Generating amplicon reads for microbial community assessment with next-generation sequencing.

Marker gene amplicon sequencing is often preferred over whole genome sequencing for microbial community characterization, due to its lower cost while still enabling assessment of uncultivable organisms. This technique involves many experimental steps, each of which can be a source of errors and bias. We present an up-to-date overview of the whole experimental pipeline, from sampling to sequencing reads, and give information allowing for informed choices at each step of both planning and execution of a microbial community assessment study. When applicable, we also suggest ways of avoiding inherent pitfalls in amplicon sequencing.

Effect of the 5-HTTLPR polymorphism on affective temperament, depression and body mass index in obesity.

BACKGROUND AND AIM: Many studies show high prevalence of affective disorders in obese patients. Affective temperament is a subclinical manifestation of such conditions. The 5-HTT gene encoding the serotonin transporter may be involved in both mood and eating dysregulation. The aim of this study was to investigate the influence of a polymorphism in the 5-HTT gene on affective temperament types, depressive symptoms and Body Mass Index (BMI) in obese patients. METHODS: This study involved 390 patients (237 females, and 153 males) with obesity. The TEMPS-A questionnaire, Beck Depression Inventory (BDI) and Hamilton Depression Rating Scale (HDRS) were used to evaluate affective temperaments and prevalence of depression. DNA was obtained for serotonin transporter gene-linked polymorphism (5-HTTLPR) genotyping. RESULTS: In obese patients S/S genotype was associated with depressive and L/L with cyclothymic temperament. Subjects with L/L genotype presented significantly higher BMI and greater intensity of depressive symptoms in BDI and HDRS. Females scored higher in anxious and depressive, while males in hyperthymic, cyclothymic and irritable temperaments. Females scored higher in BDI (subjective depression) while males in HDRS (objective depression). LIMITATIONS: TEMPS-A, BDI and HDRS are frequently used in studies on affective disorders. However, these methods do not examine all dimensions of mood and personality. CONCLUSIONS: In obese patients S allele of 5-HTTLPR was associated with development of depressive temperament while L allele corresponded with greater obesity and prevalence of depression. Different mechanisms may be involved in manifestation of depression in males and females with obesity.

The relationship between IL-28B polymorphisms and the response to peginterferon alfa-2a monotherapy in anti-HBe-positive patients with chronic HBV infection.

The impact of interleukin 28B (IL-28B) on the results of interferon (IFN)-based therapy in patients chronically infected with hepatitis B virus (HBV) is poorly understood. The aim of this study was to evaluate the relationship between IL-28B markers and the response to IFN monotherapy in Polish patients with anti-hepatitis B e (HBe)-positive chronic hepatitis B (CHB). We determined three single-nucleotide polymorphisms (SNPs) of IL-28B (rs12979860, rs12980275, and rs8099917) in 86 patients who were treated with pegylated interferon (PEG-IFN) for 48 weeks. The effectiveness of the therapy was evaluated based on the virological and biochemical response. The primary efficacy parameters were the HBV DNA viral load below 400 IU/ml and 2,000 IU/ml in combination with alanine aminotransferase (ALT) normalization (<40 IU/l), measured 24 weeks after the treatment. Viral load below 400 IU/ml or 2,000 IU/ml with ALT normalization was achieved by 37 % and 46 % of patients, respectively. It has been shown that the distribution of IL-28B genotypes in the dominant genetic model in patients with different therapeutic success differ significantly only for rs12979860. The IL-28B rs12979860 CC genotype was associated with lower treatment success [odds ratio (OR), 0.31; p = 0.025 and OR, 0.37; p = 0.044 for <400 IU/ml HBV DNA with <40 IU/l ALT, and <2,000 IU/ml HBV DNA with <40 IU/l ALT, respectively]. However, in the conditional logistic regression analysis adjusted by factors associated with combined response, rs12979860 was significantly associated only with <400 IU/ml HBV DNA with <40 IU/l ALT (OR, 0.24; p = 0.026). IL-28B polymorphisms have prognostic significance in assessing the treatment effectiveness based on the virological and biochemical response of patients with anti-HBe-positive CHB.

Impact of IL-28B polymorphisms on pegylated interferon plus ribavirin treatment response in children and adolescents infected with HCV genotypes 1 and 4.

IL-28B polymorphisms are predictors of response to therapy in adults infected with hepatitis C. We do not know whether they are markers of response to therapy in children and adolescents. The aim of this study was to determine whether single-nucleotide polymorphisms (SNPs) in the IL-28B gene could influence the probability of response to therapy compared with other known baseline prognostic factors and correlate with clinical findings in pediatric patients infected with hepatitis C virus (HCV) genotypes 1 or 4. We determined three SNPs of IL-28B (rs12979860, rs12980275, and rs8099917) in 82 patients with chronic HCV infection treated with pegylated interferon alpha and ribavirin (peg-IFNα/RBV). Treatment response and clinical data were analyzed. Overall, sustained virological response (SVR) was achieved by 45 % of patients infected with difficult-to-treat HCV genotypes 1 and 4. Except for IL-28B polymorphisms, there was no association of SVR with any other clinical data. IL-28B rs12979860 CC [odds ratio (OR), 6.81; p = 0.001] and rs8099917 TT (OR, 3.14; p = 0.013) genotypes were associated with higher SVR rates. IL-28B rs12980275 was not significantly associated with SVR (p = 0.058). Only the distribution between CC and CT-TT genotypes of rs12979860 significantly differentiated patients achieving early virological response (EVR) (OR, 10.0; p = 0.011). Children with the rs12979860 CC genotype had significantly higher baseline viral load compared with CT-TT patients (p = 0.010). In children and adolescents chronically infected with HCV genotypes 1 and 4, IL-28B rs12979860 and rs8099917 polymorphisms were the only predictors of response to peg-IFN/RBV.

Etiology of inguinal hernia: ultrastructure of rectus sheath revisited.

In the last decade, in the search for abdominal-wall hernia etiology, attention has been brought to alterations in the connective tissue ultrastructure as the probable etiological factor. These may cause weakening of connective tissue, which in turn may form ground for hernia formation. To investigate this hypothesis in depth, we compared the ultrastructure of the connective tissue in hernia patients and the control group. The study group consisted of five patients with primary inguinal hernia (Nyhus II = 4, Nyhus IIIa = 1). Another five patients posted for emergency appendectomy created the control group. Tissue specimens, harvested intraoperatively from the rectus muscle sheath (RAMS) and fixed in 4% glutaraldehyde, underwent staining by the Masson, H-E and methylene blue techniques and were assessed by microscopy (light and scanning electron). The examinations showed significant differences in the rectus sheath ultrastructure. They included altered architecture, placement and quantity of collagen and elastic fibers, differences in the caliber of individual fibers and disrupted ground matter-to-fiber ratio. In patients with hernias, chaotic arrangement of collagen fibers was seen, as well as their thinning and a decrease in the general amount of elastic fibers, replaced by ground matter. Our research has shown significant differences in the structure of the RAMS between patients with hernias and healthy individuals. This supports the theory linking connective tissue alterations with the etiology of hernia, and stating that these alterations include connective tissue at locations distant from the hernia site as well, as the rectus sheath itself does not form a hernial defect.

Calcium-dependent signal transduction pathways in plants--phytochrome mechanism of action as an example.

Higher plants appear to have some signalling molecules that are similar to those in animals. Early events in the response of plant cell to many physiological stimuli share common features, such as membrane depolarization and elevation in cytosolic free calcium level. Ca2+ has a vital role in mediating plant responses to external stimuli of both abiotic origin (e.g. light, cold, heat, movement, hypoxia and drought) and biotic origin (e.g. phytohormones, pathogens, interaction with symbionts). Recently, tremendous progress has been made in understanding the role of Ca2+ as a second messenger in plants. It has been shown that plasma membrane Ca2+ channels and vacuolar Ca2+ release channels may participate in multiple signalling pathways in higher plants. Ca(2+)-dependent modulation of cellular processes occurs via intracellular calcium-binding proteins, of which calmodulin is one of the best characterized. It is well documented that calcium is involved in light-induced, phytochrome-controlled signal transduction pathways in higher plants.

Loading and localization of Fluo-3 and Fluo-3/AM calcium indicators in sinapis alba root tissue.

Stimulus-induced changes in free cytosolic Ca2+ in different types of plant cells have been monitored with the aid of fluorescent calcium indicator dyes. However, there is no simple and convenient method for introducing these dyes into the plant cell cytoplasm. This paper reports tests of different procedures for loading either free fluorescent dyes or their acetoxymethyl esters (Fluo-3 and Fluo-3/AM, respectively) into Sinapis alba root tissue. Loading of Fluo-3 was pH and temperature dependent. Moreover, in the presence of beta-escin (saponin) in the loading medium very high fluorescent signals in root tissues were observed. The highest signals were recorded when tissue was loaded in a medium containing 0.1% beta-escin, at pH 5.0 and 30 degrees C. Only very weak fluorescence signals were found in mustard roots loaded with Fluo-3/AM. Acidity and temperature of the medium had no significant effect on the process. However, addition of eserine, a cholinesterase inhibitor led to a dramatic increase in fluorescence in the root cells. On the basis of these observations rapid and efficient methods of loading both Fluo-3 and Fluo-3/AM into mustard root tissues are proposed.

Selective binding of Ca2+, Zn2+, Cu2+ and K+ by the physodes of the green alga Mougeotia scalaris.

Cells of the zygnematophycean green alga Mougeotia contain numerous globules with polyphenolic matrix, which resemble physodes. In order to analyse the capability of this compartment to sequester various ions, trichomes of Mougeotia scalaris were either fixed for X-ray microanalysis simultaneously in 2% glutardialdehyde/1% OsO4 in phosphate buffers of different K+/Na(+)-ratios, or embedded directly (fresh material) in Nanoplast resin. In addition, fixed material was treated with potassium antimonate and Ca2+ localization was examined by electron microscopic cytochemistry. A Ca(2+)-depletion upon fixation at different K+/Na(+)-ratios resulted in selective uptake of potassium, but not sodium. Consistent with earlier findings, calcium-binding by the polyphenolic physode matrix does not depend merely on electric charge but also on the presence of protonated/deprotonated phenolic groups, together with ester-linked carbonyl oxygen, which seem to be good candidates for a co-ordinate type of calcium-binding. Nanoplast embedding turned out to be the most adequate and fastest preparation for X-ray microanalysis and, apart from retaining calcium, allowed the detection of zinc and copper inside the physodes.

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).

The role of the plasma-membrane Ca(2+)-ATPase in Ca (2+) homeostasis in Sinapis alba root hairs.

The regulation of cytosolic Ca(2+) has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca(2+)-ATPase. For this purpose, erythrosin B was used to inhibit the Ca(2+)-ATPase, and the Ca(2+) ionophore A23187 was applied to manipulate cytosolic free [Ca(2+)] which was then measured with Ca(2+)-selective microelectrodes. (i) At 0.01 µM, A23187 had no effect on the membrane potential but enhanced the Ca(2+) permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca(2+) by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca(2+) spontaneously reversed and became an increase, a process which strongly depended on the external Ca(2+) concentration, (iv) Upon removal of A23187, the cytosolic free [Ca(2+)] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca(2+) is an activation of Ca(2+) transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca(2+) from the cytosol. The two observations that after the addition of A23187, (i) Ca(2+) gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca(2+)-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH(+)/Ca(2+)-ATPase. Based on the finding that the Ca(2+)-ATPase inhibitor erythrosin B had no effect on cytosolic Ca(2+), but caused a strong Ca(2+) increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca(2+)-pump. We further conclude that this ATPase is a major Ca(2+) regulator in stress situations where the cytosolic Ca(2+) has been shifted from its steady-state level, as may be the case during processes of signal transduction.

Oscillations of acetylcholine in oat seedlings.

Using gas chromatography it was shown that acetylcholine (ACh) was present in both etiolated and green oat (Avena sativa L. cv. Diadem) seedlings. In etiolated seedlings the ACh level was low, but increased rapidly during exposure to sunlight and red light (RL). The stimulative influence of RL was reversed by far-red light (FRL). The RL- and FRL- changes in ACh level were correlated to changes in acetylcholinesterase (AChE) localization. Using Karnovsky's method, it was found that in the etiolated coleoptiles the products of enzymatic reaction showing AChE activity accumulated selectively on the external side of plasma membrane. After exposure of seedlings to RL AChE activity disappeared. Subsequent FRL made it reappear on the external side of the plasma membrane. When the plants became green, oscillations of ACh were clearly observable. For plants grown under a LD 16:8 cycle the changes were circadian.

Influence of acetylcholine agonists and antagonists on the swelling of etiolated wheat (Triticum aestivum L.) mesophyll protoplasts.

Etiolated wheat (Triticum aestivum L.) mesophyll protoplasts swell within 30 min in darkness after a red light (R) pulse or addition of acetylcholine (ACh), if 0.5 mM CaCl2 is present in the medium. In addition, ACh is also able to induce swelling in the presence of both 0.1 mM KCl or NaCl. Besides ACh, only carbamylcholine out of the choline derivatives tested was active in induction of swelling in the presence of K(+) or Na(+). The K(+)/Na(+)-dependent ACh-induced protoplast swelling was nullified by a 'calmodulin inhibitor', but not by Ca(2+)-channel blockers, Li(+) or VO 4 (3-) . The antagonists atropine (of muscarine-sensitive ACh receptors, mAChRs) andD-tubocurarine (of nicotine-sensitive ACh receptors, nAChRs) nullified the Ca(2+) - and the K(+)/Na(+)-dependent protoplast swelling responses, respectively, while having no effect on the Ca(2+)-dependent R-induced swelling response. Moreover, muscarine and nicotine mimicked ACh in the Ca(2+)- and K(+)/Na(+)-dependent swelling responses respectively. Just as is the case in animal cells, the proposed mAChRs appear to be associated with a phosphatidylinositol-dependent pathway, whereas the proposed nAChRs are phosphatidylinositol independent. Similarity between the action of ACh via the proposed mChRs and R via phytochrome in protoplast swelling indicates they share in common signal-transduction pathway.

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis.

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca(2+) was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.