All

study patients were managed according to the usual standard of care in each collaborating center. Only observational data were collected and anonymously sent to the main investigator. Only the treating physician knew the identity of his patients. This study had no interventional purpose and travel physicians were reminded, when closing the KABISA TRAVEL software, that they had the final responsibility for their patients and that the software was only an aid for diagnosis http://www.selleckchem.com/products/ldk378.html and not a decider itself. The study was designed, conducted, and analyzed independently of any sponsoring. The protocol got the ethical clearance from the review boards of the ITMA and of the University Hospital of Antwerp. Data were entered in an Access database (Microsoft Office 2003). Analysis was performed with Stata version 10 (StataCorp, USA). The chi-square test was used to

compare categorical variables. Comparison of proportions was performed with the Pearson chi-square test and the MacNemar’s test. Kruskal Wallis test was used to compare median. All tests were two-tailed, and p values <0.05 indicated statistical significance. Of 246 registered cases, 205 patients with confirmed diagnosis were included in the study. Cases were excluded because final diagnosis was not confirmed (n = 36), inclusion criteria were not respected (two patients returned from nontropical countries), MK0683 molecular weight or clinicians’ diagnoses were missing or doubtful (n = 3). The study Cyclic nucleotide phosphodiesterase population was composed of 190 adults (123 men and 67 women) and 15 children (9 boys and 6 girls); 69% of them had been admitted (Table 1). The mean age was 35 years (range 0.5–73 y). Of the 205 included patients, 98 (48%) were western travelers, 44 (21%) were travelers native of tropical countries who had visited friends and relatives in their country of

origin, 39 (19%) were migrants arriving from the tropics, and 24 (12%) were western expatriates. Sub-Saharan Africa was the most frequent place of stay (58%), followed by Southeast Asia (24%), Latin America (11%), and North Africa or the Middle East (6%). One patient stayed in more than one region. The reference (or “correct”) diagnoses are detailed per collaborating center in Table 1. Most febrile episodes were because of tropical diseases (65%), mainly malaria (40%) and dengue (12%). Among the cosmopolitan infections (33%), bacterial enteritis (7%), infectious mononucleosis-like syndrome (6%), and respiratory tract infections (5%) were the most common etiologies. Four (2%) patients had a noninfectious cause of fever. Of note, 93% (55/59) of the patients with Plasmodium falciparum malaria were hospitalized. Three deaths occurred in total: one patient with Marburg hemorrhagic fever, one with severe malaria, and one with lymphoma.

Quantitative immunoblots of rat CSF revealed

a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly Selleck CHIR-99021 elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced αII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly

suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum). “
“A proposed mechanism of neuronal death associated with a variety of neurodegenerative diseases Trametinib cell line is the response of neurons to oxidative stress and consequent cytosolic Ca2+ overload. One hypothesis is that cytosolic Ca2+ overload leads to mitochondrial Ca2+ overload and prolonged opening of the permeability transition pore (PTP), resulting in mitochondrial dysfunction. Elimination of cyclophilin D (CyPD), a key regulator of the PTP, results in neuroprotection in a number of murine models of neurodegeneration in which oxidative stress and high cytosolic Ca2+ have been implicated. However, the effects of oxidative stress on the interplay between cytosolic and mitochondrial Ca2+ in adult neurons and the role of the CyPD-dependent PTP in these dynamic processes have not been examined. Here, using primary cultured cerebral cortical neurons from adult wild-type (WT) mice and mice

missing G protein-coupled receptor kinase cyclophilin D (CyPD-KO), we directly assess cytosolic and mitochondrial Ca2+, as well as ATP levels, during oxidative stress. Our data demonstrate that during acute oxidative stress mitochondria contribute to neuronal Ca2+ overload by release of their Ca2+ stores. This result contrasts with the prevailing view of mitochondria as a buffer of cytosolic Ca2+ under stress conditions. In addition, we show that CyPD deficiency reverses the release of mitochondrial Ca2+, leading to lower of cytosolic Ca2+ levels, attenuation of the decrease in cytosolic and mitochondrial ATP, and a significantly higher viability of adult CyPD-knockout neurons following exposure of neurons oxidative stress. The study offers a first insight into the mechanism underlying CyPD-dependent neuroprotection during oxidative stress. “
“Proper distribution of axonal mitochondria is critical for multiple neuronal functions.

Quantitative immunoblots of rat CSF revealed

a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly GDC-0068 nmr elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced αII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly

suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum). “
“A proposed mechanism of neuronal death associated with a variety of neurodegenerative diseases find more is the response of neurons to oxidative stress and consequent cytosolic Ca2+ overload. One hypothesis is that cytosolic Ca2+ overload leads to mitochondrial Ca2+ overload and prolonged opening of the permeability transition pore (PTP), resulting in mitochondrial dysfunction. Elimination of cyclophilin D (CyPD), a key regulator of the PTP, results in neuroprotection in a number of murine models of neurodegeneration in which oxidative stress and high cytosolic Ca2+ have been implicated. However, the effects of oxidative stress on the interplay between cytosolic and mitochondrial Ca2+ in adult neurons and the role of the CyPD-dependent PTP in these dynamic processes have not been examined. Here, using primary cultured cerebral cortical neurons from adult wild-type (WT) mice and mice

missing Adenosine cyclophilin D (CyPD-KO), we directly assess cytosolic and mitochondrial Ca2+, as well as ATP levels, during oxidative stress. Our data demonstrate that during acute oxidative stress mitochondria contribute to neuronal Ca2+ overload by release of their Ca2+ stores. This result contrasts with the prevailing view of mitochondria as a buffer of cytosolic Ca2+ under stress conditions. In addition, we show that CyPD deficiency reverses the release of mitochondrial Ca2+, leading to lower of cytosolic Ca2+ levels, attenuation of the decrease in cytosolic and mitochondrial ATP, and a significantly higher viability of adult CyPD-knockout neurons following exposure of neurons oxidative stress. The study offers a first insight into the mechanism underlying CyPD-dependent neuroprotection during oxidative stress. “
“Proper distribution of axonal mitochondria is critical for multiple neuronal functions.

However, the increasing use of insulin analogues poses a challenge because commercially available insulin assays detect these with varying accuracy and precision. Insulin analogues are increasingly used in diabetes management and the case outlined here highlights the variations in assay. Initially, the local assay (ELISA kit – Dako, Copenhagen) failed to detect a significant concentration of insulin (<6pmol/L; range 9.6–65.4pmol/L) which an external reference laboratory selleck chemicals llc subsequently detected using the Mercodia Iso-insulin two-site

immunoassay (Uppsala, Sweden). The key analytical point is the recognition that different immunoassays detect insulin analogues to varying degrees. Clinical teams need to consider this if such cases are to be recognised. Following recent media reports where surreptitious insulin administration may be implicated in inpatient mortality, this knowledge is crucial to empower us to Talazoparib accurately diagnose all cases of unexplained hypoglycaemia. Copyright © 2013 John Wiley & Sons. Practical Diabetes 2013; 30(3): 118–120 “
“The evolution of diabetes centres in the UK, with co-location of clinical

teams, has resulted in examples of success in improving clinical efficiency, communication and patient-centred care. “
“Erectile dysfunction (ED) is expected to affect 322 million men by 2025. A number of lifestyle factors such as smoking, obesity, alcohol consumption and lack of physical activity are linked with erectile dysfunction. We reviewed the evidence in

recent studies examining the impact of weight loss upon erectile function in obese men with and without diabetes. Esposito et al. showed that weight loss through diet and increased physical activity can improve sexual function in about one-third of obese non-diabetic men with ED. Subsequently, Dallal et al. reported that the amount of surgical weight loss after gastric bypass predicted the degree of improvement in sexual function independent of improvement in glycaemic control. Wing et al. reported PD184352 (CI-1040) that weight loss in older obese diabetic subjects in the Look AHEAD trial may help in preventing the worsening of ED over time. Most recently in 2011, Khoo et al. have shown that rapid diet-induced weight loss improves sexual and endothelial function and systemic inflammation in obese diabetic men. In conclusion, the majority of recent studies show that weight loss can improve erectile function in obese men, though the beneficial effect is less profound in diabetic men. Copyright © 2012 John Wiley & Sons. “
“It is a myth that screening of type 2 diabetes is ‘a given’, that we provide adequate education for patients and that increasing physical activity by simply referring patients to a health trainer can prevent type 2 diabetes. Research in this area is often seen as an easy or soft option.

“
“1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T.

asperellum showed decreased ability of the mutants BTK assay to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum. Plant diseases are a major impediment to increasing

yields of many crops, Obeticholic Acid order and result in large economic losses. An environmentally safe strategy to control diseases is biological control, which is based on natural antagonistic interactions among microorganisms. Successful biocontrol agents (BCAs) colonize roots and suppress pathogens by mechanisms that include niche exclusion and competition, direct antagonism of pathogens by antibiosis, parasitism or predation and by triggering systemic host plant defense responses (Chet & Chernin, 2002; Harman et al., 2004). Some BCAs are plant growth-promoting rhizobacteria (PGPR) and fungi that also stimulate plant growth directly by altering plant hormone levels, facilitating iron acquisition through siderophore production, fixing atmospheric nitrogen and/or solubilizing minerals (Lugtenberg & Kamilova, 2009). Plant growth can also be stimulated by PGPR that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which cleaves ACC, the

immediate precursor of the plant hormone ethylene, to produce α-ketobutyrate and ammonia (Todorovic & Glick, 2008). Ethylene PLEK2 is an important signaling molecule in plants under pathogen attack or abiotic stresses and results in plant growth inhibition (Abels et al., 1992). Inoculation of plants with PGPR producing ACC deaminase (ACCD) lowers ethylene levels, which results in longer roots and decreased inhibition of plant growth following environmental or pathogen-induced stress (Glick et al., 1998, 2007; Farwell et al., 2007). Interestingly, it has been found that ACCD activity is not unique to bacteria. ACCD activity was detected in Penicillium citrinum (Jia et al., 2000). Two putative acdS genes were recently detected in the genome of Arabidopsis thaliana and evidence was presented supporting the hypothesis that these genes can act as regulators of ACC levels in A. thaliana and also in tomato fruit development (McDonnell et al., 2009; Plett et al., 2009). Certain Trichoderma spp.

Recently, the importance of Calothrix rhizosoleniae has been acknowledged as open ocean symbionts in a variety of diatoms (Foster et al., 2010). Nevertheless, to date no estimate of the overall influence in the C and N cycles of the genera within Rivulariaceae has been attempted and questions remain open regarding their phylogenetic organization. Strains examined in this study were isolated from natural populations such as microbial mats, microbialites and rocky shore biofilms, summarized in Table 1. Unicyanobacterial cultures were obtained from enrichment cultures, and individual tapering filaments with heterocysts were picked using light microscopy

(Axioscope 40, Carl Zeiss, Germany). Individual cultures were grown in

50- or 100-mL flasks in an incubation chamber at an average temperature of 29 °C, 14/10 light/dark cycles (Pozas Azules), 18 °C, 12/12 light/dark cycles PARP inhibitor (Askö) and 28 °C, 12/12 light/dark cycles (Heron Island). All cultures were grown in 50–100 μE m−1 s−1. Cultures were transferred to new media lacking reduced forms Selleckchem BAY 80-6946 of nitrogen every 3 weeks. DNA was extracted from individual cultures (approximately 500 μL) that were incubated overnight at 50 °C with 10 × extraction buffer (20 mM Tris-HCl, pH 7.5–8.2, 50 mM EDTA, 20 mM NaCl) and proteinase K (final concentration 0.25 mg mL−1). Proteins and lipids were separated with two phenol and one chloroform extraction and DNA was precipitated with sodium acetate (3 M) and absolute ethanol, followed by a 45-min incubation at −20 °C. DNA pellets were stained with GlycoBlue™ (Ambion, Austin, TX) and resuspended in water. A fragment consisting of almost the complete 16S rRNA gene, the intergenic transcribed spacers and part of the 23S rRNA gene was amplified from all strains using universal primer 27F (5′AGA GTT AGA GTT TGA TCM TGG CTC AG 3′) (Lane, 1991) and cyanobacteria-specific B23S (5′CTT CGC CTC TGT GTG CCT AGG T 3′) (Gkelis et al., 2005). The amplification reaction had a final volume of 50 μL with

1 × reaction buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.6 μM of each primer and 5 U Taq DNA polymerase. The thermal cycle included an initial denaturalization at 94 °C for 2 min, followed by 25 cycles of 94 °C Glycogen branching enzyme for 45 s; 54 °C for 45 s; 68 °C for 2 min and a final extension of 30 min at 68 °C. The PCR products obtained (approximately 1800 bp) were gel-extracted (Qiagen, Austin, TX) and sequenced. Sequences were obtained on a capillary sequencer (Applied Biosystems Avant-100) with five reactions including primers 27F, 1492R (5′TAC GGY TAC CTT GTT ACG ACT T 3′) (Lane, 1991) and B23S (Gkelis et al., 2005). Sequences were assembled and aligned with sequencher 3.1.1 (Gene Codes Corporation, Ann Arbor, MI), and identified with the Greengenes dataset (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) with basic local alignment search tool (blast).

Erm proteins catalyze either monomethylation (type I) or dimethylation (type II) reactions at the exocyclic N6 position of a specific adenine residue (A2058, Escherichia coli rRNA nucleotide numbering) in 23S rRNA to reduce the affinity of MLSB antibiotics to the peptidyl transferase center, the most problematic MLSB-resistance mechanism adopted by many clinically ICG-001 clinical trial isolated, resistant

pathogens (Weisblum, 1995). KsgA, another posttranscriptional rRNA methylation enzyme, catalyzes two consecutive dimethylation reactions, resulting in two adjacent, dimethylated adenines at the 3′ end of 16S rRNA in bacteria (Helser et al., 1972; Poldermans et al., 1979; O’Farrell et al., 2004). In contrast to Erm, the inactivation of the ksgA gene confers resistance to the aminoglycoside antibiotic kasugamycin. KsgA enzymes and the resulting methylated adenine bases Y-27632 order appear to be conserved

in all three domains of life (O’Farrell et al., 2004; Xu et al., 2008; Park et al., 2009), while Erm is found in limited species of microorganisms that are considered to be either the target or the producers of MLSB antibiotics (Weisblum, 1995). This finding suggests that KsgA might be an essential enzyme for survival, but Erm is necessary only in the presence of antibiotic pressure. However, KsgA is not absolutely essential in bacteria. Mutant E. coli (i.e., KsgA−) exhibits a longer doubling selleck screening library time, but survival does not appear to be affected by mutation (O’Farrell et al., 2004). Recent studies have demonstrated that KsgA binds to translationally inactive 30S ribosomal subunits and acts as a checkpoint in ribosome biogenesis by ensuring that only mature small subunits proceed to translation (Desai and Rife, 2006; Connolly

et al., 2008; Mangat and Brown, 2008; Xu et al., 2008). On the other hand, the eukaryotic ortholog of KsgA, Dim1, is found to be essential in yeast, where its most important role is the cleavage of 33S pre-rRNA rather than rRNA methylation (Lafontaine et al., 1994, 1995; Pulicherla et al., 2009). The sequence homology between Erm and KsgA was first recognized in the mid-1980s (van Buul and van Knippenberg, 1985). These two protein families also have a very similar basic architecture; both consist of two domains, a conserved Rossman-fold N-terminal domain and a less-conserved C-terminal domain, and carry out very similar catalytic reactions (Yu et al., 1997; Schluckebier et al., 1999; O’Farrell et al., 2004). Recent crystal structures of Aquifex aeolicus KsgA in complex with RNA and cofactor revealed that Erm and KsgA showed a very similar mode in cofactor binding, but a different mode in the details of RNA binding (Tu et al., 2009).

SDS-PAGE was performed to select the constructs expressing Imp or IdpA proteins of the proper size. The His-tagged Imp and IdpA proteins were purified from the E. coli cell extracts by chromatography on a nickel NTA column (Qiagen), according to previously described procedures (Kakizawa et al., 2004). The purified proteins were used to immunize rabbits for preparation of antisera. The IgG fractions were purified from the crude sera with a Protein A Sepharose CL-4B (GE Healthcare, Piscataway, NJ). Western blotting was performed according

to selleck chemicals previously described procedures (Kakizawa et al., 2009) using anti-Imp and anti-IdpA IgG purified from immunized rabbits. Immunohistochemical analysis was performed according to a previously described method (Arashida et al., 2008) with some modifications. Stem tissues were excised from PoiBI-infected ‘Jester Red’ and uninfected ‘Flaming Sphere’ poinsettias, fixed, embedded in Paraplast Plus (Sherwood Medical), and cut into 10-μm thick sections using a microtome. Anti-Imp and anti-IdpA IgG were used with an alkaline phosphatase-mediated reporter system to detect Imp and IdpA proteins in each tissue. These tissues were observed by Axio Imager microscopy (Carl Zeiss). To detect PoiBI in poinsettia plants, we extracted total DNA from 30 commercially

available poinsettia cultivars (Table 1) and amplified 1.3-kb DNA fragments containing the phytoplasma 16S rRNA gene by PCR. Of the 30 cultivars, all except ‘Annette Metabolism inhibitor Hegg Diva’, ‘Annette Hegg Marble’, ‘Eckespoint C-1 Red’,

and ‘Flaming Sphere’ yielded fragments of the expected size (Table 1). Sequencing of these fragments confirmed that their DNA sequences were identical to that of the 16S rRNA gene of PoiBI (Lee et al., 1997; GenBank Acc. No. 190223), indicating that these 26 cultivars were infected with PoiBI. Using total DNA isolated from the poinsettia cultivar ‘Primelo Jingle Bells’ as a template, we amplified a 6.0-kb DNA fragment containing the PoiBI imp gene, a 2.5-kb DNA fragment containing the PoiBI idpA gene, and a 3.3-kb DNA fragment between imp and idpA genes of the PoiBI DNA by LA-PCR (Fig. 1). Sequencing of these fragments yielded the complete DNA sequence of a 10-kb genomic region of PoiBI containing Vitamin B12 eight complete open reading frames and two partial open reading frames (Fig. 1). These genes (and their encoded proteins), listed in order, were rnc (RNAse III; partial gene only), dnaD (chromosome replication initiation protein), imp, pyrG (CTP synthase), psd (phosphatidylserine decarboxylase), pssA (phosphatidylserine synthase), rpoE (DNA-directed RNA polymerase δ subunit), dnaX (DNA polymerase III), idpA, and tRNA-Ser (serine transfer RNA; partial gene only). This gene structure is identical to that previously reported for WX strain (Liefting & Kirkpatrick, 2003; GenBank Acc. No. AF533231).

The RM5 and RM6 primers both contained a KpnI site allowing KpnI cut fragments (1282 and

1248 bp, respectively) to be ligated together. The ligation product was used as a PCR template using RM4 and RM7 primers. The resulting 2.5-kbp fragment was cut with XhoI and BamHI and cloned into XhoI and BamHI cut pMUTIN4 (Vagner et al., 1998) resulting in pMUT-LR. Finally, a tetracycline resistance cassette (from pLS1 M. Masalha and S.J. Foster, unpublished) was cloned into the KpnI site in the isdB gene of pMUT-LR creating pMUT-IsdB. Purified plasmid from Escherichia coli was transformed into S. aureus RN4220. selleck chemical Selection was made using Brain Heart Infusion (BHI) medium containing erythromycin and lincomycin, and integration was confirmed by PCR using primer pair RM4 and RM7. Phage lysates were prepared as described previously and transduced into SH1000. Tetracycline-resistant, selleck compound erythromycin/lincomycin-sensitive colonies were selected, and the mutation was confirmed

by PCR and Southern blot (results not shown). The isdH mutant was constructed using the primers shown in Table 1. Briefly, 1-kb fragments of chromosomal DNA flanking the isdH gene were amplified with the primer pairs MOL1313/MOL1314 and MOL1315/MOL1316 with SH1000 genomic DNA used as the template. The MOL1314 and MOL1315 primers both contained a KpnI site allowing KpnI cut fragments to be ligated together. The ligation product was used as a PCR template using MOL1313 and MOL1316 primers. The resulting 2-kbp fragment was cut with SalI and EcoRI and cloned into SalI and EcoRI cut pMAD (Arnaud et al., 2004). Finally, not a kanamycin resistance cassette (from pMAL7, Horsburgh et al., 2001a, b) was cloned into the KpnI site in the isdH gene creating pALX4. Purified plasmid from E. coli was transformed into S. aureus RN4220.

Selection was made using BHI containing kanamycin, and integration was confirmed by PCR using primer pair MOL1313 and MOL1316. Phage lysates were prepared as described previously and transduced into SH1000 and Newman. Kanamycin-resistant erythromycin/lincomycin-sensitive colonies were selected, and the mutation was confirmed by PCR (results not shown). The single marked isdB, and isdH mutations were then transduced into the Newman and SH1000 strain backgrounds alone and in combination with isdA (Clarke et al., 2004) using phage phi 11 as described previously (Novick, 1967). All strains are shown in Table 1. Complementation of isdA was carried out using the plasmid pSRC001 (Clarke et al., 2004). The complementation plasmid for the isdH mutant strain was created using the primers MOL1313 and MOL1316. Briefly, the whole isdH gene including its promoter and terminator was amplified by PCR and ligated into the plasmid pSK5630 using the restriction sites SalI and EcoRI. A transformant in E.

, 2002), the specificity and coverage for rumen Prevotella were confirmed by in silico analysis. Forty sequences of rumen Prevotella 16S rRNA gene including the four characterized species and 26 rumen Bacteroides sequences

were obtained from the GenBank database. The coverage and specificity of two sets of Prevotella genus-level primers, g-Prevo and PreGen4 (Stevenson & Weimer 2007), were tested in silico. The sequences were subjected to multiple alignments using the Selleck Gefitinib program clustalx to identify sequence identities with the primer sets. In addition to the exact match of the primer sequences with the Prevotella and Bacteroides sequences, the presence of consecutive matching sequences at the 3′ ends of the primer was considered to estimate the specificity. Plasmid DNA to be used as the standard in real-time PCR was obtained by cloning of 16S rRNA gene PCR products into Escherichia coli JM109 cells, as described previously (Koike et al., 2007). For click here the species-specific PCR, the 16S rRNA gene fragment of the respective target species (Table 1) was used to prepare the plasmid DNA. The strains used for plasmid preparation were as follows: P. ruminicola

ATCC19189, Ruminococcus flavefaciens ATCC19208, Ruminococcus albus ATCC27210, P. bryantii B14, Fibrobacter succinogenes ATCC19169, Streptococcus bovis ATCC33317, Treponema bryantii ATCC33254, Selenomonas ruminantium ATCC12561, Anaerovibrio lipolytica ATCC33276, Ruminobacter amylophilus ATCC29744, Succinivibrio dextrinosolvens ATCC19716 and Megasphaera elsdenii ATCC25940. The PCR primers used are shown in Table 1. PCR amplification for the quantification of target bacterial 16S rRNA gene was performed using a LightCycler 2.0 system (Roche Applied Science, Penzberg, Germany). The FastStart DNA Master SYBR Green I was used for PCR. The optimal amplification conditions for each all primer pair were achieved with 3.5 mM (final concentration) MgCl2. The reaction mixture in 20 μL of the final

volume contained 2.5 mM MgCl2, 2 μL 10 × Mastermix (containing FastStart Taq DNA polymerase, reaction buffer, dNTP mixture, 1 mM MgCl2 and SYBR Green I dye), 0.5 pmol of each primer and 10 ng template DNA. The thermal profile consisted of denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, annealing at the temperature indicated for the primer pair (Table 1) for 5 s and 72 °C for an appropriate extension time (Table 1). A 10-fold dilution series of the plasmid DNA standard for the respective target bacterial 16S rRNA gene was run along with the samples. Using standard curves obtained from the amplification profile of known concentrations of the plasmid DNA standard, the respective genes were quantified.