive action as powder, followed by dental bites and dry pet food. The data concerning its curative action are limited to one study in cats in which no beneficial action has been observed. Based on available clinical data it is recommended to administer Ascophyllum nodosum to dogs and cats after oral cavity prophylactic procedure to reduce the recurrence of plaque and calculus formation.

Reference intervals for transthoracic echocardiographic measurements in adult Dachshunds.

The aim of this study was to establish reference values for 2D and M-mode measurements in Dachshunds. Basic echocardiographic data, including M-mode, 2D and spectral Doppler mea- surements, was collected, analyzed and compared between 41 healthy Dachshunds and 50 other healthy dogs of similar weight. Echocardiographic reference intervals were prepared for Dachs- hunds. Dachshunds had a smaller left ventricular diameter in diastole and systole and a thicker septum than other dog breeds. Male Dachshunds had larger diastolic and systolic left ventricular diameter than females. Reference intervals for 2D and M-mode measurements in healthy Dachs- hunds differ from other dogs of similar weight and should be used for this breed to assess cham- ber enlargement.

Geographically predominant genotypes of Aspergillus terreus species complex in Austria: s microsatellite typing study.

Aspergillus terreus species complex is recognized as a frequent agent of invasive aspergillosis in Tyrol. The reason for this specific epidemiological situation is unclear. Aspergillus terreus strains isolated from environmental and clinical sources were genotyped using a novel panel of short tandem repeats and were evaluated for virulence. Three major endemic genotypes collected from the Inn region and its side valleys were found to cause the majority of invasive A. terreus infections. All of these genotypes were of the same mating type, which suggests that a mating barrier is present between these geographically well-adapted strains which is found to persist for at least 11 years. The three major genotypes were prevalent in both human infections and the environment. No major differences in virulence were observed using Galleria mellonella as model. Our data suggest a specific environmental exposure being responsible for the high incidence of A. terreus infections in Innsbruck, the Inn valley and side valleys (Tyrol, Austria).

Correlation between peripheral blood cell transcriptomic profile and clinical parameters of chronic mitral valve disease in Dachshunds.

Studies identifying specific pathologically expressed genes have been performed on diseased myocardial tissue samples, however less invasive studies on gene expression of peripheral blood mononucleated cells give promising results. This study assessed transcriptomic data that may be used to evaluate Dachshunds with chronic mitral valve disease. Dachshunds with different stages of heart disease were compared to a control, healthy group. Microarray data analysis revealed clusters of patients with similar expression profiles. The clusters were compared to the clinical classification scheme. Unsupervised classification of the studied groups showed three clusters. Clinical and laboratory parameters of patients from the cluster 1 were in accordance with those found in patients without heart disease. Data obtained from patients from the cluster 3 were typical of advanced heart failure patients. Comparison of the cluster 1 and 3 groups revealed 1133 differentially expressed probes, 7 significantly regulated process pathways and 2 significantly regulated Ariadne Metabolic Pathways. This study may serve as a guideline for directing future research on gene expression in chronic mitral valve disease.

Mismatch between transcriptomic and histopathologic picture of canine lymphomas.

Lymphoma is one of the most common malignant tumours occurring in dogs. Since there is a constant need for new, more comprehensive laboratory diagnostic tools which permit the precise determination of many tumour-related factors we decided to verify whether the use of microarray analysis could be helpful in classifying lymphomas. The study was performed on samples collected from 7 dogs in which multicentric lymphoma was recognized. Among this group we were able to identify one sub-cluster of transcriptionally similar tumours, which completely differed in terms of the histopathological examination. Among them there were one diffuse large B cell lymphoma, one diffuse macronucleolated medium-sized cell lymphoma and one pleomorphic mixed small and large T-cell lymphoma. The lymphomas belonging to the sub-cluster differed from other analysed tumours in the expression of more than 100 genes of which only 18 were described earlier in regard to lymphomas.

The transcriptomic signature of myostatin inhibitory influence on the differentiation of mouse C2C12 myoblasts.

GDF8 (myostatin) is a unique cytokine strongly affecting the skeletal muscle phenotype in human and animals. The aim of the present study was to elucidate the molecular mechanism of myostatin influence on the differentiation of mouse C2C12 myoblasts, using the global-transcriptome analysis with the DNA microarray technique. Treatment with exogenous GDF8 strongly affected the growth and development of C2C12 mouse myoblasts. This was manifested by the inhibition of proliferation and differentiation as well as the impairment of cell fusion. DNA microarray analysis revealed 778 genes regulated by GDF8 in differentiating myoblasts (436 down-regulated and 235 up-regulated). Ontological analysis revealed their involvement in 17 types of biological processes, 10 types of molecular functions and 68 different signalling pathways. The effect of GDF8 was mainly mediated by the disruption of the cell cycle, calcium and insulin signalling pathways and expression of cytoskeletal and muscle specific proteins. The identified key-genes that could play a role as GDF8 targets in differentiating myoblasts are: Mef2, Hgf, Ilbl, Itgb1, Edn1, Ppargc1a.

Gene expression profiling in peripheral blood nuclear cells in patients with refractory ischaemic end-stage heart failure.

Functional analysis of up- and down-regulated genes might reveal whether peripheral blood cells may be considered as a material of diagnostic or prognostic value in patients with end-stage heart failure (HF). The aim of the present study was to compare the transcriptomic profile of peripheral blood nuclear cells from 6 male patients with ischaemic end-stage HF with those of 6 male patients with asymptomatic cardiac dysfunction. The expression of genes in peripheral blood nuclear cells in both groups of patients was measured using whole-genome oligonucleotide microarrays utilizing 35 035 oligonucleotide probes. Microarray analyses revealed 130 down-regulated genes and 15 up-regulated genes in the patients with end-stage HF. Some of the down-regulated genes belonged to the pathways that other studies have shown to be down-regulated in cardiomyopathy. We also identified up-regulated genes that have been correlated with HF severity (CXCL16) and genes involved in the regulation of expression of platelet activation factor receptor (PTAFR, RBPSUH, MCC, and PSMA7). In conclusion, the identification of genes that are differentially expressed in peripheral blood nuclear cells of patients with HF supports the suggestion that this diagnostic approach may be useful in searching for the molecular predisposition for development of severe refractory HF in patients with post-infarction asymptomatic abnormalities and remodelling of the left ventricle. These results need further investigation and validation.

Transcriptional pattern of TGF-beta1 inhibitory effect on mouse C2C12 myoblasts differentiation.

The aim of the present study was to define the effect of TGF-beta1 on C2C12 myoblasts myogenesis. TGF-beta1 together with its receptor is a negative auto-paracrine regulator of myogenesis, which influences the proliferation, differentiation, and functions of muscle cells. TGF-beta1 exerts highly significant inhibitory effect on differentiation of C2C12 mouse myoblasts manifested by the impairment of cell fusion and very low expression of myosin heavy chain. The study of differentiating C2C12 mouse myoblasts treated with TGF-beta1 revealed 502 genes (436 down-regulated and 66 up-regulated) with statistically different expression. TGF-beta1-regulated genes were identified to be involved in 29 biological processes, 29 molecular functions groups and 59 pathways. The strongest inhibiting effect of TGF-beta1 was observed in the cadherin and Wnt pathways. The key-genes that could play the role of TGF-beta1 targets during myoblasts differentiation was identified such as: Max, Creb1, Ccna2, Bax, MdfL, Tef, Tubg1, Cxcl5, Rho, Calca and Lgals4.

Differences in growth and transcriptomic profile of bovine mammary epithelial monolayer and three-dimensional cell cultures.

Mammary epithelial cells (MECs) are characterized by specific spatial architecture with several distinguishing features such as: polarized morphology, specialized cell-cell contacts, and attachment to an underlying basement membrane. Three dimensional (3D) basement membrane cultures provide a unique opportunity to model the architecture of epithelium in vitro. The aim of this study was to characterize the growth of bovine mammary epithelial cell line BME-UV1 in 3D culture on Matrigel and identification of differently expressed genes in bovine MECs forming polarized structures in comparison to conventional monolayer (2D) cell culture. We demonstrate that BME-UV1 cells grown on Matrigel form polarized acinar structures during 16 days of culture. A microarray study has proven that the difference in spatial architecture between MECs cultured in monolayer and 3D system is reflected by differences in transcriptomic profile. Microarray data analysis showed 40 differentially expressed genes with statistical significance (p<0.05) and characterized biological functions. Identified genes comprised of cytoskeletal proteins, extracellular matrix components, kinases such as: Rac serine/threonine kinase, SRPK, protooncogene tyrosine-protein kinase ABL1, uridine cytidine kinase and proteins with nucleic acid binding / transcription factor activity. Products of those genes are involved in processes which are known to participate in regulating mammary gland polarization and function.

Transcriptomic index of skeletal muscle of beef breeds bulls.

In the present study cDNA microarray (18263 probes) were used for analysis of bovine skeletal muscle (m.semitendinosus) transcriptome in 12-month-old bulls of four cattle breeds: Holstein-Friesian (HF), Limousine (LIM), Hereford (HER) and Polish Red (PR), aiming to identify the genes, which expression is common for beef breed bulls. The number of transcripts significantly different from HF bulls muscle amounted to 393, 462 and 638 for LIM, HER and PR, respectively. As a result of this study the transcriptomic index was proposed, being the set of 48 genes expressed similarly in beef breed bulls in comparison to HF bulls. Classification of genes according to molecular function of their protein products has shown the highest number of genes encoding proteins involved in nucleic acid binding (10), regulatory proteins (6), kinases (4) and signaling molecules (3). Classification according to biological processes revealed the highest number of genes involved in protein metabolism i modification (14), signal transduction (5), cell cycle (4), intracellular protein traffic (4), nucleoside, nucleotide and nucleic acid metabolism (4), apoptosis (3), cell structure and motility (3), and cellular transport (3). Since the role of the most genes included to the transcriptomic index has not been described yet in bovine skeletal muscle, obtained results may be very useful in revealing new candidate genes to search a new criteria of animal selection in beef production.

Transcriptional dysregulation of skeletal muscle protein metabolism in streptozotocin-diabetic mice.

The purpose of the study was to evaluate potential changes in expression of genes involved in protein metabolism and myogenic differentiation markers in skeletal muscle of streptozotocin-diabetic mice. Microarray analysis revealed alterations in the expression of 84 gene transcripts in gastrocnemius muscle of diabetic mice. Regarding protein metabolism a marked downregulation in gene transcripts for: general transcription factor IIA1 (-1.88, P=0.016309), TATA box binding protein (-2.17, P=0.037373), eukaryotic translation initiation factor 4E nuclear import factor 1 (-1.61, P=0.037373), eukaryotic translation elongation factor Ibeta2 (-1.95, P=0.010406), ubiquitin-like 5 (-1.67, P=0.024975) and ubiquitin conjugating enzyme 7 interacting protein 1 (-1.68, P=0.016309) was observed. STZ-diabetes caused a drop in the expression of myogenin, whereas myostatin level was significantly elevated. In conclusion, 1) STZ-diabetes attenuates expression of gene transcripts involved in the process of transcription and translation, which may affect skeletal muscle protein synthesis and lead to nitrogen imbalance, 2) impaired expression of gene transcripts involved in the regulation and activity of the ubiquitin-proteasome pathway may contribute to attenuation of mechanisms eliminating damaged proteins in STZ-diabetes, 3) changes in the expression of key myogenic factors, manifested by a decrease in myogenin level and enhancement of myostatin expression may be one of the mechanisms limiting skeletal muscle growth and regeneration associated with diabetes.

Comparison of skeletal muscle transcriptional profiles in dairy and beef breeds bulls.

A cDNA microarray (18 263 probes) was used for transcriptome analysis of bovine skeletal muscle (m. semitendinosus) in 12-month-old bulls of the beef breed Limousin (LIM) and the typical dairy breed Holstein-Friesian (HF, used as a reference). We aimed to identify the genes whose expression may reflect the muscle phenotype of beef bulls. A comparison of muscle transcriptional profiles revealed significant differences in expression of 393 genes between HF and LIM. We classified biological functions of 117 genes with over 2-fold differences in expression between the examined breeds. Among them, 72 genes were up-regulated and 45 genes were down-regulated in LIM vs. HF. The genes were involved in protein metabolism and modifications (22 genes), signal transduction (15), nucleoside, nucleotide and nucleic acid metabolism (13), cell cycle (9), cell structure and motility (9), developmental processes (9), intracellular protein traffic (7), cell proliferation and differentiation (6), cell adhesion (6), lipid, fatty acid and steroid metabolism (5), transport (5), and other processes. For the purpose of microarray data validation, we randomly selected 4 genes: trip12, mrps30, pycrl, and c-erbb3. Real-time RT-PCR results showed similar trends in gene expression changes as those observed in microarray studies. Basing on results of the present study, we proposed a model of the regulation of skeletal muscle growth and differentiation, with a principal role of the somatotropic pathway. It may explain at least in part the development of muscle phenotype in LIM bulls. We assume that the growth hormone directly or indirectly (through IGF-1) activates the calcium-signaling pathway with calcineurin, which stimulates myogenic regulatory factors (MRFs) and inhibits early growth response gene. The inhibition results in indirect activation of MRFs and impaired activation of TGF-beta1 and myostatin, which finally facilitates terminal muscle differentiation.

Effect of temperature and salinity on the wastewater treatment performance of aerobic submerged fixed bed biofilm reactors.

The influence of temperature (5-35 C) and salinity (up to 20 g/l NaCl) on the wastewater purification process in completely mixed and aerated submerged fixed bed biofilm reactors (SFBBRs) was studied. C- and N-conversion in SFBBRs designed according to the DWA (German Association for Water, Wastewater and Waste) rules for carbon removal was investigated for several months on synthetic wastewater. The DOC degradation rate was even at, according to the DWA, high DOC/BOD loading rates not much affected by temperatures between 5-35 degrees C and salt contents up to 20 g/L NaCl. At these high DOC loadings an appreciable ammonium conversion could also be observed. The ammonium conversion proved to be sensitive to temperature and salinity. At 5 degrees C the ammonium removal rate decreased by a factor of five compared to 25-35 degrees C. Under many operation conditions investigated more than 50% of the converted ammonium was transformed into gaseous nitrogen. The addition of 20 g/L NaCl caused a strong inhibition of the ammonium removal rate over the whole temperature range investigated.

Age-dependent changes in bovine skeletal muscle transcriptomic profile.

The postnatal growth of muscle tissue occurs by hypertrophy comprising satellite cells proliferation, differentiation and protein turnover. The highest rate of skeletal muscle gains and protein synthesis in bulls occurs in the period between 180 and 360 days of postnatal life. However, genes which are responsible for quantitative and qualitative changes in skeletal muscle during this period are not identified up to date. The aim of our study was to compare the changes in transcriptomic profile of skeletal muscle (m. semitendinosus) in 12 Polish Black and White bulls between 6 and 12 month of life. For experimental purposes we used bovine cDNA microarray (the NBFGC EST collection) which contains 18,263 unique genes, derived from many different tissue types and various physiologically important states within these tissues. Our results revealed 53 genes which expression changed in the same manner depending on age of all examined pairs of animals. Thirty two of these genes showed at least 2-fold difference in expression between two analyzed age points. Age-dependent up-regulation was the most pronounced in the case of following genes: similar to MAD2L1 binding protein, similar to thymocyte protein thy28 isoform 1, similar to type I inositol-1,4,5-triphosphate 5-phosphatase, similar to nucleoside diphosphate kinase 6, proline rich 14, similar to transcription factor E2-alpha and phospholipase C gamma 1. The highest age-dependent decrease of the transcript was observed in the case of: similar to ubiquitin carboxy-terminal hydrolase L1, similar to latent TGF-beta binding protein 3 precursor, phospho-mannomutase 2, CD74 antigen, similar to BCL6 co-repressor-like 1, platelet/endothelial cell adhesion molecule (PECAM1), necdin, zygin, tight junction protein 3, ankyrin and apolipoprotein-L3. Although the role of the most of above genes and interactions between products of their expression is not clear at the moment, the significance of their response between 6 and 12 month of age may indicate their involvement in growth, development and metabolic changes in bovine skeletal muscle during the first year of postnatal life.

Transforming growth factor-beta1 upregulates myostatin expression in mouse C2C12 myoblasts.

Myostatin (MSTN) and transforming growth factor-beta1 (TGF-beta1) belong to the same TGF-beta superfamily of proteins. They are involved in regulation of skeletal muscle growth and development as well as muscle catabolism. The aim of the present study was to investigate the relationship between MSTN and TGF-beta1 expression in proliferating and differentiating mouse C2C12 myoblasts cultured in normal and catabolic conditions and to evaluate the effect of exogenous TGF-beta1 as well as "knock down" of TGF-beta1 receptor type II on MSTN expression in proliferating and differentiating myogenic cells. The direct effect of TGF-beta1 on myostatin was also examined. Myostatin expression increased gradually with cell confluency in proliferating cultures, while the level of TGF-beta1, detected in the form of a 100 kDa small latent complex diminished. Myostatin expression was accompanied by a partial cell cycle arrest. Three forms of myostatin were found: a 52 kDa precursor, a 40 kDa latency associated propeptide, and a 26 kDa active peptide. A decrease in myostatin and TGF-beta1 levels was observed during the first three days of differentiation, which was subsequently followed by significant increase of their expression during next three to four days of differentiation. Catabolic state induced by dexamethasone significantly increased the level of all forms of myostatin as well as latent (100 kDa) and active (25 kDa) forms of TGF-beta1 in differentiating myoblasts in a dose dependent manner. Exogenous TGF-beta1 (2 ng/ml) significantly increased myostatin levels both in proliferating and differentiating C2C12 myoblasts, whereas silencing of the TGF-beta1 receptor II gene significantly lowered myostatin level in examined cells. The presented results indicate that TGF-beta1 may control myostatin-related regulation of myogenesis through up-regulation of myostatin, predominantly in the course of terminal differentiation and glucocorticoid-dependent catabolic stimulation.

Rats with a glucocorticoid-induced catabolic state show symptoms of oxidative stress and spleen atrophy: the effects of age and recovery.

In this study we wanted to determine whether changes in antioxidant profile could follow the catabolic effects of glucocorticoids. We also wanted to compare resistance to glucocorticoid overload in young and old rats. To address these questions, whole body catabolism was induced by the administration of dexamethasone (Dex) at either 2 mg/kg bodyweight/day to young (6 weeks old) or 0.5 mg/kg body-weight/day to old (94 weeks old) rats. Bodyweight loss of pair-fed rats not given Dex was only 2% in the young rats and 8% in the old rats, whereas in Dex-treated rats the decrease in bodyweight was 22% in the young rats and 13% in the old rats after 5 days of treatment. Spleen weight decreased by 65% in the young rats and by 52% in the old rats. Additionally, in the young rats there was a 46% reduction in glutathione (GSH) in erythrocytes as well as a 36% reduction in GSH/tissue wet weight in the soleus muscle. The corresponding figures for the old rats were 35 and 26%, respectively. Taken together, these results suggest that Dex directly and/or indirectly impaired the antioxidant reactions. This was further confirmed by a significant (50%) decline in Cu-Zn superoxide dismutase (SOD-1) activity in erythrocytes isolated from the young rats treated with Dex but not the old rats as they showed a significant elevation in SOD-1 activity (by 101%). Thiobarbituric acid reactant substances were significantly higher in both young and old rats. Activity of blood plasma creatine kinase increased by 73% in the young rats and by 307% in the old rats treated with Dex. Although both the young and the old rats could recover from oxidative stress, the old rats in contrast to the young rats remained catabolic until the end of the experiment. In conclusion, we suggest that old rats are more vulnerable to the catabolic action of Dex, whereas young rats are more susceptible to the oxidative stress induced by Dex.

Creatine and beta-hydroxy-beta-methylbutyrate (HMB) additively increase lean body mass and muscle strength during a weight-training program.

We investigated whether creatine (CR) and beta-hydroxy-beta-methylbutyrate (HMB) act by similar or different mechanisms to increase lean body mass (LBM) and strength in humans undergoing progressive resistance-exercise training. In this double-blind, 3-wk study, subjects (n = 40) were randomized to placebo (PL; n = 10), CR (20.0 g of CR/d for 7 d followed by 10.0 g of CR/d for 14 d; n = 11), HMB (3.0 g of HMB/d; n = 9), or CR-and-HMB (CR/HMB; n = 10) treatment groups. Over 3 wk, all subjects gained LBM, which was assessed by bioelectrical impedance analysis. The CR, HMB and CR/HMB groups gained 0.92, 0.39, and 1.54 kg of LBM, respectively, over the placebo group, with a significant effect with CR supplementation (main effect P = 0.05) and a trend with HMB supplementation (main effect P = 0.08). These effects were additive because there was no interaction between CR and HMB (CR x HMB main effect P = 0.73). Across all exercises, HMB, CR, and CR/HMB supplementation caused accumulative strength increases of 37.5, 39.1, and 51.9 kg, respectively, above the placebo group. The exercise-induced rise in serum creatine phosphokinase was markedly suppressed with HMB supplementation (main effect P = 0.01). However, CR supplementation antagonized the HMB effects on serum creatine phosphokinase (CR x HMB interactive effect P = 0.04). Urine urea nitrogen and plasma urea were not affected by CR supplementation, but both decreased with HMB supplementation (HMB effect P < 0.05), suggesting a nitrogen-sparing effect. In summary, CR and HMB can increase LBM and strength, and the effects are additive. Although not definitive, these results suggest that CR and HMB act by different mechanisms.

Excess of glucocorticoids impairs whole-body antioxidant status in young rats. relation to the effect of dexamethasone in soleus muscle and spleen.

The action of glucocorticoids in high doses is catabolic, but not much is known about the accompanying effects on antioxidative capacity of the entire body. Animals were treated (or not) with dexamethasone (Dex) 2 mg/kg b.w. d-1 during 5 consecutive days followed by recovery, during which an additional group received 3-hydroxy-3-methylbutyrate (40 mg/kg b.w.). Animals were killed after treatment with Dex, and after 5 days of the recovery period. Dexamethasone treatment decreased appetite almost twofold (from 20 g/day to 10 g/day, P < 0.001). Feed restriction, however, seemed to have only minor impact on the effects observed since body weight loss of pair-fed rats after the 5th day of treatment was only 2% and Dex-treated rats decrease in body weight was 22% (P < 0.05). In turn, wet weight of the soleus muscle (expressed per body weight) did not significantly decrease after Dex treatment, suggesting relative resistance of oxidative type muscles to the catabolic action of dexamethasone. Spleen wet weight expressed per body weight dropped by 65% (P<0.001). Additionally, there was a 46% reduction (P<0.001) of blood glutathione (GSH/Hb), and 36% (P < 0.001) of muscle glutathione (GSH/tissue wet weight). This suggests that dexamethasone directly and/or indirectly impaired antioxidant reactions. This was further confirmed by a significant (49%) decline of SOD-1 activity in erythrocytes isolated from the group treated with dexamethasone. Another index of lipid peroxidation (TBARS) was also significantly increased. Activity of blood plasma CK increased by 73% (P<0.001) in Dex-treated rats, indicating moderate injury of muscle tissue. In conclusion, young growing rats were sensitive to the dosage of dexamethasone, but in contrast to lymphoid tissue, could easily compensate the outcomes of impaired antioxidative defence within 5 days of recovery.

Expression and biotinylation of a mutant of the transcarboxylase carrier protein from Propioni shermanii.

A deletion mutant (residues 10 to 48 cut) of the biotinyl subunit (tcc) from the enzyme transcarboxylase (EC 2.1.3.1) of Propioni shermanii was overexpressed in Escherichia coli. Complete biotinylation of the protein was achieved by addition of exogenous biotin and coexpression of the biotin holoenzyme synthetase (EC 6.3. 4.15.) from E. coli. The transcription of both genes was put under control of different operators/promoters, thus achieving independent control of expression levels and optimized yields of the holo-tcc. Bacteria were grown in a biotin-supplemented minimal medium (M9) that contained [(13)C]glucose as the carbon source and [(15)N]NH(4)Cl as the sole nitrogen source. The target protein could be purified to homogeneity by ion-exchange chromatography and concentrated to NMR-suitable concentrations (2 mM) without aggregation.