Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella.

Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ± 0.05 °C for invA, 85.56 ± 0.28 °C for ipaH, and 89.21 ± 0.24 °C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10(4) CFU g(-1); however, the limit of detection was 10(3) CFU g(-1). This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.

Effectiveness of ProTaper, D-RaCe, and Mtwo retreatment files with and without supplementary instruments in the removal of root canal filling material.

AIM: To assess the efficacy of different retreatment rotary files in removing gutta-percha and endodontic sealer from canals. METHODOLOGY: Ninety straight single-rooted premolars were prepared up to a size 30 and filled with gutta-percha and sealer and then randomly assigned to six retreatment groups (n = 15). Groups I, III, and V were retreated using rotary systems ProTaper Universal Retreatment (PTUR), D-RaCe, and Mtwo Retreatment, respectively. Groups II, IV, and VI were retreated using the additional instruments F4, size 40, .04 taper RaCe, and size 40, .04 taper Mtwo, respectively. The roots were split vertically, and images of the halves were obtained using a high-resolution scanner and evaluated with AutoCAD software to calculate the percentage of residual material. Data were analyzed with Kruskal-Wallis and Student-Newman-Keuls tests using a 5% significance cutoff (P < 0.05). RESULTS: There were no statistically significant differences (P > 0.05) between groups when additional instruments were used. The percentage of residual material was lowest in the PTUR group and was statistically significant only when compared to the D-RaCe system (P = 0.0038). CONCLUSIONS: All root canals had residual filling material after retreatment even when additional instruments were used.

Concurrent training remodels the subcutaneous adipose tissue extracellular matrix of people living with HIV: a non-randomized clinical trial.

Evaluate the effect of 12 wks of concurrent training (CT) in the extracellular matrix (ECM) of subcutaneous adipose tissue (SAT) in people living with HIV/AIDS (PLWHA). In the non-randomized clinical trial, 19 participants, 11 healthy (HIV-) and 18 PLWHA under the use of highly active antiretroviral therapy (HAART) for at least 1 year (HIV+). All participants engaged in a moderate-intensity CT program for 12 weeks, 3 times a week. Before and after CT, aerobic and strength performance were assessed, as well as anthropometric and biochemical blood profiles. In addition, SAT biopsies were performed for histologic and morphometric analyses. Statistical analysis was carried out with R Studio, using descriptive and inferential analysis, ANOVA test, and mixed-effect model (P < 0.05). HIV+ showed higher levels of very-low-density lipoproteins and triglycerides and lower levels of high-density lipoproteins at baseline than HIV- (P < 0.05). All groups showed improved aerobic and strength performances (P < 0.05). Both groups showed reduced adipocyte sizes after CT (P < 0.05). Lastly, HIV+ presented smaller adipocytes and higher elastic fiber deposition at baseline and decreased after training only in HIV+, similar to the HIV group. Thus, CT in PLWHA promoted a decrease in the size heterogeneity of adipocytes and elastic fiber deposition, remodeling the ECM, and improving the SAT fibrosis profile. Brazilian Clinical Trials Registry (ensaiosclinicos.gov.br - UTN: U1111-1214-3022). Novelty: Adipose tissue fibrosis is improved by training in people living with HIV. Concurrent training remodels adipose tissue extracellular matrix.

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli strains isolated from Peruvian children.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.

Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli.

Five Escherichia coli colonies/patient were studied to evaluate the reliability of a multiplex real-time PCR assay for detection of diarrheagenic Escherichia coli groups, using a pool of five colonies rather than individual colonies. Sensitivity and specificity were 98% and 100%, respectively, at a fifth of the cost of the individual colony analysis.

Mechanistic Projection of First-in-Human Dose for Bispecific Immunomodulatory P-Cadherin LP-DART: An Integrated PK/PD Modeling Approach.

A bispecific immunomodulatory biotherapeutic molecule (P-cadherin LP-DART) based on the Dual Affinity Re-Targeting (DART) scaffold has been developed as a potential antitumor treatment showing efficacy in preclinical testing. A minimal anticipated biological effect level (MABEL) approach was applied to project the first-in-human (FIH) dose, because of its immune agonistic properties following target engagement. The pharmacological activity of P-cadherin LP-DART is driven by binding to both P-cadherin on the tumor cells and CD3 on T cells. Therefore, the concentration of the tri-molecular synapse formed between drug, T cell, and tumor cell, rather than drug concentration, is responsible for efficacy. A mechanistic pharmacokinetic/pharmacodynamic (PK/PD)-driven approach was explored to understand the exposure-response relationship based on the synapse concentration to project the MABEL dose. Orthogonal approaches including PK-driven and receptor occupancy calculations were also investigated. This study showcases the application of PK/PD modeling in immune-oncology, and could potentially be implemented for other bispecific biotherapeutics.

YY1 represses vitamin D receptor-mediated 25-hydroxyvitamin D(3)24-hydroxylase transcription: relief of repression by CREB-binding protein.

Ying Yang transcription factor (YY1) can repress or activate transcription. 25-Hydroxyvitamin D(3)-24-hydroxylase [24(OH)ase], an enzyme involved in the catabolism of 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], is up-regulated at the transcriptional level by 1,25-(OH)(2)D(3) to self-induce its deactivation. Here we report that YY1 can repress 1,25-(OH)(2)D(3)-induced 24(OH)ase transcription in CV1 cells transfected with vitamin D receptor (VDR) expression vector or in LLCPK(1) cells that contain VDR endogenously. With increasing amounts of YY1 DNA transfected (500 ng to 2 microg), ligand-dependent VDR activation of 24(OH)ase transcription was steadily repressed (maximum repression was 10-fold). Thus, YY1 may be a key modulator preventing activation at times that do not require the enzyme to be expressed. Relief of YY1 repression was observed in the presence of TFIIB or CBP (CREB binding protein) suggesting that YY1 may exert repression, in part, by sequestering TFIIB/CBP. Glutathione-S-transferase (GST) pull-down assays identified regions in the N and C termini of CBP that can bind YY1. In addition, the N-terminal region of CBP that interacts with YY1 can inhibit YY1 from binding to TFIIB. Thus, CBP may alleviate YY1-mediated repression, in part, by preventing YY1 from binding to TFIIB, which is required for VDR-mediated transcription. In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. In addition, our findings demonstrate a role for CBP in relief of repression of VDR-mediated transcription.

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

Effectiveness of magnesium pidolate in the prophylactic treatment of primary dysmenorrhea.

To evaluate Magnesium (Mg) effectiveness in the treatment of primary dysmenorrhea, 30 volunteer dysmenorrheic women of mean age 22.6 years were selected from the out-patients of the Dept. of Obstetrics and Gynaecology of the University of Parma during the period January-December 1989. Patients affected by secondary dysmenorrhea were excluded from the trial. The women considered were asked to self-evaluate their menstrual pain for 6 subsequent cycles using the VAS (Visual Analogue Scale). In the first cycle, as control, no drug was administered; in the following ones, every woman was given 4.5 mg oral Mg Pidolate in 3 administrations daily, from the 7th day preceding the onset of menses till the 3rd day of menstruation. Data were statistically analyzed. In Mg-treated cycles compared with the control one, first day dysmenorrhea progressively decreased, with a significant drop (p < 0.05) from the 1st to the 6th cycle. A similar trend, but not statistically significant, was seen for the 2nd and 3rd day of cycle. No side effect was remarked. These data suggest Mg administration to be a reliable therapy of primary dysmenorrhea.

Hemispheric functionality patterns between dextrals and sinistrals in tactile-visual tasks at lower or higher level of mental processes.

Two different patterns of unilateral tactile-visual recognition tasks with random shapes were administered to 64 subjects, 32 right-handed (16 males, 16 females) and 32 left-handed (16 males, 16 females). The main effects were found in the over-all performance: dextral subjects performed better than sinistral subjects; males performed better than females. On the task at a lower level of mental process dextral subjects performed better over-all than the sinistral subjects; however, neither group showed superiority of one hand over the other. On the task at a higher level of mental process performance of sinistral subjects improved to a level equivalent to that of the dextral subjects. Dextral subjects tended to perform better with their left hands, whereas the sinistral subjects scored equally with both hands. The findings are discussed in terms of quantitative and qualitative differences in patterns of hemispheric functionality between dextral and sinistral subjects, and the more specific cerebral activation for tasks at a higher level of mental process is hypothesized.

A novel method for the rapid and prospective identification of Beijing Mycobacterium tuberculosis strains by high-resolution melting analysis.

Genotypic analysis of Mycobacterium tuberculosis (MTB) has enabled the definition of several lineages. The Beijing family, which is considered highly virulent and transmissible, has been associated with resistance in certain settings and involved in severe outbreaks, making it one of the most closely-monitored lineages. Therefore, rapid prospective identification of Beijing MTB strains could be relevant. In the present study, we evaluate a real-time PCR followed by high-resolution melting (HRM) based on the identification of a single nucleotide polymorphism (SNP) in the Rv2629 gene which defines Beijing lineage (A191C for Beijing genotype and A191A for non-Beijing genotype). This combined methodology efficiently differentiated Beijing and non-Beijing strains in 100% of the isolates from a collection of reference strains without requiring specific DNA probes. Additionally, HRM was able to assign a Beijing/non-Beijing genotype in 90.9% of the respiratory specimens assayed. Its applicability was tested on a Peruvian sample of circulating MTB strains, in which it identified 10.7% as belonging to the Beijing genotype; this proportion reached 20% in the North Lima area. HRM analysis of the A191C SNP is a rapid, reliable, and sensitive method for the efficient prospective survey of high-risk Beijing MTB strains, even in developing settings where MTB culture is often not available.

Effectiveness of different techniques for removing gutta-percha during retreatment.

AIM: To evaluate the effectiveness of various techniques for removing filling material from root canals in vitro. METHODOLOGY: Eighty extracted mandibular premolar teeth were selected for the study. The teeth were root filled using thermomechanical compaction of gutta-percha. After 8 months, the filling material was removed and canals were reinstrumented using the following techniques: group I - hand instrumentation with K-type files (SybronEndo, Orange, CA, USA); group II - K3 Endo System (SybronEndo); group III - M4 system (SybronEndo) with K-type files (SybronEndo); and group IV - Endo-gripper system (Moyco Union Broach, York, PA, USA) with K-type files (SybronEndo). The amount of filling debris remaining on root canal walls was assessed radiographically; the images were digitized and analysed using AutoCAD 2000 software. Total canal area, area of the cervical, middle and apical thirds, and area of remaining filling material were outlined by one operator. The ratios between these areas were calculated as percentages of remaining debris. Thereafter, data were analysed by means of one-way anova and the post-hoc Duncan test to identify differences between the four techniques. RESULTS: Multiple comparisons of the percentages of remaining filling material in the entire canal did not reveal any significant differences between the methods of removal. However, when each third was analysed separately, significant differences for remaining debris were present between groups. The apical third had the most remaining material, whilst the cervical and middle thirds were significantly cleaner (P = 0.002). Comparison of the techniques revealed that teeth instrumented with K3 rotary instruments had a lower ratio of remaining filling material in the apical third (P = 0.012). CONCLUSION: In the apical third, K3 rotary instruments were more efficient in removing gutta-percha filling material than the other techniques, which were equally effective for the other thirds.

Vitamin D target proteins: function and regulation.

Recent findings have indicated that calbindin-D(28k), the first known target of vitamin D action, is present in osteoblasts and protects against TNF and glucocorticoid induced apoptosis of osteoblastic cells. Cytokine mediated destruction of pancreatic beta cells, a cause of insulin dependent diabetes, is also inhibited by calbindin-D(28k). In calbindin-D(28k) transfected pancreatic beta cells free radical formation by cytokines is inhibited by calbindin. Thus, besides its role as a facilitator of calcium diffusion, calbindin has a major role in protecting against cellular degeneration in different cell types. Besides calbindin, the other known pronounced effect of 1,25(OH)(2)D(3) in intestine and kidney is increased synthesis of 25(OH)D(3) 24-hydroxylase (24(OH)ase) which is involved in the catabolism of 1,25(OH)(2)D(3). We have noted that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and osteoblastic cells and can enhance the transcriptional response of 24(OH)ase to 1,25(OH)(2)D(3). These studies establish C/EBPbeta as a novel 1,25(OH)(2)D(3) target gene and indicate a role for C/EBPbeta in 24(OH)ase transcription. These studies extend our previous studies related to factors that affect vitamin D receptor (VDR) mediated 24(OH)ase transcription (YY1, TFIIB, CBP) and the effect of signaling pathways on 24(OH)ase transcription and cofactor recruitment.