Governmental actions including increasing awareness of the importance of vitamin D and guidelines on how to obtain it

are necessary. Creating areas where women, particularly those of lower socio-economic status, can enjoy sun exposure as well as fortifying more foods would go some way towards tackling this problem. “
“Behçet’s disease (BD) is a systemic vasculitis disease with oral and genital aphthous ulceration, uveitis, skin manifestations, arthritis and neurological involvement. Many investigators have published articles on BD in the last two decades since introduction of diagnosis criteria by the International Study Group for Behçet’s Disease in 1990. However, there is no scientometric analysis available for this increasing amount of literature. A scientometric analysis www.selleckchem.com/products/Trichostatin-A.html method was used

to achieve a view of scientific articles about BD which were published between 1990 and 2010, by data retrieving from ISI Web of Science. The specific features such as publication year, language of article, geographical distribution, main journal in this field, institutional affiliation and citation characteristics were retrieved and analyzed. International collaboration was analyzed using Intcoll and Pajek softwares. There was a growing trend in the number of BD articles from 1990 to 2010. The number of citations to BD literature also increased around 5.5-fold in this period. The countries found to have the highest output were Turkey, Japan, the USA and England; the first two universities GSK J4 purchase were from Turkey. Most of the top 10 journals publishing BD articles were in the field of rheumatology, consistent with the subject areas of the articles. There was a correlation between the citations per paper and the impact factor of the publishing journal. This

is the first scientometric analysis of BD, showing the scientometric characteristics of ISI publications on BD. “
“The historical significance Teicoplanin of the Medici family of Florence is widely recognised, but the diseases which afflicted leading members of this family have only been scientifically studied in recent decades. Paleopathological findings on exhumed skeletons, supplemented by medical descriptions in historical documents, have permitted a retrospective diagnosis of a triple pathological syndrome in the senior branch of the Medici family. Peripheral joint and spinal conditions, with the presence of skin disease, are identified in several generations of the family in the 15th century and are presented as the ‘Medici syndrome’. Radiological findings are compared with macro- and microscopical descriptions in the diagnosis of the peripheral joint disease and spinal ankylosis/stenosis within the syndrome. “
“Objective:  To investigate the effect of Kashin-Beck disease (KBD)-affected feed and T-2 toxin on the bone development of Wistar rats.

, 1998; Holo et al., 2001; Maldonado et al., 2003; Diep et al., 2009). Strain-related differences in bactericidal activity affect the susceptibility of other microorganisms to plantaricins and organic acids (Ehrmann et al., 2000; Omar et al., 2006; Nielsen et al., 2010). None of the strains had genes for plantaricins NC8, S, or W (Table 1). With the methodology used, plantaricin A-, EF-, JK-, and N-related genes were detectable in all strains except for TO1001 (Table 1). Similar to the case of TO1001, L. plantarum strain 3.9.1, isolated from an African fermented

food, does not have any of these plantaricin genes (Omar et al., 2006). Certain L. plantarum strains show the following different types of plantaricin-related gene combinations: (1)

plnEF and plnW; (2) plnD, plnEF, plnI, and plnG; (3) plnD, plnJ, plnK, and plnG; (4) PI3K inhibitor plnD, plnEF, plnI, plnK, and plnG; (5) plnA, plnC, plnD, plnEF, plnI, plnJ, plnK, and plnN (Omar et al., 2006; Moghadam et al., check details 2010). Thus, the characteristics of the gene combinations carried for the production of plantaricins in TO1000, TO1002, and TO1003 are unique among the known L. plantarum strains isolated from fermented products. The synthesis of plantaricin A is observed from early exponential to early stationary phase. During stationary phase, the amount of plantaricin A strikingly declines (Diep et al., 1994). The addition of sucrose to the medium enhances production of nisin, another bacteriocin produced by Lactococcus lactis, (Devuyst & Vandamme, 1992). Thus, bacterial growth rate and available nutrients are associated with antimicrobial activity. In fact, the rates of fermentation differed among the four strains at 30 and 60 days of storage (Tables 3 and 4), suggesting that, in addition to the divergence in the available carbohydrates, the capacity for production of organic acids, and

the pH and temperature preferences for growth, antimicrobial activity may also be an important factor in the regulation of silage fermentation quality. Further GABA Receptor studies are needed both to elucidate the production of plantaricins by the TO strains inoculated in silage and to understand their roles in the improvement of silage quality. In conclusion, phenotypic and genotypic differences were present among LAB strains in spite of their belonging to the same species and subspecies, and the fermentation quality of silage inoculated with different conspecific strains differed significantly, supporting the idea that suitable LAB inoculants should be selected on a strain basis. Because TO1002 most effectively improved the fermentation quality in terms of pH decrease, regulation of undesirable microorganisms, and high DM recovery, this strain should be the most suitable inoculant for longer storage of paddy rice silage. The selected L. plantarum subsp.

Mechanistic investigations revealed that the neuronal and behavioral recovery produced by

exercise in the chronic parkinsonian mice was associated with an improved mitochondrial function and an increase in the brain region-specific levels of brain-derived and glial cell line-derived neurotrophic factors. Our findings indicate that exercise not only produces neuronal and mitochondrial protection, find more it also boosts nigrostriatal neurotrophic factor levels in the chronic parkinsonian mice with moderate neurodegeneration. Therefore, modifying lifestyle with increased exercise activity would be a non-pharmacological neuroprotective approach for averting neurodegenerative processes, as demonstrated in experimental chronic parkinsonism. “
“A key feature of early visual cortical regions is that they contain

discretely organized retinotopic maps. Titration of these maps must occur through experience, and the fidelity of their spatial tuning will depend on the consistency and accuracy of the eye movement system. Anomalies in fixation patterns and the ballistics of eye movements are well documented in autism spectrum disorder (ASD), with off-center fixations a hallmark of the phenotype. We hypothesized that these atypicalities might affect the development of visuo-spatial maps and specifically that peripheral inputs might receive altered processing in ASD. Using high-density recordings of visual evoked potentials CHIR-99021 datasheet (VEPs) and a novel system-identification approach known as VESPA (visual evoked spread spectrum analysis), we assessed sensory responses to centrally and peripherally presented stimuli. Additionally, input luminance was varied to bias

responsiveness to the magnocellular system, given previous suggestions of magnocellular-specific deficits in ASD. Participants were 22 ASD children (7–17 years of age) and 31 age- and performance-IQ-matched neurotypical controls. Both VEP and VESPA responses to central presentations were indistinguishable between groups. In contrast, peripheral presentations resulted in significantly greater early VEP and VESPA amplitudes in the ASD cohort. We found no evidence that anomalous enhancement was restricted to magnocellular-biased responses. The extent of peripheral response enhancement was related NADPH-cytochrome-c2 reductase to the severity of stereotyped behaviors and restricted interests, cardinal symptoms of ASD. The current results point to differential visuo-spatial cortical mapping in ASD, shedding light on the consequences of peculiarities in gaze and stereotyped visual behaviors often reported by clinicians working with this population. Atypicalities in how individuals with an autism spectrum disorder (ASD) direct their gaze to socially relevant stimuli such as faces and eyes have long been noted (Klin et al., 2002; Pelphrey et al., 2002; Hernandez et al., 2009; Kliemann et al., 2010).

Thus, cue onset introduced a large bias in microsaccade direction during this session, as documented previously (Hafed et al., 2011). During SC inactivation, and with the cued location in the same, but now affected, region (Fig. 6B), this pattern was completely reversed – the initial bias

of microsaccade directions after cue onset was now towards the foil and not the cue (red arrows). This finding demonstrates that, even though inactivation of the peripheral SC in this sample experiment did not reduce overall Selleck PLX4032 microsaccade rate or change the overall temporal pattern of microsaccade generation (Fig. 3), it did cause a large redistribution in the directions of microsaccades (Fig. 6B). When the stimulus configuration was altered such that the foil was placed in the affected region of this sample SC inactivation instead of the cue, this large redistribution of microsaccade directions caused by inactivation did not occur (compare Fig. 6C and 6D), because the cue in the unaffected region of space was as effective in inducing microsaccades toward

its location (Fig. 6D) as it was before the inactivation (Fig. 6C). The results from this sample session therefore indicate that cue-induced changes in microsaccade directions were mediated by cue-related activity in the peripheral SC; elimination of such activity through muscimol-induced Gemcitabine datasheet inactivation altered the influence of the peripheral spatial cue on microsaccade directions. We Erastin cost next confirmed that this effect was not a mechanical effect resulting from fluid injection into the neural tissue by repeating exactly the same analysis but for our saline control injection of Fig. 4. The results were very different from those in Fig. 6, because the saline injection did not cause the massive reversal

of microsaccade directions seen above with muscimol. This result is illustrated in Fig. 7, which is presented in a format identical to that of Fig. 6. Thus, the results of the two sample sessions of Figs 6 and 7 combined suggest that muscimol inactivation of the peripheral SC in our task caused a significant alteration in cue-induced microsaccade directions. The effect of peripheral SC inactivation on cue-induced changes in microsaccade directions was also observed consistently across sessions from this monkey. Figure 8 shows the results of analysing microsaccade directions before and during SC inactivation for all experimental sessions involving monkey M. This analysis follows the approach from our previous behavioral study of microsaccades during this covert attention task (Hafed et al., 2011). Figure 8A shows the data obtained prior to SC inactivation for monkey M when the cue was placed in the region soon to be affected by SC inactivation. As can be seen, Fig.

Increasing the size of the clone libraries would help provide more conclusive data on the identity of the protist species involved. The clone library analysis showed that the

Day 0 cycloheximide-treated and -untreated libraries were statistically similar as expected, validating our approach. At other time points, the treatment and control libraries were statistically AMPK inhibitor different, indicating that cycloheximide did affect the protist ecology, which correlated with the improvement in the survival of E. coli O157:H7. In conclusion, our data point toward the role played by the protists in the reduction of E. coli O157:H7. We identified a number of protists that were present in our model compost, and it remains to be determined whether any of these species were responsible for the Selleckchem Depsipeptide decline in observed E. coli O157:H7 counts. The isolation and identification of the protist(s) that mediate this effect was beyond the scope of this study; however, this is an active area of investigation in our laboratory. Whether similar protist species are present in other composts, such as cow, pig, and horse manure, or in raw manure, is poorly understood and will be investigated in the future as well. Further work is also needed to determine how different temperatures and moisture levels would affect protist-mediated killing of E. coli O157:H7.

Composting conditions designed to support the proliferation of protists, as well as bacteria and fungi, that are antagonistic to E. coli O157:H7 may provide improved methods for bioremediation. This work was supported by a United States Department of Agriculture Cooperative State Research, Education, and Extension Service (USDA-CSREES)

grant 2008-34163-19283 and by start-up Funds from the Department of Food Science and the College of Agricultural Sciences OSBPL9 at the Pennsylvania State University. We would like to thank Dr Stephen Knabel, Dr Mary Ann Bruns, Morgan Minyard and Dr Bindhu Verghese at the Pennsylvania State University for their valuable input and help with the clone library sequence data processing. We would also like to thank the Nucleic Acid Facility at the Pennsylvania State University for providing sequencing data. “
“Propionic acid bacteria (PAB) are important as starter cultures for the dairy industry in the manufacture of Swiss-type cheeses, in which they are involved in the formation of eyes and are responsible for the typical flavour and aroma. These characteristics are mainly due to the classical propionic acid fermentation, but also the conversion of aspartate to fumarate and ammonia by the enzyme aspartase and the subsequent reduction of fumarate to succinate, which occur in dairy Propionibacterium freudenreichii ssp. shermanii and ssp. freudenreichii starter strains. Additionally, the metabolism of free amino acids may be partly responsible for secondary fermentation and the subsequent split defects in cheese matrix.

8% at months 12, 24, 36 and 48, respectively). Regarding plasma lipid levels (Fig. 1d and e) we did not observe significant changes during the follow-up period. We found hypercholesterolaemia (>200 mg/dL) in 9.5, 30.4, 21.7, click here 14.3 and 13.3% of patients at months 0, 12, 24, 36 and 48, respectively, and hypertriglyceridaemia (>170 mg/dL) in 14.3, 8.3, 13, 4.5 and 0% of patients at the same time-points. Throughout follow-up, and especially at the end of the study, we found an increase in plasma resistin and significant increases in total plasminogen activator inhibitor type 1 (tPAI-1), adiponectin and leptin levels (P<0.05) (Fig.

1f–i). Regarding the leptin:adiponectin ratio, HOMA values and C-peptide levels, we observed a slight increase during the first few months on HAART followed by a moderate decrease or stabilization after 24 months on HAART (Fig. 1j–l). The median BMI did not change significantly during follow-up, with values being between 17.32 and 16.42. There were no children in the overweight and low-weight BMI categories. www.selleckchem.com/products/AZD8055.html Concerning diagnoses of lipoatrophy, 17 children had no lipoatrophy, three had mild lipoatrophy, five had moderate lipoatrophy and two had severe lipoatrophy. Overall, seven of the 27 patients (25.9%) had lipoatrophy with scores ≥2. Concerning lipohypertrophy, 16 children did not have lipohypertrophy, three had mild lipohypertrophy, five had moderate lipohypertrophy and three had severe

lipohypertrophy. Overall, eight of the 27 patients (29.6%) had lipohypertrophy with scores ≥2. By the end of the study, 12 of the 27 children (44.4%) had lipodystrophy. However, only three of the 27 children (11.1%) had both lipoatrophy and lipohypertrophy scores ≥2. We carried out a follow-up study in PI-naïve HIV-infected find more children on HAART for 4 years, and found an increase in adipokine levels. This increase could be related to the

direct effect of PIs on adipose tissue, which could contribute to an imbalance in lipid metabolism and spatial development of lipodystrophy and metabolic syndrome in HIV-infected patients [21]. The metabolic pathway and the cytokine profile accomplices in the development of lipodystrophy and lipoatrophy is very complex. Thus, we did not find any significant trend in adipokine kinetics that may be associated with the onset of lipodystrophy at the end of the study. Moreover, results did not differ between patients with complete HIV suppression and those failing therapy. Therefore, we cannot definitely conclude from these results that there is a direct effect of HAART on adipose tissue, but there is a trend that warrants further investigation in studies with another design. In the present study we used clinical assessments of lipodystrophy; Dual Energy X-ray Absortiometry (DEXA) scanning would have provided a more quantitative assessment of lean vs. fat mass (particularly visceral fat) and may have provided better insights into the potential relationship between fat changes and adipokine levels.

16–18,24–31 The role GSK-3 cancer of the rapid diagnostic test (RDT) is well defined and its use is promoted by the World Health Organization for the diagnosis of this disease in endemic countries which have no access to microscopic evaluation. However, not all hospitals of industrialized countries have microbiologists on call 24 hours per day to do the peripheral blood examination. Rapid tests are therefore useful, especially for the diagnosis of significant parasitemia of P falciparum that is the one that conveys significant risk to the patient. Nevertheless, clinical examination is essential and it is the clinician who decides

whether or not to initiate antimalarial treatment if the patient is sick despite a negative RDT test. On the other hand, RDTs have less sensitivity for the diagnosis of low and mixed parasitemia, which is more frequent in recent immigrants. VFRs rarely use the Primary Health Care

Services possibly due to the fact that they are often symptomatic and go directly to the Emergency Department. As recent immigrants might have more cultural and language barriers and unfamiliarity with Western Health Care systems, delay in treatment may be exacerbated.18,32 However, no differences between groups were observed possibly due to the fact that most recent immigrants had relationship with relatives already living in our country and so barriers are lessened and they seek early attention MTMR9 requiring “infectious diseases screening. Fever Selleckchem CX 5461 was present at the time of diagnosis in 75% (45 of 60) of patients, and in 87% of patients (52 of 60) it was the main reason for consultation, similar to the proportion described in previous series (80%–100%).14,16,18,24–37 Fever, thrombocytopenia, and visceromegaly were more frequent in VFRs than in recent immigrants at the time of diagnosis (p < 0.05). Mascarello et al.9 found that VFRs had lower average platelet count and longer

fever duration in a subgroup of 43 children with imported malaria. Thrombocytopenia in children with fever is highly predictive of malaria following travel to a malaria-endemic area.9,38 Due to their semi-immunity,24,31,33 recent immigrants with malaria may be asymptomatic. In fact, seven cases in our series (11.6%) did not refer any related symptoms, which is in line with previously reported data (7%–36%).18,34,39,40 P falciparum was the most prevalent species in both groups. The percentage of mixed parasite infestations (5 of 60) was higher than other series.14,16,25,26,31 However, this greater percentage may be due to the use of the PCR for Plasmodium sp. in a high proportion of patients. All cases with mixed infections were detected in recent immigrants, perhaps due to an increased exposure time in the endemic areas. Previously described risk factors for imported severe malaria include young age (less than 5 y), delayed diagnosis, and lack of immunity to malaria.

Our data agree with a transcriptome study of osmo-adaptation in S. meliloti (Dominguez-Ferreras et al., 2006), which showed that many genes involved in flagellum biosynthesis and function are repressed in response to increased osmolarity and that transcription of ndvB is not significantly regulated by the osmotic strength of

the medium. Interestingly, in response to an osmotic downshift, the S. meliloti CβG transporter ndvA was induced, however (Dominguez-Ferreras et al., 2006), suggesting that although CβG synthesis is not regulated, the transport of CβG from the cytoplasmic compartment to the periplasmic space is osmo-regulated. The capacity of NGR∆ndvB to attach to the roots and develop a functional symbiosis with legume plants producing either determinate HDAC inhibitor (V. unguiculata) or indeterminate (L. leucocephala) types of nodules was compared to that of the wild-type strain. As expected, we found that adhesion to the roots and nodulation of both plant species were strongly affected by mutation of ndvB (Table 2). These results are consistent

with previous studies made with CβG mutants in other rhizobia (Breedveld & Miller, 1994; Crespo-Rivas et al., 2009). When L. leucocephala which forms indeterminate nodules was tested, the mutant produced mostly pseudonodules and one pink nodule for every 20 plants indicating that nodulation was not fully inhibited. On the other hand, neither nodules nor pseudonodules were observed on V. unguiculata roots when inoculated with the CβG mutant, suggesting that nodule development is impaired at an early stage in this plant. These results confirm that click here in V. unguiculata, nodulation is aborted early in the nodulation process when a CβG mutant is tested as showed for S. fredii (Crespo-Rivas et al., 2009). To further investigate the importance of cyclic glucans in the symbiosis, the transcriptional activity of ndvB was studied during nodule development, and the early infection process was followed using GFP-tagged strains. Roots of V. unguiculata and L. leucocephala were inoculated Rapamycin clinical trial with NGR234 carrying the ndvB promoter

cloned upstream of gfp. ndvB expression was observed in both young/developing nodules as well as mature (nitrogen-fixing) nodules (Fig. 3a, b, d, and e). This suggests that CβG of NGR234 are produced in nodules, supporting a role for cyclic glucans in invaded nodule cells, as suggested for B. japonicum (Gore & Miller, 1993). However, the pleiotropic effects shown by the mutant and the expression of ndvB in all conditions tested make it difficult to assess the role of CβG at this later stage of symbiosis development and during the functional symbiosis. We wanted to explore the effect cyclic glucans had on the early stage of symbiosis development. To know whether the nodulation defect was directly linked to the low plant root adhesion capacity of the ndvB mutant (Table 2) or if the mutation altered the normal infection process notably in V.

For example, the genome of Pectobacterium carotovorum SCRI1043 contains a gene cluster for the biosynthesis and transport of the siderophore enterobactin, which has been shown to be regulated by quorum sensing (Bell et al., 2004; Monson et al., 2012). Genes encoding the transport machinery, but not biosynthesis of achromobactin Selleck Z-VAD-FMK are also present, suggesting it may be utilized as a xenosiderophore (Franza & Expert, 2010). The role of these systems in virulence is yet to be tested and as Pectobacterium can adopt a saprophytic, soil-dwelling lifestyle, iron acquisition during infection may not be their

prominent role (Toth et al., 2006). Iron-uptake systems more likely to be involved in virulence are a ferric citrate uptake system and the HasA/HasR system discussed earlier. Plants utilize citrate to transport ferric iron to photosynthetic tissues via the xylem, suggesting uptake of this complex GSK3235025 cost may be important during vascular colonization by the pathogen (Thomine & Lanquar, 2011). As our understanding of pathogensis-related iron-uptake systems in Pectobacterium is still limited, it is quite possible that the genus may have evolved unique mechanisms to obtain iron from its host. Two bacteriocins Pectocin M1 and M2 from Pectobacterium were recently characterized by our laboratory (Grinter

et al., 2012). The cytotoxic domain of these proteins is homologous to that of colicin M, which functions by cleaving the peptidoglycan precursor lipid II (El Ghachi et al., 2006; Zeth et al., 2008; Barreteau et al., 2009; Fig. 1). We identified these proteins bioinformatically based on similarity to colicin M and this similarity was also noted by Helbig et al. (Helbig & Braun, 2011). Due to its low abundance and key role in cell-wall synthesis, lipid II constitutes a common vulnerability

among bacteria and is also targeted by a number of peptide-antibiotics (Breukink & de Kruijff, 2006; Schneider et al., 2010). Based on homology to the catalytic domain of colicin M, putative colicin M-like bacteriocins have been identified in a number genera of the γ-proteobacteria (Barreteau et al., 2004). Pectocin M sequence homology with colicin M is confined to the minimum C-terminal region of colicin M required for cytotoxic activity (Barreteau et al., 2009). Strikingly, Reverse transcriptase the remainder of the protein, which in colicin M consists of a helical receptor-binding domain and unstructured N-terminus, has been replaced through recombination with a plant-like [2Fe-2S] ferredoxin domain with an intact iron–sulphur cluster (Palmer et al., 1967; Grinter et al., 2012; Fig. 2). [2Fe-2S] ferredoxins represent a super family of small (≈100 amino acid) soluble proteins, which contain a single [2Fe-2S] cluster coordinated by four conserved cysteine residues and are predominantly found in the chloroplasts of plants and cyanobacteria (Fukuyama, 2004).

mirabilis ATCC 29906 were submitted to GenBank and assigned the accession numbers AF397169 (gyrA), AF503506 (gyrB) and AF363611 (parC). CIP uptake was assayed by the method of Giraud et al. (1999) with some modifications. Bacteria suspended in PBS to OD600 nm ~1.2 were equilibrated for 10 min at 37 °C. After the addition of CIP to a final concentration of 10 μg mL−1, 0.5 mL samples were removed at different time intervals. Five minutes after this addition,

the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) 100 μM was added to the reaction mixture. The samples were diluted in 1 mL of ice-cold PBS and centrifuged for 5 min at 5600 g. The pellet was washed once with 1 mL of ice-cold PBS and

resuspended in 1 mL of 0.1 M glycine hydrochloride (pH 3.0) for 1 h at room temperature. The samples were then centrifuged at 5600 g for 10 min and the fluorescence VE-822 solubility dmso of the supernatant was measured with a YASCO FP-777 spectrofluorimeter at excitation and emission wavelengths of 278.5 and 448.5 nm, respectively. The concentration of CIP in the supernatant was calculated by comparison with a standard curve for CIP in 0.1 M glycine hydrochloride. The results were expressed as nanograms of CIP incorporated mg−1 of protein. The FRAP assay (Benzie & Strain, 1999) was adapted to measure the antioxidant capacity of P. mirabilis. A volume of 100 μL of bacterial suspensions (OD600 nm ~1) was incubated with 125 μL of 3.1 mg mL−1 of 2,4,6-tripyridyl-1,3,5-triazine (TPTZ) in 40 mM HCl, 125 μL of FeCl3·6H2O 5.4 mg mL−1 and 1.25 mL of 300 mM acetate buffer (pH 3.6). Absorbances FK506 were

determined at 593 nm and expressed as μM of FeSO4 mg−1 protein. The concentration of proteins in bacterial suspension was determined by Folin–Ciocalteau assay (Stauffer, 1975). Bacterial suspensions of 1 mL were incubated with CIP or using PBS (control). The incubations were stopped P-type ATPase at 2 h with 1 mL of TCA 35% (p/v) in the absence of light. After 20 min, 1 mL of 0.5% (p/v) thiobarbituric acid was added and the samples were heated to 80 °C for 30 min. An ice bath was used to cool the samples, which were centrifuged at 1500 g and the absorbance of the supernatant was determined at 535 nm. A calibration curve of malondialdehyde (MDA) solutions was applied to estimate lipid oxidation. MDA levels were expressed as nmol MDA mg−1 protein. Bacterial suspensions of 3 mL were incubated with 0.5 mL of CIP or PBS (control) for 2 h. After that, 1 mL of the samples was treated with 1 mL of 0.1% 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl for 1 h. The proteins were precipitated in 5% trichloroacetic acid (TCA), centrifuged 20 min at 10 000 g, and the supernatant discarded. Samples were extracted three times with 1 mL ethanol/ethylacetate (1 : 1, v/v) to remove any remaining residual of DNPH. The precipitate was dissolved in 6 M guanidine hydrochloride solution in PBS and incubated for 30 min at 37 °C.