Differentiation of HELLP patients from healthy pregnant women by proteome analysis--on the way towards a clinical marker set

As the pathogenesis of the HELLP-syndrome is unknown, a complex consideration regarding the changes in the plasma as the main transport medium in the body is of great benefit because it is well available and can rapidly be investigated in the clinics. Besides that, the liver which is early affected in HELLP-syndrome produces the main part of plasma proteins. For the purpose of our study plasma protein abundances from patients with HELLP-syndrome and from control individuals were determined before and after delivery. In the differential analysis using two-dimensional gel electrophoresis, six areas with variable protein spot intensities were detected. The reference gel that we developed for HELLP plasma samples integrates the changes of plasma proteins when comparing HELLP patients to healthy women prior to and after delivery. A specific plasma protein profile for the HELLP-syndrome was generated involving protein areas that contain inter-alpha-trypsin inhibitor heavy chain H4, kininogen 1, fibrinogen gamma chain, transthyretin, haptoglobins, and serum amyloid A with statistically significant expression differences when compared to controls. The most striking difference between the majority of the gels from HELLP patients and the gels from non-HELLP samples were clearly overexpressed protein spots at about 11 kDa which were identified as serum amyloid A (SAA). This differential expression was validated and quantitatively assayed by ELISA measurements against human SAA in plasma. Our results show that significant differences in SAA expressions between healthy controls and HELLP patients were obtained, that could function as markers for the HELLP-syndrome. According to our data it is possible to draw a line of separation with no overlap between the HELLP group for which SAA plasma levels were found to be above 3.51 mg/L and the non-HELLP groups in which SAA plasma levels were below 3.51 mg/L. It now is possible to clinically elucidate if the differentially expressed proteins are suited for longitudinal studies concerning both, to function as markers or perhaps even as disease predictors that might become relevant for diagnostic tests.     

Molecular cloning of low-temperature-inducible ribosomal proteins from soybean

Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.     

Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The epidemiology of soil-transmitted helminth and protozoan infections in south-west Cameroon

A cross-sectional study of the prevalence, intensity and effects of soil-transmitted helminth and protozoan infections was undertaken among patients at the Buea Hospital Annex located in Buea sub-division of Cameroon. Stool samples from 356 subjects (174 males and 182 females) were collected and processed using standard concentration methods. Our results showed that 31.0% of subjects were infected with intestinal helminths and the prevalence was higher in females (32.4%) than in males (30.5%). A significantly higher prevalence was observed in rural (47.2%) than in urban areas (21.0%); significance < 0.1%. Prevalence was highest among those aged between 6 and 12 years (41.4%). The total prevalence of intestinal helminth infections were 19.3% for Ascaris lumbricoides, 14.0% for hookworm and 11.8% for Trichuris trichiura. The intensity of infection was unevenly distributed, with very heavy loads concentrated in a few individuals. Data also showed that 28.1% (100/356) of the subjects were infected with protozoans. Females showed a higher prevalence (28.6%; 52/182) than males (20.7%; 36/174). Also, there was a significantly higher prevalence in rural (34.0%; 49/144) than urban areas (18.4%; 39/212); significance < 0.1%. The age group 6-12 years again had a higher prevalence (37.1%; 26/70). The total prevalence of intestinal protozoans was: Entamoeba histolytica (24.4%), Entamoeba coli (11.2%) and Giardia lamblia (0.6%). These relatively heavy prevalences in patients may be reduced by appropriate medication and maintaining strict personal hygiene. Health education, clean water supply, good sewage management and a congenial environment will all help to minimize infection.     

Glycomics analyses of tear fluid for the diagnostic detection of ocular rosacea

A Glycomics approach to detect disease is illustrated in the analyses of human tear fluid for rosacea. The diagnosis of ocular rosacea is particularly challenging in a subgroup of patients that do not present with typical facial skin findings but have ocular signs and symptoms. Indeed, up to 90% of patients with ocular rosacea may have neither obvious roseatic skin changes nor a previous diagnosis of rosacea. Tear fluid was collected from 37 subjects (21 controls and 16 patients with ocular rosacea) after conjunctival stimulation with filter (Schirmer) paper. O-linked oligosaccharides were released from tear fluid by beta-elimination and then purified using solid-phase extraction. Mass spectra were recorded on an external source HiResMALDI with a 7.0 T magnet. Mass spectra were obtained in both the positive and negative modes. However, signals were stronger in the negative mode. Tear fluid samples from rosacea patients yielded distinctive clusters of peaks that extend to higher masses. Patients with rosacea presented several oligomeric series that were not found in the controls. To discriminate the ocular rosacea cases from the normal controls, the sum of absolute intensities of 13 series corresponding to nearly 50 identified mass spectrum peaks was used. Thirty-six out of the 37 samples were correctly classified. This yields a sensitivity of 100% (95% CI 79.5-100) and specificity of 95.2% (95% CI 76.2-99.9). The high abundance of oligosaccharides in the tear fluid of patients with rosacea may lead to an objective diagnostic marker for the disease.     

Diagnosis of active tuberculosis using MPB64, a specific antigen of Mycobacterium bovis

Because the incidence of tuberculosis (TB) is still high in developing countries, an inexpensive and rapid diagnostic test for this infection is needed. To develop a screening test for TB, MPB64 antigen was produced by recombinant technology and purified with a polyhistidine tag. Next, serum and urine samples from patients with TB and uninfected individuals were examined by the dot‐blot assay method using this purified antigen. Serum samples from patients with TB reacted more strongly with MPB64 antigen than did those from uninfected individuals. In addition, serum samples from TB patients with active infection reacted more strongly with the antigen than did samples from patients with inactive TB. When urine samples were assessed using this assay, similar results were obtained. Correlations between the data obtained from serum and urine samples were analyzed for all subjects, including uninfected individuals, and a strong positive correlation between the results of serum and urine tests (n = 36, r = 0.672) was found. The sensitivity and specificity of this assay for serum samples was 85.7 % and 85.0 %, and for urine samples 75.0 % and 85.0 %, respectively. These results suggest that dot‐blot assay with MPB64 antigen could be a useful screening test for active TB. Because urine samples can be obtained more easily than serum samples and because urine is less contagious, urine testing should probably be employed for screening purposes.     

Application of enzyme-linked immunosorbent assay for mass examination of strongyloidiasis in okinawa, Japan

The enzyme-linked immunosorbent assay was tested to evaluate whether it could be applicable in screening for mass examination of strongyloidiasis. A total of 2906 inhabitants in three areas (858 in Gushikawa Village, 849 in Nakazato Village and 1199 in Sashiki Town) were screened by the enzymatic assay and approximately 11–30% (11.8% in Gushikawa, 17.0% in Nakazato and 27.7% in Sashiki) were considered to be antibody positive. In the parasitological follow-up examinations of those who were antibody positive, actual infection was found in more than half (51%) the subjects. The overall infection rates estimated from the results reached 5.8% in Gushikawa, 9.1% in Nakazato and 14.0% in Sashiki (mean = 10.4%). The infection rates were significantly higher than those in previous surveys conducted in the same areas. The ELISA technique was found to be useful for strongyloidiasis screening and for seroepidemiological purposes in Okinawa.     

A mass-spectrometric approach to primary screening of collagenolytic enzymes

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestesfrischi and D. maculates) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks ("fingerprint") characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.     

Characterization of humic materials extracted from hazelnut husk and hazelnut husk amended soils

This study was carried out to determine effects of composted hazelnut husk (CHH) on some chemical properties of soil and soil humic acid (HA). Compost application increases organic matter (OM) content of the soil considerably, OM value of 3.18% became 3.89% in 3 years time interval. Before application of compost, the soil pH was found to be 5.37, while after compost application it became 5.61. FTIR characteristics of humic acid/humic acid-like materials extracted from the original hazelnut husk, composted hazelnut husk and composted hazelnut husk amended soil have been investigated. C and O content of humic acid-like/humic acid materials were in the range of 41.4–50.8% and 37.8–50.5%, respectively. The N content of the humic acid/humic acid-like materials are in the expected range for humic materials which is 2–6%. Comparison of FTIR spectra of hazelnut husk and composted hazelnut husk humic acid-like fractions shows that both exhibit similar but not identical series of IR bands indicating the presence of the same functional groups in both samples. The only difference in the spectra seemed to be a decrement in the peak intensities of composted sample compared to uncomposted one. The similar differentiation of the intensities of IR bands of compost applied soil sample has also been observed. The FTIR spectral results show that the characteristics of composted material tend to become similar to that of soil humic acids characteristics in time.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.     

Algorithms for alignment of mass spectrometry proteomic data

MOTIVATION: The analysis of biological samples with high-throughput mass spectrometers has increased greatly in recent years. As larger datasets are processed, it is important that the spectra are aligned to ensure that the same protein intensities are correctly identified in each sample. Without such an alignment procedure it is possible to make errors in identifying the signals from peptides with similar molecular weight. Two algorithms are provided that can improve the alignment among samples. One algorithm is designed to work with SELDI data produced from a Ciphergen instrument, and the other can be used with data in a more general format. RESULTS: The two algorithms were applied to samples drawn from a common pool of reference serum. The results indicate substantial improvement in consistently identifying peptide signals in different samples.     

Characterisation of extracellular polysaccharides produced by Crypthecodinium cohnii

The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6-48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via alpha-(1-3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.     

Muscle sound and electromyogram spectrum analysis during exhausting contractions in man

The changes in the soundmyogram (SMG) and electromyogram (EMG) frequency content during exhausting contractions at 20%, 40%, 60% and 80% of the maximal voluntary contraction (MVC) were investigated by the spectral analysis of the SMG and EMG detected from the biceps brachii muscles of 13 healthy men. The root mean squares (rms) of the two signals were also calculated. Throughout contraction the EMG rms always increased while this was true only at 20% MVC for the SMG. A marked decrease was detected at 60% and 80% MVC. With fatigue the EMG spectra presented a compression towards the lower frequencies at all exercise intensities. The SMG showed a more complex behaviour with a transient increase in its frequency content, followed by a continuous compression of the spectra, at 60% and 80% MVC, and a nearly stable frequency content at lower contraction intensities. This study suggested that different aspects of the changes in the motor unit's activation strategy at different levels of exhausting contractions can be monitored by SMG and EMG signals.     

Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.     

Urinary proteome profiling using microfluidic technology on a chip

Clinical diagnostics and biomarker discovery are the major focuses of current clinical proteomics. In the present study, we applied microfluidic technology on a chip for proteome profiling of human urine from 31 normal healthy individuals (15 males and 16 females), 6 patients with diabetic nephropathy (DN), and 4 patients with IgA nephropathy (IgAN). Using only 4 microL of untreated urine, automated separation of proteins/peptides was achieved, and 1-7 (3.8 +/- 0.3) spectra/bands of urinary proteins/peptides were observed in the normal urine, whereas 8-16 (11.3 +/- 1.2) and 9-14 (10.8 +/- 1.2) spectra were observed in urine samples of DN and IgAN, respectively. Coefficient of variations of amplitudes of lower marker (1.2 kDa), system spectra (6-8 kDa), and upper marker (260.0 kDa) were 22.84, 24.92, and 32.65%, respectively. ANOVA with Tukey post-hoc multiple comparisons revealed 9 spectra of which amplitudes significantly differed between normal and DN urine (DN/normal amplitude ratios ranged from 2.9 to 3102.7). Moreover, the results also showed that 3 spectra (with molecular masses of 12-15, 27-28, and 34-35 kDa) were significantly different between DN and IgAN urine (DN/IgAN amplitude ratios ranged from 3.9 to 7.4). In addition to the spectral amplitudes, frequencies of some spectra could differentiate the normal from the diseased urine but could not distinguish between DN and IgAN. There was no significant difference, regarding the spectral amplitude or frequency, observed between males and females. These data indicate that the microfluidic chip technology is applicable for urinary proteome profiling with potential uses in clinical diagnostics and biomarker discovery.     

Structure of valinomycin-K+ complex in solution by extended x-ray absorption fine structure.

We used synchrotron radiation to measure the K-edge absorption spectra of the potassium ion in valinomycin-K+ complexes dissolved in ethanol and methanol. Our motivation is to study the structure of valinomycin around the potassium ion and the effect of solvents. From the extended x-ray absorption fine structure, we found that the mean distance from potassium to its coordination atoms, oxygen, is the same for both solvents, 2.79 +/- 0.02 A, compared with 2.76 A in crystal. The K-edge threshold spectra of the two solutions are almost identical but have a small difference in their relative peak intensities. The coincidence of their corresponding peak positions indicates that the strength of ligand field is about the same in these two samples. This agrees with the known binding energies of potassium ion to valinomycin in solutions. The difference in the relative peak intensities suggests a perturbation of ligand symmetry by solvents.     

Cracking the molecular weight barrier: fragment screening of an aminotransferase using an NMR-based functional assay

NMR-based screening of protein targets has become a well established part of the drug discovery process especially with respect to fragments. However, as target size increases the two-dimensional spectra typically used for such screening become more crowded due to the increased number of signals, and the individual signals broaden due to the decreased rotational correlation time of the protein. Here we present an NMR-based functional assay for the branched-chain aminotransferase BCATc, a dimer with a total molecular weight of 88 kDa, which overcomes the limitations of the typical protein-based NMR screening method. BCATc is involved in glutamate production in the brain and is a therapeutic target for neuronal disorders involving a glutamatergic mechanism. Several fragments which inhibit BCATc were discovered using this assay and these may serve as novel cores for the development of potent BCATc inhibitors.     

Towards fruitful metabolomics: high throughput analyses of polyphenol composition in berries using direct infusion mass spectrometry

Tannin-enriched extracts from raspberry, cloudberry and strawberry were analysed by liquid chromatography-mass spectrometric (LC-MS) techniques. The raspberry and cloudberry extracts contained a similar mixture of identifiable ellagitannin components and ellagic acid. However, the strawberry extract contained a complex mixture of ellagitannin and proanthocyanidin components that could not be adequately resolved to allow identification of individual peaks. Nevertheless, the negative ESI-MS spectra obtained by direct infusion mass spectrometric (DIMS) analysis described the diversity of these samples. For example, the predominance of signals associated with Lambertianin C in cloudberry and Sanguiin H6 in raspberry tannin extracts could be discerned and the diversity of signals from procyanidin and propelargonidin oligomers could be identified in the strawberry extract. The dose response for the main ellagitannin-derived signals in the raspberry tannin sample revealed a saturation effect probably due to ion suppression effects in the ion trap spectrometer. Nevertheless, DIMS spectra of whole berry extracts described qualitative differences in ellagitannin-derived peaks in raspberry, cloudberry and strawberry samples. In addition, positive mode DIMS spectra illustrated qualitative differences in the anthocyanin composition of berries of progeny from a raspberry breeding population that had been previously analysed by LC-MS. This suggests that DIMS could be applied to rapidly assess differences in polyphenol content, especially in large sample sets such as the progeny from breeding programmes.     

A mass-spectrometric approach to primary screening of collagenolytic enzymes

A comparative analysis of MALDI TOF mass spectra of low-molecular products resulting from the hydrolysis of native collagen I by collagenases of various classes (bacterial metallocollagenase from Clostridium histolyticum, serine collagenase from the Morikrasa commercial preparation, cysteine collagenase from Serratia proteomaculans, and cysteine collagenases from larvae of beetles Dermestes frischi and D. maculatus) was carried out. The spectra contain a number of ion peaks common for all collagenases; nevertheless, the mass spectra of each hydrolysate contains a unique set of peaks (“fingerprint”) characteristic of each enzyme. This is especially true for the peaks of major products with relative intensities of more than 50%. At the same time, the enzymes of one class (cysteine collagenases) exhibit in their mass spectra peaks of identical major products. The results show a potential opportunity for MALDI TOF application in the primary screening of collagenases according to the fingerprints of collagen hydrolysis products.