PCR and real-time PCR primers developed for detection and identification of Bifidobacterium thermophilum in faeces

BACKGROUND: Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora. RESULTS: Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/microl to 200 ng/microl and showed a detection limit of 10(5) cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples. CONCLUSION: A new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA:rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C:N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

Urinary metabolite profiling reveals CYP1A2-mediated metabolism of NSC686288 (aminoflavone)

NSC686288 [aminoflavone (AF)], a candidate chemotherapeutic agent, possesses a unique antiproliferative profile against tumor cells. Metabolic bioactivation of AF by drug-metabolizing enzymes, especially CYP1A monooxygenases, has been implicated as an underlying mechanism for its selective cytotoxicity in several cell culture-based studies. However, in vivo metabolism of AF has not been investigated in detail. In this study, the structural identities of 13 AF metabolites (12 of which are novel) in mouse urine or from microsomal incubations, including three monohydroxy-AFs, two dihydroxy-AFs and their sulfate and glucuronide conjugates, as well as one N-glucuronide, were determined by accurate mass measurements and liquid chromatography-tandem mass spectrometry fragmentation patterns, and a comprehensive map of the AF metabolic pathways was constructed. Significant differences between wild-type and Cyp1a2-null mice, within the relative composition of urinary metabolites of AF, demonstrated that CYP1A2-mediated regioselective oxidation was a major contributor to the metabolism of AF. Comparisons between wild-type and CYP1A2-humanized mice further revealed interspecies differences in CYP1A2-mediated catalytic activity. Incubation of AF with liver microsomes from all three mouse lines and with pooled human liver microsomes confirmed the observations from urinary metabolite profiling. Results from enzyme kinetic analysis further indicated that in addition to CYP1A P450s, CYP2C P450s may also play some role in the metabolism of AF.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.

Comprehensive MicroRNA expression profiling identifies novel markers in follicular variant of papillary thyroid carcinoma

BACKGROUND Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data. METHODS Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study. RESULTS Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan-Meier (p < 0.05) and multivariate Cox regression analysis (p < 0.05). CONCLUSIONS Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.

Noninvasive Urinary Metabolomic Profiling Identifies Diagnostic and Prognostic Markers in Lung Cancer

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients.

BACKGROUND A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0-40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events. METHODS We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated. RESULTS The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8). CONCLUSION In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score. TRIAL REGISTRATION Clinicaltrials.gov identifier NCT00761787.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

Integrated mRNA and miRNA expression profiling in blood reveals candidate biomarkers associated with endurance exercise in the horse.

The adaptive response to extreme endurance exercise might involve transcriptional and translational regulation by microRNAs (miRNAs). Therefore, the objective of the present study was to perform an integrated analysis of the blood transcriptome and miRNome (using microarrays) in the horse before and after a 160 km endurance competition. A total of 2,453 differentially expressed genes and 167 differentially expressed microRNAs were identified when comparing pre- and post-ride samples. We used a hypergeometric test and its generalization to gain a better understanding of the biological functions regulated by the differentially expressed microRNA. In particular, 44 differentially expressed microRNAs putatively regulated a total of 351 depleted differentially expressed genes involved variously in glucose metabolism, fatty acid oxidation, mitochondrion biogenesis, and immune response pathways. In an independent validation set of animals, graphical Gaussian models confirmed that miR-21-5p, miR-181b-5p and miR-505-5p are candidate regulatory molecules for the adaptation to endurance exercise in the horse. To the best of our knowledge, the present study is the first to provide a comprehensive, integrated overview of the microRNA-mRNA co-regulation networks that may have a key role in controlling post-transcriptomic regulation during endurance exercise.

Ectopic Meis1 expression in the mouse limb bud alters P-D patterning in a Pbx1-independent manner

During limb development, expression of the TALE homeobox transcription factor Meis1 is activated by retinoic acid in the proximal-most limb bud regions, which give rise to the upper forelimb and hindlimb. Early subdivision of the limb bud into proximal Meis-positive and distal Meis-negative domains is necessary for correct proximo-distal (P-D) limb development in the chick, since ectopic Meis1 overexpression abolishes distal limb structures, produces a proximal shift of limb identities along the P-D axis, and proximalizes distal limb cell affinity properties. To determine whether Meis activity is also required for P-D limb specification in mammals, we generated transgenic mice ectopically expressing Meis1 in the distal limb mesenchyme under the control of the Msx2 promoter. Msx2:Meis1 transgenic mice display altered P-D patterning and shifted P-D Hox gene expression domains, similar to those previously described for the chicken. Meis proteins function in cooperation with PBX factors, another TALE homeodomain subfamily. Meis-Pbx interaction is required for nuclear localization of both proteins in cell culture, and is important for their DNA-binding and transactivation efficiency. During limb development, Pbx1 nuclear expression correlates with the Meis expression domain, and Pbx1 has been proposed as the main Meis partner in this context; however, we found that Pbx1 deficiency did not modify the limb phenotype of Msx2:Meis1 mice. Our results indicate a conserved role of Meis activity in P-D specification of the tetrapod limb and suggest that Pbx function in this context is either not required or is provided by partners other than Pbx1.