Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.     

Discrimination of microbial diversity by fatty acid profiles of phospholipids and lipopolysaccharides in differently cultivated soils

The effects of long-term management practices on the diversity of the microbial community were examined by analyzing the composition of fatty acids (FAs) in phospholipids (PL) and lipopolysaccharides (LPS). According to the Principal Component Analysis (PCA) of total fatty acids the soils were divided in two groups: a) Black fallow soil (1) and soils cropped with potatoes (3, 4), and b) green fallow soil (2), soils cropped with wheat (5, 6), crop rotation (7) and grassland (8). The PCA for saturated FAs and for hydroxy FAs of both PL and LPS shows that the green fallow soil (2) can be distinguished from the other soils. For monounsaturated FAs the grassland soil (8) and for polyunsaturated FAs the wheat with vetch soil (6) clearly differed from the other soils. Fatty acids with biomarker quality such as 15:0 for bacteria and 18:26 for fungi were used for determining the ratio between bacteria and fungi: the black fallow soil (1) and the soil managed with crop rotation (7) contained significantly higher proportions of bacteria than the other soils. The largest proportion of the indicator fatty acid il5:0 for Gram-positive bacteria was measured in the black fallow soil (1), while the-hydroxy FAs indicative of Gram-negative bacteria most frequently occurred in manured potato cropped soil (4). Both indicator fatty acids 18:26 for fungi and cy19:0 for anaerobic bacteria had their highest concentrations in the manured potato cropped soil (4).     

Simultaneously quantitative measurement of comprehensive profiles of esterified and non-esterified fatty acid in plasma of type 2 diabetic patients

Fatty acids, having intimate relationship with type 2 diabetes mellitus (DM2), are not only the main energy source as nutrients, but also signaling molecules in insulin secretion. In this work, we developed a two-step rapid method to comprehensive profiling of esterified fatty acid (EFA) and non-esterified fatty acid (NEFA) using KOH–CH₃OH to methylate EFA followed by H₂SO₄–CH₃OH to methylate NEFA. Its applications to fatty acids profiling of type 2 diabetic patients and health controls were also presented. The t-test results informed that 16 NEFAs and 7 EFAs had distinct differences between type 2 diabetes and health controls. Furthermore, quantitative alterations of fatty acids in plasma of type 2 diabetic patients treated with rosiglitazone were obtained by this method. Our research results indicated that the dynamic changes of NEFAs are various. Some decreased linearly, such as C18:0, C18:3n-6 and C22:6, and some changed nonlinearly, such as C18:3n-3 and C22:4. All results informed that fatty acid profiles could provide comprehensive and accurate information for not only discrimination between DM2 patients and health controls, but also evaluation alterations of fatty acids during therapeutic process.     

Discrimination of Golgi type II membrane proteins based on their hydropathy profiles and the amino acid propensities of their transmembrane regions

Membrane proteins in the Golgi apparatus play important roles in biological functions, predominantly as catalysts related to post-translational modification of protein oligosaccharides. We succeeded in extracting the characteristics of Golgi type II membrane proteins computationally by comparison with those of Golgi no retention proteins, which are mainly localized in the plasma membrane. Golgi type II membrane proteins were detected by combining hydropathy alignment and a position-specific score matrix of the amino acid propensities around the transmembrane region. We achieved 96.2% sensitivity, 93.5% specificity, and a 0.949 success rate in a self-consistency test. In a 5-fold cross-validation test, 88.0% sensitivity, 85.5% specificity, and a 0.867 success rate were achieved.     

Mass spectrometry as a tool for identifying group D2 corynebacteria by their fatty acid profiles

Corynebacterium group D2 (CGD2) are lipophilic antibiotic-multiresistant bacteria involved in some infections of immunocompromised patients. The fatty acid composition and structure of different strains was established by several mass spectrometric methods, particularly negative ion tandem mass spectrometry coupled with capillary gas chromatography. Non-hydroxylated fatty acid profiles of three strains of CGD2 (ATCC 43042, ATCC 43043, ATCC 43044) were almost identical and revealed the presence of several straight chain unsaturated fatty acids from the omega-9 series, with even carbon numbers ranging from 14 to 24. Branched saturated fatty acids were mainly anteiso-heptadecanoic acid and tuberculostearic acid. Surprisingly, a relatively large quantity of 10-methylene octadecanoic acid was found. The non-hydroxylated fatty acid profile of one rare beta-lactam susceptible strain (SC1) was different; 10-methylene octadecanoic acid was lacking whereas tuberculostearic acid was much more abundant. In contrast, the four CGD2 strains displayed highly similar mycolic acid patterns. The major mycolic acid species corresponded to C32, C30 and C28 bis-unsaturated with a double bond on each branch at the omega-9 position. The comparison of the mycolic acid composition and structure with those of other medically important corynebacteria strains, revealed a characteristic pattern for CGD2 strains, and CGD2 strains were easily distinguished from Corynebacterium jeikeium (CIP 82.51).     

Computational prediction of CRISPR cassettes in gut metagenome samples from Chinese type-2 diabetic patients and healthy controls

Background

CRISPR has been becoming a hot topic as a powerful technique for genome editing for human and other higher organisms. The original CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats coupled with CRISPR-associated proteins) is an important adaptive defence system for prokaryotes that provides resistance against invading elements such as viruses and plasmids. A CRISPR cassette contains short nucleotide sequences called spacers. These unique regions retain a history of the interactions between prokaryotes and their invaders in individual strains and ecosystems. One important ecosystem in the human body is the human gut, a rich habitat populated by a great diversity of microorganisms. Gut microbiomes are important for human physiology and health. Metagenome sequencing has been widely applied for studying the gut microbiomes. Most efforts in metagenome study has been focused on profiling taxa compositions and gene catalogues and identifying their associations with human health. Less attention has been paid to the analysis of the ecosystems of microbiomes themselves especially their CRISPR composition.

Results

We conducted a preliminary analysis of CRISPR sequences in a human gut metagenomic data set of Chinese individuals of type-2 diabetes patients and healthy controls. Applying an available CRISPR-identification algorithm, PILER-CR, we identified 3169 CRISPR cassettes in the data, from which we constructed a set of 1302 unique repeat sequences and 36,709 spacers. A more extensive analysis was made for the CRISPR repeats: these repeats were submitted to a more comprehensive clustering and classification using the web server tool CRISPRmap. All repeats were compared with known CRISPRs in the database CRISPRdb. A total of 784 repeats had matches in the database, and the remaining 518 repeats from our set are potentially novel ones.

Conclusions

The computational analysis of CRISPR composition based contigs of metagenome sequencing data is feasible. It provides an efficient approach for finding potential novel CRISPR arrays and for analysing the ecosystem and history of human microbiomes.

     

Serum nickel levels of diabetic patients and healthy controls by AAS with a graphite furnace

In this study, serum nickel levels of diabetic patients and healthy controls were determined by AAS with a graphite furnace. The serum nickel concentrations were found to be 1.15 +/- 1.89 micrograms/L in healthy controls and 0.82 +/- 0.74 microgram/L in diabetics. There was, however, no statistically significant difference between the two groups (p = 0.13). The relationship of nickel levels to diabetes type and duration, diabetic complications and treatment, sex, age, and heredity was investigated. However, again no significant differences were found, nor was there any correlation between serum nickel levels and blood sugar, HBa1c, fructosamine, sialic acid levels, and age.     

Haemagglutination-inhibiting variola antibodies in blood serum of former smallpox patients,their healthy siblings,and unvaccinated controls from other areas

Summary Haemagglutination-inhibiting variola antibodies were compared in 50 smallpox survivors (P), 50 siblings who had shown no signs of smallpox (CI) and 44 persons living in villages which had apparently been free of the infection during recent years (CII). Only unvaccinated persons were examined. Distribution of titres was identical in groups P and CI, whereas the average values in group CII were lower.

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Zusammenfassung 50 früher an Pocken erkrankte Inder, 50 Geschwister dieser Patienten, die selbst nie an Pocken erkrankt waren, sowie 44 Personen aus Dörfern, die in den letzten Jahren keine Pockenepidemie durchgemacht hatten, wurden bezüglich der Variola-Antikörper miteinander verglichen. Alle untersuchten Personen waren niemals gegen Pocken geimpft worden. Die Verteilung der Titerwerte bei früher Erkrankten und bei ihren nicht erkrankten Geschwistern stimmte überein, während die dritte Gruppe im Durchschnitt geringere Werte zeigte.

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This study was supported by the Fischer-Stiftung, Heidelberg.     

Predictions of methane emission levels and categories based on milk fatty acid profiles from dairy cows

Milk fatty acid (MFA) have already been used to model methane (CH₄) emissions from dairy cows. However, the data sets used to develop these models covered limited variation in dietary conditions, reducing the robustness of the predictions. In this study, a data set containing 140 observations from nine experiments (41 Holstein cows) was used to develop models predicting CH₄ expressed as g/day, g/kg dry matter intake (DMI) and g/kg milk. The data set was divided into a training (n=112) and a test data set (n=28) for model development and validation, respectively. A generalized linear mixed model was fitted to the data using the marginal R²_(m) and the Akaike information criterion to evaluate the models. The coefficient of determination of validation (R²_(v)) for different models developed ranged between 0.18 and 0.41. Form the intake-related parameters, only inclusion of total DMI improved the prediction (R²_(v)=0.58). In addition, in an attempt to further explore the relationships between MFA and CH₄ emissions, the data set was split into three categories according to CH₄ emissions: LOW (lowest 25% CH₄ emissions); HIGH (highest 25% CH₄ emissions); and MEDIUM (50% remaining observations). An ANOVA revealed that concentrations of several MFA differed for observations in HIGH compared with observations in LOW. Furthermore, the Gini coefficient was used to describe the MFA distribution for groups of MFA in each CH₄ emission category. The relative distribution of the MFA, particularly of the odd- and branched-chain fatty acids and mono-unsaturated fatty acids of observations in category HIGH differed from those in the other categories. Finally, in an attempt to validate the potential of MFA to identify cases of high or low emissions, the observations were re-classified into HIGH, MEDIUM and LOW according to the proportion of each individual MFA. The proportion of observations correctly classified were recorded. This was done for each individual MFA and for the calculated Gini coefficients, finding that a maximum of 67% of observations were correctly classified as HIGH CH₄ (trans-12 C18:1) and a maximum of 58% of observations correctly classified as LOW CH₄ (cis-9 C17:1). Gini coefficients did not improve this classification. These results suggest that MFA are not yet reliable predictors of specific amounts of CH₄ emitted by a cow, while holding a modest potential to differentiate cases of high or low emissions.     

Differentiation between Candida species isolated from diabetic foot by fatty acid methyl ester analysis using gas chromatography

Gas chromatography (GC) was used to differentiate 100 isolates of Candida species (Candida parapsilosis, Candida albicans, Candida tropicalis, Candida famata and Candida glabrata) from 22 of 509 diabetic patients in whom the same species had been isolated from ulcer and interdigital spaces of the same and/or the other foot. All clinical isolates were identified by quantitative differences in the composition of six cell fatty acids (CFA). The values of the coefficients of variability (CV) of CFA show that the isolates from foot ulcers and interdigital spaces of the same diabetic patient probably belong to different chemotypes of the same Candida species.     

Identification of high affinity fatty acid binding sites on human serum albumin by MM-PBSA method

Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the “three-trajectory method” and the “single-trajectory method”. The former, which is less precise than the latter but may be more accurate as it includes the effects of conformational change upon binding, was used to classify high and low affinity sites. As a result, Sites 2, 4, and 5 were identified as high affinity sites for both FAs. The latter method, which is precise because energies are calculated from snapshots of the same trajectory for HSA-FAcomplex, was performed to compare the magnitude of binding free energy for these sites. The order of magnitude was 5 > 4 > 2, identical to that of a previous publication by others. In this way, a combination of the two methods was effectively used to identify high affinity sites. This study therefore provides an insight into the quantitative identification of high affinity FA binding sites on HSA.     

Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements

Abstract The importance of protein breakdown and amino acid fermentation in the overall economy of the large intestine has not been quantitated. We have therefore measured the production of branched chain-fatty acids (BCFA) both in vitro and in vivo in order to estimate the contribution of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids (SCFA) were the principal end products formed during the degradation of protein by human colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA. Branched-chain fatty acids (BCFA) constituted 16% of the SCFA produced from bovine serum albumin and 21% of the SCFA generated when casein was the substrate. BCFA concentrations in gut contents taken from the human proximal and distal colons were on average, 4.6 and 6.3 mmol kg⁻¹ respectively, corresponding to 3.4% and 7.5% of the total SCFA. These results suggest that protein fermentation could potentially account for about 17% of the SCFA found in the caecum, and 38% of the SCFA produced in the sigmoid/rectum. Measurements of BCFA in portal and arterial blood taken from individuals undergoing emergency surgery indicated that net production of BCFA by the gut microflora was in the region of 11.1 mmol day⁻¹, which would require the breakdown of about 12 g of protein. These data highlight the role of protein in the colon and may explain why many colonic diseases affect mainly the distal bowel.     

Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements

Abstract The importance of protein breakdown and amino acid fermentation in the overall economy of the large intestine has not been quantitated. We have therefore measured the production of branched chain-fatty acids (BCFA) both in vitro and in vivo in order to estimate the contribution of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids (SCFA) were the principal end products formed during the degradation of protein by human colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA. Branched-chain fatty acids (BCFA) constituted 16% of the SCFA produced from bovine serum albumin and 21% of the SCFA generated when casein was the substrate. BCFA concentrations in gut contents taken from the human proximal and distal colons were on average, 4.6 and 6.3 mmol kg⁻¹ respectively, corresponding to 3.4% and 7.5% of the total SCFA. These results suggest that protein fermentation could potentially account for about 17% of the SCFA found in the caecum, and 38% of the SCFA produced in the sigmoid/rectum. Measurements of BCFA in portal and arterial blood taken from individuals undergoing emergency surgery indicated that net production of BCFA by the gut microflora was in the region of 11.1 mmol day⁻¹, which would require the breakdown of about 12 g of protein. These data highlight the role of protein in the colon and may explain why many colonic diseases affect mainly the distal bowel.     

Obese type 2 diabetics and obese patients have comparable plasma phospholipid fatty acid compositions deviating from that of healthy individuals

There exist controversial reports regarding the differences in phospholipid fatty acids in type 2 diabetic and obese patients as compared to controls. The study was aimed at assessing the combined effect of type 2 diabetes and obesity on the fatty acid composition of plasma phospholipids. The experimental group consisted of 23 Belgian obese type 2 diabetics on Metformin. Two control groups were used: healthy lean and obese individuals in the same BMI range as the diabetics. Plasma phospholipids were isolated and their fatty acids and vinyl ether moieties were determined. Significance was set at P < 0.01. Plasma phospholipid fatty acids and plasmalogen-derived dimethyl acetals in diabetics deviated in many respects from these of lean controls but were not significantly different from those of obese non-diabetic patients. Therefore, the deviations of the fatty acid pattern of plasma phospholipids in type 2 diabetes may be attributed to obesity rather than to diabetes itself.     

Mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice

The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.     

Reliable identification of Prevotella and Butyrivibrio spp. from rumen by fatty acid methyl ester profiles

Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera,Prevotella andButyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzedPrevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The strains ofPrevotella could be well identified on species level by the characteristic ratios among major fatty acids and by acids unique fatty for each species. The 45Butyrivibrio strains were grouped into 4 major and 2 minor groups according to FAMEs profiles. The major fatty acids for the bulk of theButyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups corresponded to those based on 16S rDNA sequences.     

Reduced Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels from arteries of Type 2 diabetic Zucker diabetic fatty rats

Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.     

Correlation of the fatty acid composition and fluid property of the cholesteryl esters in the serum of Nigerian children with sickle cell disease and healthy controls

In a previous study conducted in Nigeria, we found that children with sickle cell disease (SCD) had exceedingly low total serum cholesterol levels (mean=100-102mg/dl). The fact that significant reductions in the levels of certain polyunsaturated fatty acids (PUFA) have been documented in the serum phospholipids of these same SCD subjects led us to inquire as to the fatty acid composition of the cholesteryl esters (CE) in their serum. Lecithin:cholesterol acyl transferase (LCAT), the enzyme in blood that catalyzes the reaction in which tissue cholesterol is acylated prior to its removal from cell membranes, is relatively specific for certain PUFA. CE in blood serum from 43 male and 42 female children with SCD, ages 4-18 years, and equal numbers of age- and gender-matched controls were analyzed for their fatty acid composition. Relative to the non-SCD controls, the CE of the SCD subjects contained 9% less linoleic acid, 16% less arachidonic acid, 40% less alpha-linolenic acid, 50% less eicosapentaenoic acid, and 36% less docosahexaenoic acid, but 15% more palmitic acid and 10% more oleic acid. Overall, the acyl chains of the CE of the SCD subjects were less fluid than those of the controls, as determined by comparison of their mean melting points (MMP) and double bond indices (DBI). MMP and DBI were both estimated from the individual constituent fatty acids comprising the CE acyl chains. The strongest correlations between MMP and fatty acid mole percent were seen with palmitic acid and linoleic acid. These results show that the fatty acid composition of the serum CE of children with SCD is abnormal relative to controls who do not have this hematologic disorder. We speculate that suboptimal fatty acid nutrition in Nigerian children with SCD compromises their ability to remove cholesterol from their tissues due to preference of the LCAT enzyme for PUFA, thereby accounting, in part at least, for the low total serum cholesterol levels one finds in children with SCD.