Performance of the 47-Kilodalton Membrane Protein versus DNA Polymerase I Genes for Detection of Treponema pallidum by PCR in Ulcers.

Treponema pallidum PCR (Tp-PCR) is a direct diagnostic method for primary and secondary syphilis, but there is no recommendation regarding the best choice of target gene. In this study, we sequentially tested 272 specimens from patients with sexually transmitted ulcers using Tp-PCR targeting the tpp47 and then polA genes. The two methods showed similar accuracies and an almost-perfect agreement.

Multiplex blood PCR in combination with blood cultures for improvement of microbiological documentation of infection in febrile neutropenia.

The frequent lack of microbiological documentation of infection by blood cultures (BC) has a major impact on clinical management of febrile neutropenic patients, especially in cases of unexplained persistent fever. We assessed the diagnostic utility of the LightCycler SeptiFast test (SF), a multiplex blood PCR, in febrile neutropenia. Blood for BC and SF was drawn at the onset of fever and every 3 days of persistent fever. SF results were compared with those of BC, clinical documentation of infection, and standard clinical, radiological, and microbiological criteria for invasive fungal infections (IFI). A total of 141 febrile neutropenic episodes in 86 hematological patients were studied: 44 (31%) microbiologically and 49 (35%) clinically documented infections and 48 (34%) unexplained fevers. At the onset of fever, BC detected 44 microorganisms in 35/141 (25%) episodes. Together, BC and SF identified 78 microorganisms in 61/141 (43%) episodes (P = 0.002 versus BC or SF alone): 12 were detected by BC and SF, 32 by BC only, and 34 by SF only. In 19/52 (37%) episodes of persistent fever, SF detected 28 new microorganisms (7 Gram-positive bacterial species, 15 Gram-negative bacterial species, and 6 fungal species [89% with a clinically documented site of infection]) whereas BC detected only 4 pathogens (8%) (P = 0.001). While BC did not detect fungi, SF identified 5 Candida spp. and 1 Aspergillus sp. in 5/7 probable or possible cases of IFI. Using SeptiFast PCR combined with blood cultures improves microbiological documentation in febrile neutropenia, especially when fever persists and invasive fungal infection is suspected. Technical adjustments may enhance the efficiency of this new molecular tool in this specific setting.

PHYTOAVAILABILITY OF COPPER IN INDUSTRIAL BY-PRODUCTS AND MINERAL FERTILIZERS

Alternative copper (Cu) sources could be used in fertilizer production, although the bioavailability of copper in these materials is unknown. The objective of this study was to evaluate the extractants neutral ammonium citrate (NAC), 2 % citric acid, 1 % acetic acid, 10 % HCl, 10 % H2SO4, buffer solution pH 6.0, DTPA, EDTA, water, and hot water in the quantification of available Cu content in several sources, relating them to the relative agronomic efficiency (RAE) of wheat grown in a clayey Latossolo Vermelho eutrófico (Oxisol) and Neossolo Quartzarênico (Typic Quartzipsamment). Copper was applied at the rate of 1.5 mg kg-1 as scrap slag, brass slag, Cu ore, granulated copper, and copper sulfate. The extractants 10 % HCl, 10 % H2SO4, and NAC extracted higher Cu concentrations. The RAE values of brass slag and Cu ore were similar to or higher than those of Cu sulfate and granulated Cu. Solubility in the 2nd NAC extractant, officially required for mineral fertilizers with Cu, was lower than 60 % for the scrap slag, Cu ore, and granulated copper sources. This fact indicates that adoption of the NAC extractant may be ineffective for industrial by-products, although no extractant was more efficient in predicting Cu availability for wheat fertilized with the Cu sources tested.

Occurrence and Structure of Arbuscular Mycorrhizal Fungal Communities in Cassava after Cultivation of Cover Crops as Observed by the “PCR-DGGE” Technique

ABSTRACT Cassava (Manihot esculenta Crantz) is a highly mycotrophic crop, and prior soil cover may affect the density of arbuscular mycorrhizal fungi (AMFs), as well as the composition of the AMFs community in the soil. The aim of this study was to evaluate the occurrence and the structure of AMFs communities in cassava grown after different cover crops, and the effect of the cover crop on mineral nutrition and cassava yield under an organic farming system. The occurrence and structure of the AMFs community was evaluated through polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A randomized block experimental design was used with four replications. Six different cover crop management systems before cassava were evaluated: black oats, vetch, oilseed radish, intercropped oats + vetch, intercropped oats + vetch + oilseed radish, plus a control (fallow) treatment mowed every 15 days. Oats as a single crop or oats intercropped with vetch or with oilseed radish increased AMFs inoculum potential in soil with a low number of propagules, thus benefiting mycorrhizal colonization of cassava root. The treatments did not affect the structure of AMFs communities in the soil since the AMFs communities were similar in cassava roots in succession to different cover crops. AMFs colonization was high despite high P availability in the soil. The cassava crop yield was above the regional average, and P levels in the leaves were adequate, regardless of which cover crop treatments were used. One cover crop cycle prior to the cassava crop was not enough to observe a significant response in variables, P in plant tissue, crop yield, and occurrence and structure of AMFs communities in the soil. In the cassava roots in succession, the plant developmental stage affected the groupings of the structure of the AMF community.

Biomarker analysis of stored blood products: emphasis on pre-analytical issues.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

Multiplex PCR of sonication fluid accurately differentiates between prosthetic joint infection and aseptic failure.

OBJECTIVE: Cultures have limited sensitivity in the diagnosis of prosthetic joint infection (PJI), especially in low-grade infections. We assessed the value of multiplex PCR in differentiating PJI from aseptic failure (AF). METHODS: Included were patients in whom the joint prosthesis was removed and submitted for sonication. The resulting sonication fluid was cultured and investigated by multiplex PCR, and compared with periprosthetic tissue culture. RESULTS: Among 86 explanted prostheses (56 knee, 25 hip, 3 elbow and 2 shoulder prostheses), AF was diagnosed in 62 cases (72%) and PJI in 24 cases (28%). PJI was more common detected by multiplex PCR (n=23, 96%) than by periprosthetic tissue (n=17, 71%, p=0.031) or sonication fluid culture (n=16, 67%, p=0.016). Among 12 patients with PJI who previously received antibiotics, periprosthetic tissue cultures were positive in 8 cases (67%), sonication fluid cultures in 6 cases (50%) and multiplex PCR in 11 cases (92%). In AF cases, periprosthetic tissue grew organisms in 11% and sonication fluid in 10%, whereas multiplex PCR detected no organisms. CONCLUSIONS: Multiplex PCR of sonication fluid demonstrated high sensitivity (96%) and specificity (100%) for diagnosing PJI, providing good discriminative power towards AF, especially in patients previously receiving antibiotics.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Measurement of total estrone content in foods. Application to dairy products.

Estrone is a powerful growth-inducing hormone that is present in milk, mainly in the form of fatty acid esters, at concentrations that promote growth in experimental animals. We present here a method useful for the measurement of this natural hormone in foods and applied it to several common dairy products. Samples were frozen, finely powdered, and lyophilized then extracted with trichloromethane/methanol; the dry extract was saponified with potassium hydroxide. The free estrone evolved was extracted with ethyl acetate and was used for the estimation of total estrone content through radioimmunoassay. Application of the method to dairy products showed high relative levels of total estrone (essentially acyl-estrone) in milk, in the range of 1 ¿M, which were halved in skimmed milk. Free estrone levels were much lower, in the nanomolar range. A large proportion of estrone esters was present in all other dairy products, fairly correlated with their fat content. The amount of estrone carried by milk is well within the range, where its intake may exert a physiological response in the sucklings for which it is provided. These growth-inducing and energy expenditure-lowering effects may affect humans ingesting significant amounts of dairy products.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Use of palm-oil by-products in chicken and rabbit feeds: effect on the fatty acid and tocol composition of meat, liver and plasma

This study was undertaken in the framework of a larger European project dealing with the characterization of fat co- and by-products from the food chain, available for feed uses. In this study, we compare the effects, on the fatty acid (FA) and tocol composition of chicken and rabbit tissues, of the addition to feeds of a palm fatty acid distillate, very low in trans fatty acids (TFA), and two levels of the corresponding hydrogenated by-product, containing intermediate and high levels of TFA. Thus, the experimental design included three treatments, formulated for each species, containing the three levels of TFA defined above. Obviously, due to the use of hydrogenated fats, the levels of saturated fatty acids (SFA) show clear differences between the three dietary treatments. The results show that diets high in TFA (76 g/kg fat) compared with those low in TFA (4.4 g/kg fat) led to a lower content of tocopherols and tocotrienols in tissues, although these differences were not always statistically significant, and show a different pattern for rabbit and chicken. The TFA content in meat, liver and plasma increased from low-to-high TFA feeds in both chicken and rabbit. However, the transfer ratios from feed were not proportional to the TFA levels in feeds, reflecting certain differences according to the animal species. Moreover, feeds containing fats higher in TFA induced significant changes in tissue SFA, monounsaturated fatty acids and polyunsaturated fatty acids composition, but different patterns can be described for chicken and rabbit and for each type of tissue.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.