Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a ∼2–3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased ∼3–4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1α-1 (eEF1α1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids ∼10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.
Reactive oxygen species (ROS)1and reactive nitrogen species are generated as a result of normal metabolic processes in the cell, including the uncoupling of the electron transport chain in the mitochondria and the oxidation of excess NADPH by NADPH oxidase (1). Oxidative stress occurs due to increased pro-oxidative conditions or the decline of antioxidant systems and is highly correlated with a wide variety of inflammatory and metabolic disease states, including Alzheimer disease, macular degeneration, lung dysfunction, and obesity-linked insulin resistance (2–5). Although the precise role of oxidative stress in disease mechanisms is not completely understood, ROS are known to be highly reactive with proteins, DNA, carbohydrates, and lipids in the cell. The ROS-initiated peroxidation of polyunsaturated acyl chains of membrane phospholipids can result in a Hock cleavage and the subsequent formation of lipid-derived reactive aldehydes (6).
Of the variety of reactive aldehydes formed from lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal (4-ONE) have significant contributions to oxidative disease due to their high abundance and strong reactivity (6–9). 4-HNE specifically is metabolized (detoxified) by being reduced to an alcohol (aldehyde reductase), oxidized to a carboxylic acid (aldehyde dehydrogenase), or conjugated to glutathione (glutathione S-transferase) (10–12). However, some fraction of 4-HNE escapes metabolism and forms adducts with other nucleophiles including both protein and DNA. In the case of protein, 4-HNE reacts avidly with the side chains of cysteine and histidine residues as well as lysine albeit to a far lesser extent (13). Although the reactive lipid is formed in membranes, it can diffuse into the cytoplasm and nucleus due to its relatively high solubility, allowing it to react with proteins localized far away from the initial site of oxidative formation (14).
4-HNE modification of protein has been shown to have a variety of effects on its targets. Alkylation by 4-HNE frequently inhibits the activity of enzymes; examples include the Na+-K+-ATPase and NADP+-dependent isocitrate dehydrogenase (15, 16). In some cases, however, 4-HNE modification has been suggested to increase the activity of key regulatory proteins such as the dimerization and ligand-independent activation of the epidermal growth factor receptor (17) or the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor, leading to increased expression of genes implicated in the antioxidant response (18–20). In addition to altering the activity of enzymes, 4-HNE alkylation has been shown to alter the rate of degradation of some proteins (alcohol dehydrogenase and αB-crystallin) (21, 22).
Obesity-linked insulin resistance has recently been causally linked to the development of ER stress and an increase in ROS (5, 23, 24). Adipocytes isolated from C57Bl/6J mice with diet-induced obesity have increased reactive oxygen species (25). Oxidative stress in human and murine adipose tissue as well as in cultured 3T3-L1 adipocytes affects the secretion of adipokines such as adiponectin and tumor necrosis factor α (5, 26, 27). As adipokines secreted by adipose tissue have dramatic effects on insulin sensitivity in liver and muscle, determining the effects of oxidative stress in adipose tissue is an important step in understanding the molecular mechanisms of type 2 diabetes and systemic energy metabolism (28). The modification of adipose proteins with lipid peroxidation products could be one contributing factor linking oxidative stress to insulin resistance.
In this study we investigated the modification of adipose proteins by 4-HNE and other aldehydes in lean and obese C57Bl/6J mice. To accomplish this, hydrazide chemistry was used for coupling with aldehyde adducts on proteins (29). Biotin hydrazide tagging allowed evaluation of the relative extent of carbonylation as well as the identification of novel targets of such modifications, including the adipocyte fatty acid-binding protein (A-FABP).
EZ-link biotin hydrazide, FITC-streptavidin, poly-HRP streptavidin, and ImmunoPure Immobilized Monomeric Avidin kit were purchased from Pierce. 4-HNE was purchased from Cayman Chemicals (Ann Arbor, MI). Rabbit anti-4-HNE antibody was purchased from Alpha Diagnostic International (San Antonio, TX). Mouse anti-glutathione S-transferase A4 (GSTA4) antibody was purchased from Abnova Corp. (Taipei, Taiwan). Sequencing grade modified trypsin was purchased from Promega. Endoproteinase Glu-C was purchased from Roche Applied Science. 1-Anilinonaphthalene 8-sulfonic acid (1,8-ANS) was purchased from Molecular Probes, Inc. ECL Western blotting detection reagents were purchased from Amersham Biosciences. C4 and C18 Zip Tip pipette tips and YM10 ultrafiltration membranes were purchased from Millipore (Billerica, MA). C18 SepPak cartridges were purchased from Waters (Milford, MA). High fat, high carbohydrate diet F3282 was purchased from Bioserve Industries (Frenchtown, NJ). (R)-4-HNE and (S)-4-HNE were synthesized as described previously (10).
C57Bl/6J mice were weaned onto high fat or chow diet at 3 weeks of age to induce obesity-linked insulin resistance (30). Using this feeding regimen, at 12 weeks of age the animals exhibit impaired insulin and glucose tolerance as evaluated using glucose and insulin tolerance tests as well as clamping studies (31). The animals were housed on a 12-h light/dark cycle and fed ad libitum with continual access to water. The mice were sacrificed by cervical dislocation at 12 weeks of age, and epididymal adipose tissue was recovered. Tissue samples were dissected and stored at −80 °C until needed. The University of Minnesota Institutional Animal Care and Use Committee approved all experiments.
Adipose tissue samples were homogenized in 100 mm sodium acetate, 20 mm NaCl, 0.1 mm EDTA, pH 5.5 (coupling buffer) supplemented with 0.1 mm PMSF, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 2 μg/ml leupeptin. The extract was subjected to centrifugation (100,000 × g for 1 h) at 4 °C, the floated lipid cake was removed, and the soluble protein fraction was frozen at −80 °C until needed. Protein concentration was determined using the BCA assay. For aldehyde detection, extracts (50 μg/sample) were incubated with a final concentration of 0.5 mm EZ-link biotin hydrazide (5 mm stock prepared fresh in DMSO) in coupling buffer for 2 h at room temperature, resolved by SDS-PAGE (14% acrylamide), and transferred to PVDF membrane. The membranes were blocked overnight at 4 °C in PBS containing 0.05% Tween 20 (PBST) with 10 mg/ml BSA, washed in PBST, and incubated for 1 h at room temperature with either FITC-conjugated streptavidin (1:50 dilution) or HRP-conjugated streptavidin (1:10,000 dilution). The biotin-avidin interaction was detected using a Fuji imager or by ECL.
For immunodetection of 4-HNE modification of proteins, membranes were blocked in PBST with 10 mg/ml BSA at 4 °C overnight and incubated with anti-4-HNE rabbit polyclonal antibody (1:1,000 dilution) at 4 °C for 16 h in PBST with 1 mg/ml BSA. The membranes were washed in PBST and incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution) at room temperature for 1 h, and immunoreactivity was detected via ECL. For detection of A-FABP or GSTA4, anti-A-FABP rabbit polyclonal antibody (1:10,000 dilution) or anti-GSTA4 mouse polyclonal antibody (1:2,500) were used for the primary antibody incubation. HRP-conjugated goat anti-mouse secondary antibody (1:10,000 dilution) was used for GSTA4 detection specifically. For both A-FABP and GSTA4 detection, blocking conditions of 10 mg/ml BSA were used throughout the procedures.
Soluble adipose tissue protein (10 mg) was incubated with 0.5 mm EZ-link biotin hydrazide in coupling buffer for 2 h at room temperature and dialyzed exhaustively against PBS, pH 7.4, at 4 °C. To remove proteins that bound to Sepharose nonspecifically, the extract was initially passed through a 1-ml column of Sepharose 4B at room temperature, and the flow-through was applied to a monomeric avidin column for 1 h to maximize binding. The column was washed with PBS containing 0.5 m NaCl to remove nonspecifically bound proteins, and specifically bound protein was eluted by the addition of 2 mm d-biotin. The eluted proteins were precipitated with chloroform and methanol, dried under nitrogen, and stored at −20 °C until used (32).
The carbonylated proteins were resolubilized in 250 μl of 50 mm NH4HCO3, 1 mm CaCl2, pH 8.5 (trypsin digestion buffer) and digested with 10 μg of trypsin for 15 h at 37 °C. The digestion was stopped by acidification with TFA, and the samples were applied to SepPak C18 cartridges to desalt and remove undigested protein. The peptides were eluted with 80% acetonitrile, 0.1% TFA in water; dried; and reconstituted in 20 μl of 0.1% formic acid.
Analysis of peptides by LC-MS/MS was achieved using the methods described previously (33, 34). Briefly each 20-μl sample was loaded onto a precolumn for concentrating and desalting followed by a C18 in-line analytical capillary column (75-μm inner diameter × 12 cm). The peptides were eluted over a linear gradient of 10–35% acetonitrile in 0.1% formic acid over 60 min at a flow rate of ∼250 nl/min followed by isocratic elution at 80% acetonitrile in 0.1% formic acid. Tandem MS analysis of the eluted peptides was performed using either an LCQ classic ion trap mass spectrometer (Finnigan Mat, San Francisco, CA) or an LTQ linear ion trap mass spectrometer system (Thermo Fisher Scientific, Waltham, MA). For each MS survey scan, the top three most abundant precursor ions were selected for MS/MS on the LCQ (33), and the top four most intense precursor ions were selected on the LTQ (34). The instrument settings used for data collection on each platform were identical to those published previously (33, 34), and Xcalibur 2.0 software was used to create the peak lists.
Proteins were identified by analyzing fragment and parent ions using BioWorks 3.2 software and the SEQUEST search algorithm (35) using the International Protein Index (IPI) mouse database, version 3.03 (the database contained 42,402 protein entries at the time it was searched), appended with a reversed database containing reversed sequences of each protein to facilitate false positive rate estimations (36). Oxidation of methionine was included as a variable modification. Mass tolerance was set at 2.0 for parent ions and 1.0 for fragment ions, and trypsin was specified as the enzyme allowing for two internal missed cleavages. Each peptide match was assigned a probability score (p score) using the program Peptide Prophet (37). The probability score cutoff value was set at 0.2 for proteins identified from multiple hits (two or more unique peptides) and at 0.90 for single hits. The false positive rate using these thresholds was below 1%, as estimated by reverse database searching, and consistent with our previous studies (38).
Recombinant epithelial fatty acid-binding protein (E-FABP) and A-FABP were expressed in Escherichia coli and purified as described previously (39, 40) using a combination of acid and protamine sulfate fractionation followed by gel filtration chromatography using Sephadex G-75. For E-FABP an additional cation exchange chromatography step was utilized. Histidine-tagged A-FABP (His-A-FABP) was expressed in E. coli and purified by nickel affinity chromatography and gel filtration chromatography as described previously (41). A-FABP was purified from mouse adipose tissue by methods similar to those described previously for purification from 3T3-L1 adipocytes (42).
Purified E-FABP was incubated with or without 0.5 mm 4-HNE for 20 min at 22 °C in 10 mm potassium phosphate, 150 mm NaCl, pH 7.4 (standard 4-HNE adduction buffer). The reaction was quenched with 100 mm β-mercaptoethanol (βME), and the protein was dialyzed exhaustively to remove excess 4-HNE and βME. C4 ZipTips were used to desalt the sample, and the presence of a 4-HNE adduct on E-FABP was confirmed by MALDI-TOF MS. For A-FABP, the protein was incubated with 4-HNE in 10 mm potassium phosphate, 10 mm NaCl, pH 7.4 (low salt 4-HNE adduction buffer), and the protein was spotted on a MALDI-TOF target without the use of a ZipTip after the reaction was stopped by acidification with TFA.
MALDI-TOF spectra for undigested proteins were collected as described previously (43). Briefly the samples were spotted with sinapinic acid as the matrix (3,5-dimethoxy-4-hydroxycinnamic acid) in 50:50 acetonitrile:nanopure water, 0.1% TFA. A Bruker Biflex III (Bruker Biosciences, Billerica, MA) equipped with a N2 laser (337 nm, 3-ns pulse length), and a microchannel plate detector was used to collect data in the linear mode, positive polarity, with an accelerating potential of 19 kV. Each spectrum was obtained by averaging ∼200 laser shots. External calibration was performed using singly and doubly charged peaks for horse heart cytochrome c (average mass [MH+], 12,361 Da). The relative amount of protein modified was estimated by dividing the peak intensity for the 4-HNE-modified protein by the sum of the peak intensities for the modified and unmodified protein.
His-tagged A-FABP was incubated with 4-HNE for 15 h at room temperature in standard 4-HNE adduction buffer. The protein was dialyzed at 4 °C against 25 mm ammonium bicarbonate, pH 8.0. The protein was incubated with 6 m guanidine HCl, 10 mm EDTA, 10 mm DTT for 1 h at 56 °C to denature and reduce the protein. The protein was cooled to room temperature and incubated with iodoacetic acid at a final concentration of 20 mm for 30 min at room temperature in the dark. The protein was dialyzed into trypsin digestion buffer and incubated with trypsin for 15 h at 37 °C or dialyzed against 25 mm sodium phosphate, pH 7.8 (Glu-C digestion buffer) and incubated with Glu-C at room temperature for 15 h. For both enzymes, the ratio of 1:20 protease:protein was used. Digestion was stopped by acidification with TFA. The peptides were desalted with SepPak C18 cartridges according to the manufacturer's instructions and spotted with α-cyano-4-hydroxycinnamic acid. Full scans of the peptide mixture from 800 to 3500 m/z and tandem mass spectral data of select ions were collected on a QSTAR XL quadrupole time-of-flight mass spectrometer (Applied Biosystems Inc., Foster City, CA). The TOF region acceleration voltage was 4 kV, and the injection pulse repetition rate was 6.0 kHz. Laser pulses were generated with a nitrogen laser at 337 nm, ∼9 μJ of laser energy using a laser repetition rate of 20 Hz. Mass spectra were the average of ∼50 laser shots collected in positive mode. External calibration was performed daily using human angiotensin II (monoisotopic [MH+] m/z, 1046.5417; Sigma) and adrenocorticotropin hormone fragment 18–39 (monoisotopic [MH+] m/z, 2465.1989; Sigma). Noise was subtracted from MS/MS spectra using the periodic noise filter in Analyst QS, and the centroid peak lists were submitted to Mascot and searched against the National Center for Biotechnology Information non-redundant (NCBInr) (December 1, 2006) Mus musculus database (the database contained 107,811 protein entries at the time it was searched) including 4-HNE as a variable modification with mass tolerances set to 0.5 for precursor ions and 0.5 for fragment ions. Centroid peak masses were exported from Analyst QS and graphed using GraphPad Prism software.
A-FABP was incubated with and without 0.5 mm 4-HNE at room temperature for 5 h, incubated with 100 mm βME, and dialyzed extensively against 25 mm Tris-HCl, pH. 7.4 (1,8-ANS binding buffer). The 1,8-ANS binding affinity of native and 4-HNE-modified A-FABP was determined by monitoring changes in fluorescence upon titrating the protein into 500 nm 1,8-ANS in 1,8-ANS binding buffer as described previously (44). Excitation and emission wavelengths of 376 and 472 nm, respectively, were used with slit widths of 4 nm for each. Nonlinear regression was carried out using GraphPad Prism software to fit the data to one-site binding isotherms. The isotherm for the 4-HNE-treated protein was modeled as a two-component system to produce a theoretical binding isotherm for the 4-HNE-modified species specifically. Dissociation constant (Kd) values were determined from nonlinear regression.
All values are expressed as mean ± S.E. Statistical significance was determined by performing the two-tailed Student's t test assuming unequal variances. p values <0.05 are considered significant.
To assess the relative extent of carbonylation on adipose tissue proteins in lean and obese mice, biotin hydrazide was used as a chemical tag to couple proteins with free carbonyl groups. As an initial means of verifying the ability of biotin hydrazide to detect carbonylated proteins, purified E-FABP, a known target of 4-HNE modification, was evaluated (43). 4-HNE modification was confirmed by the presence of a mass shift of 156 Da when the protein was analyzed by MALDI-TOF MS (Fig. 1). The peak corresponding to a mass of 156 Da larger than that of E-FABP was not detected in the absence of incubation with 4-HNE (Supplemental Fig. 1). After 4-HNE-modified and unmodified E-FABP were incubated with biotin hydrazide, samples were resolved by SDS-PAGE, transferred to PVDF membrane, and incubated with FITC-streptavidin. As shown in Fig. 1, inset, biotin hydrazide modified only 4-HNE-modified E-FABP and not the native form.
Detection of 4-HNE modification of E-FABP. Purified recombinant mouse E-FABP was incubated with or without 0.5 mm 4-HNE in vitro, and the extent of modification was evaluated using MALDI-TOF MS. Inset, 10 μg of 4-HNE-modified and unmodified E-FABP were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to a PVDF membrane, and blotted with FITC-streptavidin.
Using the methods initially developed for detecting 4-HNE modification of E-FABP, soluble adipose protein extracts were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to membranes, and blotted with HRP-streptavidin. Reaction variables such as biotin hydrazide concentration, temperature, protein amount, and reaction time were altered to optimize the detection of endogenous carbonylation (Supplemental Fig. 2). The reaction was relatively insensitive to temperature or biotin hydrazide concentration but was dependent upon the amount of protein in the reaction. Standard conditions of 0.5 mm biotin hydrazide, 50 μg of protein, 2 h, and 22 °C were chosen for all subsequent biotin hydrazide/HRP-streptavidin blotting experiments. To confirm that biotin hydrazide is highly specific for aldehydes, as has been demonstrated previously for dinitrophenylhydrazine (45), soluble adipose protein was incubated with NaBH4 to reduce aldehydes to primary alcohols. After dialysis, the protein was coupled with biotin hydrazide and blotted with HRP-streptavidin (Supplemental Fig. 3). The signal intensity for the reduced protein was decreased significantly, confirming that the signal intensity from the unreduced protein coupled with biotin hydrazide was primarily from aldehyde groups on the proteins.
To examine the profile of carbonylated proteins in various tissues as well as to confirm that the endogenous carbonylation detected by biotin hydrazide included 4-HNE-modified proteins, soluble protein extracts were prepared from mouse eye, lung, tongue, and adipose tissues. The protein samples were coupled with biotin hydrazide using the standard conditions, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin. In addition, parallel samples were subjected to immunochemical analysis using an anti-4-HNE antibody. As shown in Fig. 2, different patterns of protein carbonylation were detected in the various tissues, and in general, the profiles of modified proteins were similar, although the biotin hydrazide method appeared to be more sensitive and detected more proteins than did the anti-4-HNE antibody. As hydrazide chemistry has been shown previously to be specific for carbonyl groups (45), the additional bands detected by biotin hydrazide may arise from other carbonyl modifications besides 4-HNE that are known to be prominent (6, 46). Moreover some bands detected by the anti-4-HNE antibody were not observed with the biotin hydrazide method and may be the result of nonspecific reactivity often observed with antibody detection (dark smear at ∼55 kDa seen in all samples).
Detection of carbonylated proteins in different murine tissues. Soluble extracts (50 μg) from eye (E), lung (L), tongue (T), or adipose tissue (A) were either coupled with biotin hydrazide (using the standard conditions) and detected by blotting with HRP-streptavidin (left panel) or immunoblotted with anti-4-HNE antibodies (right panel) to detect carbonylated proteins. An additional sample that had not been coupled with biotin hydrazide (−BH) was subjected to HRP-streptavidin blotting as a control. Molecular mass (kDa) of protein standards is indicated on the left.
To investigate the extent of carbonylation of proteins in obesity, equal amounts (50 μg) of soluble proteins from epididymal adipose tissue of lean and obese mice were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF membrane, and blotted with HRP-streptavidin (Fig. 3 A). Quantification of signal intensities by densitometry (normalized to total protein) indicated increased (p < 0.05) carbonylation of proteins (∼2–3-fold) in the samples from the obese mice relative to the lean counterparts (Fig. 3B). This finding agrees well with those reported by Talior et al. (25) who measured a 2-fold increase in ROS in adipocytes from high fat-fed mice compared with control chow-fed animals. Consistent with increased ROS in adipose tissue of high fat-fed mice, extracts from obese animals exhibited a 3–4-fold decrease (p < 0.05) in GSTA4 protein compared with lean animals (Fig. 4). GSTA4 catalyzes the Michael addition of 4-HNE to glutathione (with high specificity for 4-HNE as a substrate), and its decreased abundance may contribute to the increased reactive aldehydes in adipose tissue from high fat-fed mice (47, 48).
Carbonylation of proteins in adipose tissue from lean and obese mice.A, soluble proteins (50 μg) from adipose tissue of lean (L) and obese (O) mice were coupled with biotin hydrazide, resolved by SDS-PAGE, transferred to PVDF, and blotted with HRP-streptavidin. Molecular masses (kDa) of the proteins are indicated on the left. B, relative carbonylation (indicated as percentage of the average value for obese mice) was determined from the blot in A by densitometry (normalized to total protein). Values are expressed as mean ± S.E. (n = 3; *, p < 0.05).
Protein levels of GSTA4 in lean and obese mice. Soluble protein (50 μg) from adipose tissue of lean and obese mice was resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with anti-GSTA4 antibody. Relative abundance (indicated as percentage of the average value for lean mice) was determined by densitometry (normalized to total protein). Values are expressed as mean ± S.E. (n = 8; *, p < 0.05). Inset, representative lean (L) and obese (O) samples from anti-GSTA4 immunoblot.
To identify proteins that are targets of aldehyde modification, soluble adipose proteins from obese mice were coupled with biotin hydrazide and enriched via affinity chromatography using immobilized monomeric avidin. Bound proteins were eluted with biotin, digested with trypsin, and subjected to LC-ESI MS/MS. Proteins were identified by analyzing parent and fragment ions (Supplemental Fig. 4) using SEQUEST. All data were filtered using the SEQUEST parameters described under "Experimental Procedures." The experiment was performed multiple times and repeated in the absence of biotin hydrazide coupling to control for nonspecific enrichment by the avidin column. After removing all proteins that were enriched without biotin hydrazide coupling, a list of soluble proteins identified as targets of aldehyde modification in adipose tissue was generated (Table I). Peptides matched to keratins, serum proteins, trypsin, and hypothetical proteins were also removed from the list. The identified proteins sorted into the following clusters: carbohydrate and lipid metabolism, signal transduction, antioxidant enzymes/cell stress response, nucleic acid metabolism, protein synthesis/degradation, and structural/motor proteins. Of particular note was A-FABP/aP2, a cytoplasmic fatty acid carrier protein and a member of the multigene family of lipid-binding proteins that includes previously characterized E-FABP (43).
Soluble proteins from adipose tissue of obese mice were coupled with biotin hydrazide, enriched via affinity chromatography using immobilized monomeric avidin, and digested with trypsin. Peptides were subjected to LC-ESI MS/MS, and proteins were identified using SEQUEST software. The identified proteins are listed along with their accession number. The peptides sequenced for each protein are indicated along with the Peptide Prophet probability score (P score), the charge of the peptide (z), the singly charged mass ([M + H]+1]) with the experimental error shown in parentheses, the cross-correlation value (Xcor), delta correlation value (Δcor), and preliminary score rank (Rsp).
Because A-FABP was found to be an in vivo target of aldehyde modification in adipose tissue by LC-ESI MS/MS, adipose extracts from A-FABP-null mice were used to confirm that A-FABP was modified with 4-HNE (Fig. 5). When soluble adipose protein extracts from wild type mice were subjected to aldehyde detection via biotin hydrazide coupling/HRP-streptavidin blotting, a prominently modified protein was detected at ∼15 kDa that was absent in protein extracts from A-FABP-null mice. When these same samples were subjected to immunoblotting with an anti-4-HNE antibody a similar result was observed. To further verify that A-FABP is a target of 4-HNE modification in vivo, the protein was purified from mouse adipose tissue. Both biotin hydrazide/streptavidin blotting and immunoblotting with anti-4-HNE antibody confirmed that A-FABP was modified with 4-HNE in vivo (Fig. 5).
In vivo carbonylation and 4-HNE modification of A-FABP.Left, soluble protein (50 μg) from adipose tissue of A-FABP null (−/−) and wild type (Wt) mice was evaluated using either biotin hydrazide coupling or immunochemically using anti-4-HNE and anti-A-FABP antiserum. Right, A-FABP was purified from murine adipose tissue and subjected to a parallel analysis.
To determine the stoichiometry of in vivo 4-HNE modification of A-FABP, quantitative immunoblotting using antibodies directed to 4-HNE and A-FABP was utilized (Supplemental Fig. 5). The fraction of A-FABP modified with 4-HNE in adipose tissue of obese mice was calculated by determining the concentrations of total A-FABP and 4-HNE-modified A-FABP in tissue extracts. Through immunodetection of either A-FABP or 4-HNE-modified A-FABP, it was estimated that 6–8% of total A-FABP is modified by 4-HNE in adipose tissue of obese mice. Given the total abundance of A-FABP is ∼250 μm in adipocytes (42), the concentration of 4-HNE-modified protein was estimated to be 15–20 μm.
To further characterize the modification of A-FABP with 4-HNE, purified protein was incubated with 0.5 mm (R)- or (S)-4-HNE or a 50:50 racemic mixture for various lengths of time, and the extent of modification was assessed using MALDI-TOF mass spectrometry (linear mode). As shown in Fig. 6, there was no statistical difference between the extent of modification of the protein by (R)- or (S)-4-HNE compared with the racemic mixture.
In vitro modification of A-FABP by 4-HNE enantiomers. Purified A-FABP was incubated with 0.5 mm (R)-4-HNE, (S)-4-HNE, or a 50:50 racemic mixture. After various time points, the protein was subjected to MALDI-TOF MS analysis. The relative extent of 4-HNE modification was estimated from spectral peak intensities. The experiment was done in triplicate with error bars shown. Squares represent (R)-4-HNE, circles represent (S)-4-HNE, and diamonds indicate 50:50 racemic mixture.
To identify the site of 4-HNE modification of A-FABP in vitro, His6-tagged A-FABP was incubated with 4-HNE and enzymatically digested with either trypsin or Glu-C, and the resulting peptides were subjected to MALDI-TOF MS/MS analysis (41). The m/z values of abundant ions in the initial MS spectra demonstrated A-FABP sequence coverage of 47.7% for Glu-C and 73.5% for trypsin (data not shown). For both trypsin and Glu-C digestions, parent ions with m/z values corresponding to a peptide with Cys-117 modified by 4-HNE (1076.5 for trypsin and 1628.8 for Glu-C) were isolated and subjected to fragmentation to generate MS/MS spectra (Fig. 7). Many of the ions in the MS/MS spectra observed corresponded to those in the predicted fragmentation pattern for the peptides with Cys-117 modified by 4-HNE (Table II). The 4-HNE modification itself appeared to undergo significant neutral loss fragmentation during the collision-induced dissociation process (labeled as [M + H]+1 − 156 for parent ion and with * for fragment ions in Fig. 7), a commonly observed event during MS/MS analysis of 4-HNE-modified peptides (49, 50) resulting in parent and fragment ions with 4-HNE modification intact in some cases and absent in others. The presence of b ions at 104.0 and 260.1 m/z in the spectrum for the Glu-C-digested peptide (Fig. 7A, labeled as b1 and b1*) verify that the 4-HNE modification is on Cys-117 as these m/z values correspond to N-terminal b1 ions consisting only of cysteine with and without the addition of 4-HNE. The 104.0 m/z peak represents the unmodified Cys-117 with the 260.1 m/z peak representing the same cysteine with the 4-HNE modification intact, giving it an additional 156 m/z units. Furthermore the additional 156 m/z units was only seen on b ions for the tryptic peptide in which Cys-117 is part of the given fragment (ions indicative of 4-HNE modification at Cys-117 are shown in bold in Table II). No fragments besides those containing Cys-117 were found to have 4-HNE adducts, and no other 4-HNE-modified peptides were detected for either digest. A-FABP contains no histidine residues and only one additional cysteine, Cys-1, making it the only other strong candidate as a potential site of in vitro 4-HNE modification. Although ions with m/z values corresponding to peptides with Cys-1 in the unmodified (alkylated with iodoacetic acid) state were detected for both trypsin and Glu-C, the corresponding ion for a 4-HNE-modified peptide was absent in each case. The reactivity of lysine with 4-HNE is much weaker than that of cysteine or histidine, and we found no evidence for a lysine modification on A-FABP at a detectable level. Furthermore the MS/MS spectra in Fig. 7 were submitted to Mascot to search against the mouse database in an unbiased way using 4-HNE as a variable modification. For both the trypsin and Glu-C digests, the Cys-117 HNE-modified peptide derived from A-FABP was the top scoring sequence match with Molecular Weight Search scores of 14 for trypsin and 39 for Glu-C (where a Molecular Weight Search score of greater than 32 indicates identity or extensive homology at p < 0.05) (51). Together this evidence demonstrates that Cys-117 is the site of 4-HNE modification on A-FABP in vitro.
MS/MS spectra of 4-HNE-modified A-FABP peptides. A-FABP was modified with 0.5 mm 4-HNE in vitro and digested with either Glu-C protease (A) or trypsin (B). In each case, the ion with m/z corresponding to the predicted 4-HNE-modified peptide was isolated and subjected to fragmentation. Both the [M + H]+1 parent ion peak corresponding to the 4-HNE-modified peptide as well as the peak corresponding to the neutral loss of 4-HNE ([M + H]+1 − 156) are labeled. The b and y ions observed are labeled as well as several observed a ions, an immonium ion of an arginine residue (Imm. Arg.), and ions corresponding to the loss of water (−H2O). Fragment ions demonstrating neutral loss of 4-HNE are indicated with an asterisk (*).
Ions with m/z corresponding to predicted 4-HNE-modified A-FABP peptides were isolated and fragmented using an Applied Biosystems QSTAR XL instrument. The observed MS/MS ions agree with theoretical fragmentation patterns for an A-FABP peptide modified on Cys-117, generated by digestion with both trypsin and Glu-C. Peptide fragments indicative of cysteine modification with 4-HNE are indicated in bold.
To verify that Cys-117 is the in vivo site of modification (and in agreement with the reactivity of the protein with 4-HNE in vitro), A-FABP purified from mouse adipose tissue was digested with Glu-C and subjected to MALDI-TOF MS. The spectrum obtained had 67.9% sequence coverage for A-FABP. Peaks at 1641.8 and 1530.6 m/z were detected, corresponding to unmodified peptides containing Cys-1 (acetylated) and Cys-117 alkylated with iodoacetic acid. Although no peak was observed corresponding to a peptide with Cys-1 modified with 4-HNE or 4-ONE, a cluster of peaks at 1626.6, 1627.6, and 1628.6 m/z (signal:noise between 4 and 5 for each peak) was observed (Supplemental Fig. 6) and is in agreement with the theoretical monoisotopic m/z values of the predicted Glu-C peptide with Cys-117 modified with 4-ONE (1626.8) and 4-HNE (1628.8). Although it is unclear whether the 1628.6 m/z peak is part of the isotope envelope for a monoisotopic peak at 1626.6 m/z (as the 1627.6 peak likely is) or whether it is the result of an independent modification, these m/z values are in agreement with the modified Glu-C peptide observed in the in vitro studies (Table II). In addition, this peak cluster was absent in a control Glu-C digest of E. coli-expressed recombinant A-FABP, which had 74.0% sequence coverage for A-FABP and contained the peak corresponding to the peptide with Cys-117 alkylated with iodoacetic acid (data not shown).
To investigate the effect of 4-HNE modification of A-FABP on its fatty acid binding activity, recombinant protein was modified with 4-HNE, and the binding of the surrogate fluorescent ligand 1,8-ANS was monitored (44). Fig. 8 shows the binding isotherm for the native and 4-HNE-modified protein with native A-FABP exhibiting a Kd of 2.05 ± 0.17 μm and the 4-HNE-modified A-FABP exhibiting a Kd of 23.20 ± 4.18 μm (p < 0.05).
Effect of 4-HNE modification of A-FABP on 1,8-ANS binding. After modification with or without 0.5 mm 4-HNE, A-FABP was titrated into 1,8-ANS, and the binding isotherm was calculated from monitoring changes in fluorescence. The experiment was done in triplicate, and the results shown are representative of the three trials. Squares represent untreated A-FABP, and triangles represent 4-HNE-modified A-FABP.
Obesity and type 2 diabetes are correlated with an increase in ER stress (5, 23) and increased ROS (5, 23). One possible contributing factor of obesity to insulin resistance is oxidative stress-induced lipid peroxidation and the modification of proteins by reactive aldehydes such as 4-HNE and 4-ONE. To gain insight into the extent of carbonylation of adipose proteins, biotin hydrazide was used as an affinity tag for proteins with free aldehyde groups (29). Whereas other studies have demonstrated an increase in oxidative stress markers such as malondialdehyde and ROS measurements (5, 25), to our knowledge this is the first report of direct protein modification in adipose tissue linked to obesity. The ∼2–3-fold increase in protein carbonylation accompanying high fat feeding agrees with other measures of oxidative stress (25). In addition, 37 targets of aldehyde modification were identified in adipose tissue of the obese mice.
Several proteins we identified in this study have been reported previously to be carbonylated or 4-HNE-modified specifically in other systems. Glyceraldehyde-3-phosphate dehydrogenase and glutathione S-transferase M1 have been shown to be targets of 4-HNE (52). In addition, dihydropyrimidinase-related protein 2 has been shown to be carbonylated in brain of Alzheimer disease patients, and triose-phosphate isomerase 1 has been found to be carbonylated in yeast upon treatment with hydrogen peroxide (53). As we found similarities between the detection of proteins with biotin hydrazide and anti-4-HNE antibody in adipose and other tissues, we presume that many of the proteins identified by enrichment via biotin hydrazide coupling are 4-HNE-modified (Fig. 2). However, as amino acid side chain oxidation and other lipid peroxidation products can also result in aldehyde groups on proteins (6, 46), additional studies need to be done to confirm the type of modification present on each specific protein of interest as we have done for A-FABP.
Work from this laboratory has shown previously that E-FABP is a target of 4-HNE modification in retina and that the site of modification is Cys-120 (corresponding to Cys-117 in A-FABP) (43). A-FABP and E-FABP are members of the intracellular fatty acid-binding protein multigene family, share ∼60% amino acid identity, and have essentially superimposable secondary and tertiary structures (54, 55). The defining feature of the FABP multigene family is a large interior water-filled cavity that serves as the hydrophobic ligand-binding domain. Lipid binding to A-FABP occurs within a defined fatty acid binding site with the carboxyl group coordinated by Arg-106, Arg-126, and Tyr-128 (56). The decreased binding affinity of 4-HNE-modified A-FABP for the hydrophobic probe 1,8-ANS (Fig. 8) agrees with the finding of Cys-117 as the site of modification (Fig. 7) and is consistent with a variety of crystallographic and NMR studies on the A-FABP apo- and holoprotein forms. X-ray crystallographic analysis of A-FABP in a variety of fatty acid-bound forms (palmitate, stearate, and arachidonate) have shown that the C3 to C6 region of a bound lipid lies in close proximity to Cys-117 (57–59). Moreover chemical modification of Cys-117 in vitro with a battery of sulfhydryl reagents decreased the affinity of A-FABP for fatty acids, and fatty acid binding attenuated Cys-117 modification (60). Prior to this study, only long and very long chain fatty acids were believed to bind within the cavity (44). This report implies that the ligand binding specificity of FABPs is broader than previously considered and extends to medium chain aldehydes. It will be interesting in the future to determine whether testis FABP, which exhibits the same structurally conserved cysteine residue, is also 4-HNE-modified.
The presence of a cluster of peaks containing ions with m/z 1626.6 and 1628.6 in the MALDI-TOF MS spectrum of in vivo purified A-FABP digested with Glu-C (Supplemental Fig. 6) is consistent with the finding of Cys-117 as the in vitro site of modification (Fig. 7). As further support for in vivo modification at Cys-117, digestion of recombinant E. coli-derived A-FABP (E. coli lacks large amounts of polyunsaturated fatty acid substrates for 4-ONE and 4-HNE synthesis) revealed no peak cluster at these m/z values. It is not surprising that a significant portion of the modified species of A-FABP in vivo may be alkylated with 4-ONE (consistent with 1626.6 peak) at Cys-117 in addition to 4-HNE (consistent with 1628.6 peak). Because 4-ONE contains a more electronegative keto function relative to the hydroxyl group of 4-HNE it has been measured to be significantly more reactive than 4-HNE (61).
It has been shown previously that A-FABP has a role in insulin resistance in mice (31), and A-FABP-null mice are relatively insulin-sensitive compared with wild type C57Bl/6J littermates. In light of this, covalent binding of 4-HNE may promote the holoprotein structure, mimicking the fatty acid-bound state. As the interaction of A-FABP and hormone-sensitive lipase has been shown previously to be fatty acid-dependent (41), the level at which A-FABP is modified with 4-HNE could have significant effects on lipolysis in the adipocyte. In this study it was estimated that 6–8% of A-FABP is modified with 4-HNE in adipose tissue of obese mice. Because of the high level of expression of A-FABP in adipose tissue (250 μm), this seemingly modest fraction that is modified would accumulate to relatively high concentration (15–20 μm HNE-modified A-FABP) in obese mice. The relative extents of modification of A-FABP in lean and obese mice was consistent with the ∼2–3-fold increase in total protein carbonylation (Fig. 3), and increased modification of A-FABP may in part contribute to insulin resistance in obesity through altering hormone-sensitive lipase activity.
A potential role for A-FABP is as an antioxidant protein, scavenging 4-HNE in the cell (43). Glutathione, a well characterized antioxidant against 4-HNE, has been shown to be enantioselective toward (S)-4-HNE in its GST-catalyzed Michael adduct formation (62). The fact that A-FABP forms adducts with both enantiomers of 4-HNE suggests that such a 4-HNE-scavenging role would not be completely overlapping with that of glutathione. As both stereoisomers of 4-HNE are formed in the cell, the ability of FABP to form adducts with (R)-4-HNE could be an important way of regulating concentrations of free 4-HNE.
Several other proteins identified in this study as targets of carbonylation may contribute to insulin resistance and inflammation. The well characterized roles of peroxiredoxin 1 and glutathione peroxidase 1 are in reducing hydroperoxides in the cell, including lipid hydroperoxides (63, 64). However, peroxiredoxin and glutathione peroxidase enzymes have also been shown to be important for the regulation of signal transduction pathways through controlling the redox state of proteins and lipids. Specifically the nuclear localization of NFκB, a stress-activated transcription factor known to contribute to insulin resistance in adipose tissue through the control of adipokine expression, has been shown to be negatively regulated in part by peroxiredoxin and glutathione peroxidase proteins (65–67). Furthermore the presence of active site cysteine (peroxiredoxin 1) and selenocysteine (glutathione peroxidase) residues makes these enzymes strong candidates for being inactivated by alkylation with 4-HNE, which could play a role in activating the NFκB stress response in obesity. In addition, glutathione S-transferase and aldehyde dehydrogenase family members are important antioxidants against 4-HNE and other electrophilic lipids (6). Their potential inactivation through modification could contribute to cellular stress by amplifying oxidative stress in obesity.
The actin filament-binding protein filamin A has been shown to interact with the insulin receptor and inhibit insulin signal transduction (68). The modification of filamin A with a lipid such as 4-HNE possibly could aid in its localization to the plasma membrane, increasing interactions with the insulin receptor and inhibiting insulin action. Calumenin, a protein involved in the regulation of calcium flux out of the ER, is also an intriguing target of carbonylation as it has been shown that ER stress is activated in obesity (23, 69). During conditions of ER stress, misfolded proteins accumulate in the ER lumen and initiate the unfolded protein response and the activation of JNK, which is known to increase serine phosphorylation of insulin receptor substrate-1 (23). As ER stress can be initiated by calcium release from the ER, the potential inactivation of calumenin by 4-HNE could contribute to the unfolded protein response and JNK activation in an indirect manor (70). In addition, the modification of eukaryotic elongation factor 1α-1 (eEF1α1) could contribute to insulin resistance through the activation of a similar stress response. This protein has been shown to be involved in activating apoptosis under lipotoxic conditions in non-adipose cells (71). Although adipocytes do not undergo apoptosis in response to high amounts of lipid, it is possible that eEF1α1 is involved in activating other stress responses in adipocytes in obesity. Although these proteins are intriguing candidates for being targets of modification by 4-HNE or other lipid peroxidation products, additional studies are necessary to determine the specific modifications and potential effects in each case.
The decreased amount of GSTA4 protein (∼3–4-fold) in adipose tissue of obese mice seen in this study (Fig. 4) is consistent with published microarray data demonstrating decreased GSTA4 mRNA expression in adipose tissue of obese C57Bl/6J mice (72). GSTA4 has been shown previously to be the primary GST isozyme responsible for metabolizing 4-HNE (47, 48). As such, a 3–4-fold decrease in GSTA4 in adipose cells would presumably contribute to increased accumulation of 4-HNE and other lipid peroxidation products, resulting in elevated protein carbonylation as observed in this study. Further investigation is needed to determine whether protein carbonylation and GSTA4 abundance are components linking obesity to insulin resistance.
We thank all members of the Bernlohr and Griffin research groups who provided helpful suggestions regarding this work and specifically Lisa Smith and Wendy Wright for maintenance and care of the animals and Mikel Roe for assistance with instrument methods. We also thank Dr. LeeAnn Higgins and the staff of the University of Minnesota Center for Mass Spectrometry and Proteomics for instruction on instrument operation and the Minnesota Supercomputing Institute for maintenance of the SEQUEST cluster.
↵ 1 The abbreviations used are: ROS, reactive oxygen species; A-FABP, adipocyte fatty acid-binding protein (also referred to as aP2); E-FABP, epithelial fatty acid-binding protein (also referred to as KLBP or Mal-1); 4-HNE, trans-4-hydroxy-2-nonenal (also referred to as 4-hydroxynonenal); 4-ONE, trans-4-oxo-2-nonenal; 1,8-ANS, 1-anilinonaphthalene 8-sulfonic acid; PBST, PBS containing 0.05% Tween 20; ER, endoplasmic reticulum; HRP, horseradish peroxidase; GSTA4, glutathione S-transferase A4 (also referred to as GSTA4-4 or GSTa4); βME, β-mercaptoethanol; JNK, c-Jun N-terminal kinase; eEF1α1, eukaryotic elongation factor 1α-1.
↵* This work was supported by National Institutes of Health Grant DK053189, the Minnesota Agricultural Experiment Station, and the Minnesota Obesity Center (to D A B.); by NHLBI, National Institutes of Health Grant T32 HL07741 (to P A G.); by National Institutes of Health Grant AG025371 (to T J G.); and by National Center for Research Resources Grant P20 RR17699-01 from the Center of Biomedical Research Excellence (to M J P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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redpajama_set_name: RedPajamaCommonCrawl


boobookittyann24 AylaAmadiMuslim livejasmin. CaseyBelle. SoSkinnyBoy. AshleyLeee.
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A nice upgrade to your old Egan's Blaster!
Interestingly enough, there is no graphic for this item when wielded by a female draenei, male tauren, and possibly others.
Instead of the usual "Invalid Target" error when you try to use this on something other than the quest target, you get "What are you doing? Only aim that thing at Wilhelm!"
I can finally use a ranged weapon as a Druid! WOOT!
For the screenshot of the engineering gnome; Yo Dawg, I heard you like zooming so we put a scope ON your scope -ON- your scope so you can Zoom while you zoom, zoom, zoom.
Most likely a reference to an SKS rifle. A gun known for having a free floating firing pin, which can fire on its own under the wrong circumstances.
"Now accurate to 15 yards!"


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Clear Pond
763 Putts Pond Road, Ticonderoga
Clear Pond is as quiet a pond as you will find anywhere, and although many hikers use the trails surrounding the pond, very few access it by boat. Use the area trails to extend your adventure, and even consider a second carry to Rock Pond to the north.
From the intersection of Route 9 and 74 near Schroon Lake, follow Route 74 toward Ticonderoga. Continue to Putts Pond Road on the right — this is also the entrance road for Putnam Pond State Campground. Drive this to the end to access the campground and the pond. A day use fee is required.
You will need to paddle through Putnam Pond to the narrows that separate it and North Pond. From the narrows you can access the 0.6 mile portage trail that leads to Clear Pond. This is a well marked trail with decent footing. No motorboats are allowed on Clear Pond, but they are allowed on Putnam Pond. The waters are typically calm. Clear Pond is relatively small, with only approximately 1-mile of shoreline.
Listed as one of the top brown trout waters in Essex County by the New York State Department of Environmental Conservation, Clear Pond is also home to rainbow and brook trout, as well as their cousin, the kokanee salmon.
At 26 acres, Clear Pond is not large, but it still manages to draw anglers at ice out. The use and possession of baitfish is prohibited. Access is via a trail from Putnam Pond Campground or paddle/ portage from Putnam Pond itself. A lean-to provides shelter at the pond.
The Clear Pond Trail extends 3.8 miles from the Putnam Pond Campground Trailhead North looping around Putnam Pond to the Grizzle Ocean Trail at a location approximately 1.2 miles from the Putnam Pond Campground Trailhead South. The trail drops 100 feet in 0.1 mile to the shore of North Pond, after passing along the northern shore of the pond it ascends 170 feet in 0.1 mile. Between Rock Pond and Clear Pond the trail ascends 265 feet and descends 135 feet in a 0.5 mile. The trail descends 120 feet in the 1.1 miles from Clear Pond to the Grizzle Ocean Trail. Clear Pond and the Clear Pond Trail can also be accessed from the western shore of Putnam Pond via a 0.6-mile spur trail.
A day use fee is required to park a vehicle at the Putnam Pond Campground when the campground is open. The trailhead may not be plowed in winter.
Brook Trout, Brown Trout, Land-Locked Salmon, Rainbow Trout
Clear Pond contour map


Colorado Trail Segment 8 Camp Hale to Copper Mountain.
Our trip on the Colorado Trail in 2010 left us skipping the main portion of Segment 8 due to weather. So in 2011 we were excited to return and ride from Camp Hale to Copper Mountain. Make sure you click on the link and read the interesting history of this area, which has been so instrumental to the Ski industry of Colorado. Once again we find ourselves in the amazing high-country! It is hard to describe the solitude and wonder that you feel when on top of the world!
For nearly 23 years I have been using EasyBoots on my horses. About 8 years ago I started keeping my horses barefoot 100% of the time. At that same time I also started trimming my horse feet with what is called a barefoot or natural trim. EasyCare the company that makes EasyBoots, regularly improves their boots and comes out with new boot styles for different kinds of riding. I have recently put together a couple of videos about a new boot product called the Glove Back Country. As you know I do a bit of Back Country riding among other things , so I was pretty excited to try these boots out. I was especially excited when the folks at EasyCare decided to feature my videos on their web page and in their newsletter! Here are those links so you can check them out. Thanks!
"The Easyboot Glove Back Country First Impressions"
As you know every year, as soon as the snow melts I begin training for my Colorado Trail ride. This spring EasyCare announced a new Back Country Glove boot. It combines the lightness of the Glove and the practicality of the Trail boots. So of course I was really excited to try this on Smoke. Smoke has had some trouble with the Glove gator giving him rubs during long assents and descents. This I determined was due to the angle of his hoof and pastern, by the end of the summer his hair gets so thin he has little hair to cushion his pastern. So day 1, Hills!
The boots really did great. No twisting , no rubbing. I did notice a mark on the front of Smokes hoof wall on one front and one back hoof. With closer inspection I realized the one of the screws holding the velcro enclosure was extended past the lock nut on the inside, and was rubbing on the hoof wall. That was an easy fix with a file. So I will add that to my check list when I get new boots. Also at the end of the ride there was some sand and small gravel sitting just above the heel bulbs. After looking at the video, I realize this probably happened when I came down the really sandy hill at the very end. I think for the next ride I will play around with the velcro closures. I'll keep you posted.
Being a Therapeutic riding instructor has been an amazing blessing in my life. I get to witness miracles everyday I teach. One very special day was with a beautiful young woman, Amy and her horse Knickee. Amy is one of my students with Cerebral Palsy. She has been riding most of her life, and now in just the last 6 months has made great strides! Most of her progress is due to her own determination and hard work. Another great factor is her wonderful partnership with Knickee. The communication between Amy and Knickee is apparent. So much emotion and love passes between them! A truly healing energy is radiated, even to those of us blessed to associate with both of them.
I taught Amy when she was a little girl. An adorable blond youngster with a contagious smile , she would just make you feel good meeting her. After 20 years or so I get to teach Amy again as a beautiful young woman. She is now an ambitious and inspiring adult. She enjoys life, and helps educate people about disabilities and how to over come and work with them.
Knickee is one of the most skilled therapy horses I have ever worked with. Her wisdom and understanding astound me. She not only adapts easily to all her riders with varied disabilities, but also gives lessons to able bodied riders, and is a very knowledgeable jumper. Patience and love is express though her big brown eyes, truly a priceless animal partner!
Here is another amazing segment of the Colorado Trail. I know, it is out of order. We did this segment in 2010. We drove up to do Segment 8 out of Copper Mt. but the weather had been bad and with driving from Denver we did not want to chance getting caught in a thunder storm over Searle Pass. Our thoughts turned then to Segment 9 starting at Tennessee Pass. I was pretty familiar with this trail. I had ridden this segment with my husband Larry, my son Tyler, and my nephew Zack many years before. We then hiked it with the 40 or so 14 year old girls from out church. You might hear me mention several times in the video the changes that I notice in the route. I must say it is much improved, and easier to follow. The last few miles were much steeper, and routed more to the west than before and it played on our stamina for sure.
Hay Problems , Hay Solutions.
Going into this fall the hay situation looked bleak. The drought in Texas, and the rising gas prices were both a clue that the price of hay would skyrocket, and at best it would be very difficult to get. Here in Colorado I was able to buy small bales (about 65lbs.) from a local grower for $7 a bale. That was picking it up and loading it myself. I was happy to get it and stock piled as much as a could. Just after Christmas my pile was looking more like a mole hill ,and I knew I needed to secure more hay, and I knew I would pay much more. Small bales are near impossible to find and if you do, you are paying anywhere from $12 to $15 a bale. Solution: Big Bales. Big square bales come in two sizes 850lbs and 1100lbs. I have several friends who had started buying the big bales and their stories of loading and unloading these monsters helped reassure me. Unloading was my biggest concern. One day I watched in amazement as one gal I know arrived at her barn with two 850lb bales straddling her truck bed. With out as much as a warning she started backing up very fast slammed on her brakes and the two bales tumbled off the back and landed perfectly side by side on the pallets at my feet! I knew I would not be doing that as my barn is on a hill and to back my truck into it requires one to be slow and careful. But as the post in my barn are set in concrete I was pretty sure I could rig a pulley to drag the bale off as I pulled forward. I was able to find the largest bales at the local feed store for $174 per bale , not a great price but fare for the cost in our area. This is equivalent to 17 to 20 small bales. Also it is a very nice Timothy hay. I have to admit it is a bit nerve racking to watch as the fellow loads it in the back of my truck with a front loader. No problem driving it home and the unloading worked like a charm! I was able to put 3 of these big boys in the aile way of my barn. The next challenge was to figure out what a flake of this weighed. It flakes off nice but way to heavy to hold the whole thing. I pulled it apart and stepped on the scale, but had to have someone else read it for me as I could not read the scale over the pile in my arms. One flake appears to be anywhere from 40lbs. to 45lbs. I was not looking forward to tackling this unruly stuff everyday! I decided I would try the hockey net. I have seen info on using nets to hold hay and get horses to slow down their eating. So off to the local hockey store, you can also order them on-line. A 72inch heavy duty net cost me $26. I wove the tie strings that came with the net to tie together the sides and made a big bag. Ran a rope around the top as a draw string and filled it with one whole flake of hay. First I hung it in the stall and watched . They seemed to be eating it soooo slow. They would take turns going in and out of the stall all day. I was worried because the volume of hay in the bag did not seem to be diminishing ,so the first day I threw a bit of loose hay out to them, and it didn't seem like they where starving. Here is a link to a site that shows several different methods : http://www.naturalhorseworldstore.com /slowfeed-hay-saver-net/ Today I dragged the bag out and tied it to a tree.They seem to like this better as they both eat at the same time now. So far so good, and I think it will be far healthier for them to eat this way. Let me know if you have tried this and how it is working for you. I'll keep you posted.
I took the tail gate off my truck so then bale would fit down in the bed.
Could fit up to 6 bale in my barn .
I like that I can look out my window and see them eating.
My most amazing and creative son made a video book trailer for my dear friend M. J. Evans' first book in her trilogy "Behind the Mist". A thrilling story of a boy and his horse who find them selves in the mystical world of Celestia, where great and noble horses earn their Unicorn horns! So guess who in the trailer plays the part of the Unicorn Urijah,? Phlame of course! Phlame loved learning his part and now every time he steps up on a rock he asks "Where is my glowing horn?" If you ever need video work contact Neil Crisp. neilacrisp@gmail.com Here is a demo reel of his work.
I took Smoke and Phlame out for their New Year stroll, we avoided the ice and trotted when we could. They tossed their heads and pranced in place wishing they could break out and stretch their winter muted muscles. I would have allowed more romping , but just ahead of me tossing sticks and snowballs where my two equally frisky Grandsons.When we would get to close, I would tell the horses to whoa, and wait for the 4 and 6 year old to get a bit further ahead. I was amazed how quickly Smoke and Phlame learned to keep the youngsters ahead of them. When they boys took off in a run the horses would ask to trot and catch-up and as soon as we did they would slow to a walk. If they stopped suddenly to ask me a question, or point out some "deer poop" (they love to say that), Smoke and Phlame would stop in their tracks and listen to the playful chatter.
My days lately have been more Grandma than horsewoman. The horses were happy to get out, but they seem to understand I have other boys in my life and are, greatfully willing to share me.
I would like to recommend a great Face Book Page called Horse and Man, I have read so many wonderful stories on it and here is another one! I love the connections that are made between horses and their owners. I also have a soft spot in my heart for our wonderful veterans. Hope this will bring a smile to your face and a tear in your eye, as it did for me.
Colorado Trail Segment 7 "The Climb"
I just finish working on the video for Segment 7 of the Colorado Trail Debbie and I rode the summer of 2010. The memory of this segment is still fresh in my mind! The climb was so dramatic, the views so spectacular and Smoke and Flame were simply amazing. We climbed to nearly 12,500 ft on a fairly narrow ridge of mountains called the Ten Mile Range. Even though it was August, snow still hung along the highest ridge lines. We mingled with the clouds looking down on Lake Dillon and Breckenridge. After reaching the top-out point at 12,494 ft. we could look just to the west and see Copper Mountain Ski Resort! Some trails will aways remain with me. This is one that had so many metaphors for difficult challenges, for over coming fears and not giving in to life's worries. I have climbed a few 14-teen-er's on foot and this was every bit as challenging and exhilarating! The fact that your horse and you are a partnership, he carries you, and you must make good decisions in his behalf, has the effect of forever melding your hearts, and minds together.


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Miquel Boixó i Geli (Banyoles, 27 de setembre de 1910 - Shúbino, Crimea, Unió Soviètica, 13 de març de 1943) va ser un militant comunista que va lluitar a la Guerra Civil Espanyola i la Segona Guerra Mundial. Va lluitar al Front d'Aragó i a la Batalla de l'Ebre de la Guerra Civil Espanyola i arran de la victòria franquista va decidir allistar-se com a voluntari al Front Oriental de la Segona Guerra Mundial. Boixó va arribar al grau de major de l'exèrcit soviètic amb el nom de guerra Guiorgui Bobrov i va ser declarat Heroi de la Unió Soviètica i condecorat amb la Medalla dels Partisans de Crimea. Va morir el 13 de març de 1943 en una acció guerrillera a Crimea darrera les línies alemanyes, juntament amb un grup de paracaigudistes catalans. El seu nom està inscrit en dos monòlits, un a Moscou, al monument als soldats espanyols que van morir durant la guerra a la Unió Soviètica, i l'altre a Shúbino, poble on va caure en combat. Davant d'aquest darrer monument, el poble celebra cada any un homenatge a la unitat de partisans morts en combat.

Referències

Comunistes catalans del sud
Militars soviètics de la Segona Guerra Mundial
Herois de la Unió Soviètica
Comunistes soviètics
Militars catalans del sud contemporanis
Polítics banyolins
Morts el 1943
Naixements del 1910

redpajama_set_name: RedPajamaWikipedia


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Arise School: Helping Families Achieve Financial Independence.
In January, 2020, Arise set up a new initiative to try both to empower local women and to provide an opportunity for some of the poorest families in the community to achieve financial independence.
Grace, the grandmother and carer of one of the children sponsored through ACE, was given a nanny goat along with the short term loan of a billy goat. The agreement was that Grace would look after the nanny goat and bring back three kids to Arise. After that, she would be able to permanently keep the nanny goat and any other kids born. The three kids returned to Arise would be raised at the school and then given out to other members of the community in need in a similar way.
Goat breeding is commonplace in Tanzania, providing both milk and meat. There are various breeds. The one issued to Grace was the kind that gives birth to twins. Therefore, it was hoped that the three kids would be born and returned to Arise to raise within the year.
Twelve months on, we can report that the nanny goat has produced one set of twins…one male, one female. These kids have already been returned to Arise and, when there is another 'happy event', one of the twins will join them. The second kid, along with her mother, the original nanny goat, will then belong to Grace. We were delighted to learn just this week that the nanny goat is pregnant again and expecting twins sometime in April.
In the same twelve month period, since January 2020, Arise has issued a further twelve nanny goats to women in the community. Six billy goats are on 'standby'!
Whilst Arise Community School exists primarily to provide education for some of the poorest children, it is also playing an essential role in the economic development of the community by empowering some of its poorest members to achieve financial independence.


... is an active scientific society, which is located at the Geography Research Unit, University of Oulu. It has been an essential part of the identity of academic geographers of Northern Finland since the year of 1963. In accordance with Following its basic objectives, the Society serves as a link between geographers and other scientists representing adjacent fields of science. The society arranges meetings and other happenings, publishes two scientific series (Nordia Geographical Publications and Nordia Tiedonantoja), and, if financially possible, aims to support the activities of young geographers in particular.
The members of the Society convene twice a year for a general assembly. The Nordia DayEvening, which is held every year, is an important geographical meeting in Northern Finland giving opportunities for both experienced and young geographers to present their research to the public and publish an overview of their work. The Society also seeks to organise excursions for its members. For instance, in 2003 a group of geographers visited one week in Romania, in 2006 another group travelled in Norway, and in 2011 geographers visited Kostamus, Russia. Recently, the Society has specialized in short excursions within Northern Finland to foster co-operation networks with alumni geographers and institutions with geographical interests.
The executive committee board of the Society consists mainly of the personnel and students of the Geography Research Unit. The membership fee of the Society is 8 euros per year for students and 18 euros per year for others. In return, all members will receive an announcement letter on a regular basis and Nordia Yearbook at the end of each year. Furthermore, membership will give the opportunity to take part in various activities arranged by the Society.
Since 1967 the Society has been engaged in scientific publishing activity. In autumn 1994, the Geographical Society of Northern Finland and the Geography Research Unit at the University of Oulu made a decision decided to publish in co-operation, and thus were born two series of publications, "Nordia Geographical Publications" and "Nordia Tiedonantoja".
The NGP YEARBOOK is published each year. There are three major aims for the YEARBOOKThe YEARBOOK has three main aims: firstly to present a cross-section of the works in the field of geography in Northern Finland, secondly to publish collections of articles dealing with various current areas of interest and themes in geography, and thirdly to publish papers presented in the symposia arranged by the Geography Research Unit of the University of Oulu or by the Geographical Society of Northern Finland.


Who we are. What drives us.
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Joe takes a multi-disciplinary approach to online marketing. Working extensively in social media, SEO, blogging, copywriting and analytics, his focus always remains on finding the right answers to the questions of branding and growth. Creative skills with a commercial application.
Having worked in strategy consulting since 2013, he is now sharing his marketing knowledge to help make sure as many people as possible see White Heat Design's beautifully-crafted websites.
In his spare time he likes to sit around watching movies until it gets dark outside and occasionally leaves the house to listen to podcasts.
Jason brings ingenuity and another fresh perspective to web design and development. He applies his skills and passions for creativity and pragmatic problem solving to all digital projects for White Heat Design.
His extensive experience with independent creative agencies gives him the vision and fluidity to flourish when it comes to meeting the diverse needs of clients - keeping their businesses competitive and ahead of the curve in the rapidly evolving frontier that is the internet.
When not hunched over his laptop enjoying playing with technology all day, Jason likes to stretch himself out in nature with a spot of wild camping, fishing and hiking.
White Heat Design are an agency enamoured with the power of aesthetics. We aim to create beauty using both words and imagery and marry that with effective business strategy. We are a full service agency.
To ensure a smooth working relationship.
Creating and designing anything to a high standard takes time, patience and expertise. We don't take on work with overly tight deadlines or restrictive budgets as they don't allow for us to produce our best work.
You'll never get the best out of a project if you're not honest with both yourself and your designer. We're open about everything we do and we expect the same from our clients.
Every project is different. Every client's expectations are different. We have to learn the ins and out of each as we go. We need you to trust us and work with us in order to achieve the best possible result.
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Putin says Russia will produce new missiles after demise of nuclear pact
Published: SEPTEMBER 5, 2019 15:26
VLADIVOSTOK - President Vladimir Putin said on Thursday that Russia would produce missiles that were banned under a landmark Cold-War era nuclear pact that ended last month, but that Moscow would not deploy them unless the United States did so first.
Speaking at an economic forum in Russia's Far East, Putin said he was concerned by U.S. talk of deploying missiles in Japan and South Korea, a deployment he said would cover parts of Russian territory.
Washington pulled out of the Intermediate-Range Nuclear Forces Treaty (INF) pact last month accusing Russia of violating it, allegations Moscow denied.


The document is an assimilation of materials and tools developed to assist operators and stakeholders make sound decisions based on effective mine closure planning. It takes a multi-disciplined approach to closure planning, with the guidance of toolkits to enable adequate implementation of the said objectives.
Key to any mine closure planning is understanding the needs and objectives of the various stakeholders, both internal and external to the mining operation. The engagement of these stakeholders should be undertaken throughout the life cycle of the mining operation. The toolkit indicates apparatus needed for community engagement, this includes ways to conduct community impact assessment, community profiling and mapping, gap analysis, active listening, risk management, partnerships and facilitation. A closure plan should ideally capture or attempt at a balancing the expectations and viewpoints of the various stakeholders.
The closure plan should also be conducted in several key stages, at the beginning of mining operations a conceptual closure plan should be developed (this would involve risk and opportunity assessment and management and target goals and outcomes). The conceptual phase should be goal oriented and involve as many practical outcomes as necessary to capture the life of the project. The conceptual plan, over the life cycle of the operation, would be transformed into a more detailed closure plan using contextual information derived from stakeholder engagement and detailed project feasibility studies. The detailed closure plan should consist of action plans for each goal envisaged with probability cost estimates which allows room for annual review and updating. This detail should also capture scenarios for dealing with project risks and sudden closure of operations.
The decommissioning and post closure phases should be conducted with active participation and input from the closure planning group so as to ensure not only focus on the social and environmental issues but also to ensure that the years of closure planning are not undermined and objectives during the planning phases are met.
Finally, any effort to develop a mining closure plan should be mindful of the various challenges that operators might face. For instance, during exploration it might not be financially feasible for the exploration companies to plan on mining closure especially where mineable resources have not yet been identified. Also managers need to keep in mind the likelihood of change in ownership structures where the burden of closure changes plus any costs associated, this corresponds with a change in management structures with the challenge being able to maintain a consistent effort in closure practices. The document is designed to assist at arriving at a mutually beneficial closure outcome, where diligent thought and application of best practices are utilised to arrive at the desired outcome.


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How to Treat Betta Fish Swim Bladder Disease or Disorder (SBD)
Many beginner fish keepers have felt the shock of approaching their aquarium, only to see one of their fish floating upside down or swimming sideways.
Your first reaction might be to think your fish is dead or getting close to it, but on closer inspection, you'll hopefully see this isn't the case.
Anyone who's seen it before will know what's going on: Betta fish swim bladder disease.
While it sounds serious, swim bladder disease – or SBD -is extremely common in betta fish and, in many cases, is easily remedied.
This article will cover all you need to know about betta swim bladder disease, what to do if it happens to your fish, and how to prevent it.
What is Swim Bladder Disease?
What are the Signs of Betta Fish Swim Bladder Disease?
What Causes Swim Bladder Disease in Betta?
Can Swim Bladder Disorder Spread?
How Can You Identify the Cause of Your Betta's SBD?
Is Swim Bladder Disease Fatal?
Do Bettas Recover From Swim Bladder Disorder?
How Do You Treat Swim Bladder Disease in Betta Fish?
Overfeeding
How Can You Care for a Betta with Chronic SBD?
Is There a Way to Prevent Swim Bladder Disease?
Despite the name, it isn't actually a disease. More accurately, it's a generic term for a range of issues with the swim bladder of a fish. Often, it's a symptom of an underlying condition, rather than a standalone problem.
To understand the issue, you first need to know more about the swim bladder.
Betta, along with most other bony fish, have a gas-filled organ inside them called a swim bladder. Its purpose is to control a fish's level of buoyancy, allowing them to easily move up and down in the water, staying afloat wherever they are.
However, when a betta has SBD, the organ is malfunctioning, so the fish is no longer able to easily move around in their tank.
Image Credit: Vilasack Southisane, Shutterstock
If you notice any of these signs, your betta probably has SBD.
Your betta fish swimming upside down
Floating right at the top of the tank
Sinking down to the bottom of the tank
Swimming upside down
Developing an S-shaped spine
There are a number of causes of SBD in betta. Let's look at the most common.
Constipation: This is the most common cause. Not soaking dry pellets and freeze-dried food can be the culprit as they expand within the stomach.
Overfeeding: Most people will identify with feeling exceptionally bloated after a heavy meal, but for betta, this can cause issues with their swim bladder.
Injury: A betta that has suffered an injury could have damaged their swim bladder.
Bacterial infection: Some types of bacterial infection can cause SBD.
Birth defect: Some betta with swim bladder problems are just born that way.
Poor water quality: High nitrate levels have been known to lead to swim bladder disorder.
Image Credit: UNIKYLUCKK, Shutterstock
Swim bladder disorder itself is not contagious, but the cause of it can be.
If the SBD is caused by a bacterial infection or parasites, then this can spread to other fish. If it's due to constipation or a genetic disorder, then no it cannot.
In all cases, swift treatment is recommended, despite what the cause may be.
To treat swim bladder disorder, you first need to know what's causing it and it can be tough to identify, sometimes requiring a bit of trial and error.
That said, we do have some tips to help you uncover the underlying cause. First of all, you must make sure your overall care of your betta is up to scratch, then work through the following checks.
The most common cause of swim bladder disorder in betta is constipation. If they are suffering from this, they'll look fairly healthy overall, but you'll notice a lack of defecation and a bloated stomach.
The signs of overfeeding are similar to the symptoms of constipation, so it can be tough to tell the two apart. And, constipation may be caused by overfeeding, anyway, so the two are linked.
If the SBD is down to an injury, it's likely you'll see some external damage. Signs to look out for that would indicate a bacterial infection include dull coloring, general lethargy and a refusal to eat.
If poor water quality is the cause, then it's important to do a water change to remove the chemicals from the water,t hen establish a good water care routine going forward, including frequent use of a water parameter test kit and frequent partial water changes.
Finally, if a birth defect is the cause, it's likely that your betta will have always had this issue, so if you've ever known them to swim normally, it's probably not a birth defect.
Image credit: Thassaphong Jarung, Shutterstock
Generally speaking, it's not fatal, no. But it certainly can be, particularly if left untreated.
The main thing to keep in mind is that SBD is usually an outward sign of another issue affecting your fish, from digestive issues, to bacterial infection. The underlying cause must be treated, otherwise it can lead to fatality.
However, very often it can be cured.
Sadly, SBD is a very common ailment in betta fish. Luckily, it's rarely fatal.
Most commonly, SBD is due to digestive problems, constipation being the main culprit. If this turns out ot be the cause, then it's very easily treatable as we shall discuss below.
If, however, the SBD is due to a bacterial infection, is genetic in nature or due to permanent damage to the swim bladder, then it can be permanent I'm afraid. However, even in these cases it's not common to be fatal, and many fish can still live long lives with some degree SBD.
No matter the cause, the first thing to do for swim bladder treatment – if possible – is to move them to a smaller hospital tank.
It should be adequately heated, filtered and treated like your main aquarium, but with a bare bottom. This helps to remove any potential environmental issues in your main tank, but also helps confine your betta, so they don't get exhausted trying to swim too much.
Once in the hospital tank, you should treat your betta according to what's causing their SBD. If you're unsure of the cause, start at the top of the list and work your way down.
The first step to treating constipation is to fast your fish for 1 to 3 days. This means no feeding at all. Often the blockage will clear out on its own, and the swim bladder disorder will disappear.
If your fish still hasn't righted themselves in the water after their fast, try feeding either a small portion of daphnia – which acts as a laxative – or a quarter of a blanched, frozen pea – which is mostly indigestible to a betta fish and may help to clear their system out. Feeding boiled, shelled peas is a well known and common way to clear constipation in many fish.
There's also some evidence to suggest Epsom salts can help. Add one tablespoon of Epsom salts to the aquarium for every 5 gallons of water.
The only way to cure swim bladder disorder caused by overfeeding is to fast your betta until they start swimming normally again, but for no more than three days. If caused by overfeeding, it will often go away in just a few hours.
If you've fasted them for three days and they still aren't back to normal, overfeeding probably isn't the cause.
Image Credit: Buddy BIGPhotographer, Shutterstock
If suffering from a bacterial infection, they will need to be medicated with antibiotics. Try treatments like Seachem Kanaplex or API Sulfa for an external infection. For an internal infection, you'll need medicated food or to soak your betta's food in medication yourself.
Remember to remove any carbon filters from your aquarium before medicating, as they'll filter out the medication.
If in doubt about how to treat bacterial infections, consult a veterinarian with expertise in pet fish, or failing that a knowledgeable staff member at a reputable local fish store.
Sometimes swim bladder disorder caused by an injury will get better with time, but sometimes the damage will sadly be permanent.
The good news is, SBD isn't painful or fatal on its own, so you can make modifications to keep your fish happy. More on that below!
There's no cure for a swim bladder issue caused by a birth defect. However, like an injured betta, it's possible to keep them happy in a suitable environment.
Image Credit: Agusngidem, Shutterstock
Just because a betta has chronic swim bladder disease due to an injury or birth defect, it doesn't mean they can't live a full and happy life, it just means you must tailor their environment to fit their needs.
A wide, shallow tank is best as they don't have to do so much work to swim to the top for air, or to the bottom for any food that may have settled there.
It's also advisable to place live or silk plants with wide, flat leaves in their tank, as they can rest on them when necessary. You can also buy "betta hammocks" which serve the same purpose.
There's no 100 percent effective way of preventing it, but there are several things you can do that will significantly decrease the chances of having issues.
Never overfeed and ideally feed two smaller meals a day rather than a single larger one.
Don't feed freeze dried foods or dried pellets unless you soak them in a little tank water before feeding, as they'll expand in this time instead of expanding in the stomach.
If you need to net or handle your betta, be extremely gentle to avoid injury.
Make sure the tank contains clean water with appropriate filtration and cycling in place.
Keep an eye on water parameters and temperature.
Swim bladder disease might look serious, but in most cases, it's nothing more than the result of your betta overeating.
That said, it certainly shouldn't be ignored, because she can be a sign of something more serious.
This post has given you all the information you need to treat and prevent betta swim bladder disease, so now you'll know what to do should you start to see the signs.
Featured Image Credit: ivabalk, Pixabay
Betta Fish Care Guide
Written By: Lindsey Stanton
Lindsey discovered her passion for fish keeping after a junior high school field trip to the Shedd Aquarium in Chicago. Prior to becoming Editor-in-Chief of It's a Fish Thing, Lindsey studied marine biology at the University of California-Santa Cruz. She loves goldfish, tetras, and mystery snails, and recently began experimenting with a saltwater aquarium.
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