Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR^® Green qPCR method, and two TaqMan^® qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.     

Management of small building works

This paper focuses on the management dimension of small building works. The paper presents a subset of findings from a major research study, supported by governmental and industry based sources, which investigated the efficiency and effectiveness of procurement methods, organizational systems and management approaches to small works and minor maintenance projects. The findings indicate that management for small works must be exerted at two levels. One is within the organization of the works themselves. The second is the development of a framework and organization which considers the implication of the works upon the organization's ongoing business. Effective small works management requires that an organization responds promptly to an identified situation, determines the cost of the work, ensures that the work is undertaken successfully, and gives value for money within the context of the organization's current business circumstance.     

MSC Pretreatment for Improved Transplantation Viability Results in Improved Ventricular Function in Infarcted Hearts

Many clinical studies utilizing MSCs (mesenchymal stem cells, mesenchymal stromal cells, or multipotential stromal cells) are underway in multiple clinical settings; however, the ideal approach to prepare these cells in vitro and to deliver them to injury sites in vivo with maximal effectiveness remains a challenge. Here, pretreating MSCs with agents that block the apoptotic pathways were compared with untreated MSCs. The treatment effects were evaluated in the myocardial infarct setting following direct injection, and physiological parameters were examined at 4 weeks post-infarct in a rat permanent ligation model. The prosurvival treated MSCs were detected in the hearts in greater abundance at 1 week and 4 weeks than the untreated MSCs. The untreated MSCs improved ejection fraction in infarcted hearts from 61% to 77% and the prosurvival treated MSCs further improved ejection fraction to 83% of normal. The untreated MSCs improved fractional shortening in the infarcted heart from 52% to 68%, and the prosurvival treated MSCs further improved fractional shortening to 77% of normal. Further improvements in survival of the MSC dose seems possible. Thus, pretreating MSCs for improved in vivo survival has implications for MSC-based cardiac therapies and in other indications where improved cell survival may improve effectiveness.     

Urea-N uptake by dasheen (Colocasia esculenta L. Schott) in relation to the fertilizer placement method

Urea has become the most important N carrier in many parts of the world and its reaction when added to soil is unique in many ways. Two field experiments were therefore undertaken using¹⁵N to investigate the uptake efficiency of the added urea-¹⁵N which was banded in Experiment I and broadcast in Experiment II. In both experiments the uptake efficiencies were not affected by N-rate and cropping system (Exp. I) or crop residue management (Exp. II) and averaged 17.4 and 16.9% respectively. These low values were supported by evidences of high losses; high pH increases following urea application (volatilization), downward movement of N (leaching), and cycles of waterlogged and well drained conditions in the soil (de-nitrification). Evidence of leaching at least down to 30 cm in the profile was observed in the first experiment where urea was banded but not in experiment II where it was broadcast. The proportion of N in the crop that was derived from added urea (%Ndff) was 57.7% and 36.4% in experiments I and II respectively, suggesting that band application resulted in a higher proportion of the added N in the root zone compared to that for broadcast application. The results indicate the need to investigate other management strategies, such as higher application frequencies and placement closer to the root zone, in order to improve the uptake efficiency of added urea-N in upland rainfed dasheen.     

Bacteroidales terminal restriction fragment length polymorphism (TRFLP) for fecal source differentiation in comparison to and in combination with universal bacteria TRFLP

Terminal restriction fragment length polymorphism (TRFLP) is an attractive community analysis method for microbial source tracking (MST) because it is accessible, relatively inexpensive, and can discern multiple fecal sources simultaneously. A new Bacteroidales TRFLP (Bac-TRFLP) method was developed and its source identification performance was evaluated by itself, in comparison to, and in combination with an existing universal bacterial TRFLP method in two laboratories. Sixty-four blind samples from 12 fecal sources (sewage, septage, human, dog, horse, cow, deer, pig, chicken, goose, pigeon, and gull) were used for evaluation. Bac- and Univ-TRFLP exhibited similarly high overall correct identification (>88% and >89%, respectively), excellent specificity regardless of fecal sources, variable sensitivity depending on the source, and stable performance across two laboratories. Compared to Univ-TRFLP, Bac-TRFLP had better sensitivity and specificity with horse, cow, and pig fecal sources but was not suited for certain avian sources such as goose, gull, and pigeon. Combining the general and more targeted TRFLP methods (Univ&Bac-TRFLP) achieved higher overall correct identification (>92%), higher sensitivity and specificity metrics, and higher reproducibility between laboratories. Our results suggest that the Bac-TRFLP and Univ&Bac-TRFLP methods are promising additions to the MST toolbox and warrant further evaluation and utilization in field MST applications.     

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.     

Analysis of lossy multiconductor transmission lines using theasymptotic waveform evaluation technique

A method is described for the transient analysis of lossy coupled transmission line networks with nonlinear elements. The method combines the asymptotic waveform evaluation technique with a piecewise decomposition algorithm. Two to three orders of magnitude speedup can be achieved relative to previously published methods with comparable accuracy. The method is useful for delay and crosstalk simulation of high speed VLSI interconnects     

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.     

Experience with reduction mammaplasty following breast conservation surgery and radiation therapy

Little is known about the outcome of breast reduction in the previously radiated breast. With the increased popularity of breast conservation in the management of breast cancer, it is inevitable that more women with breast cancer who have had a breast radiated will be seeking breast reduction. Although it would be expected that reduction of the radiated breast would be more challenging and would yield less-pleasing results, it has been unclear whether reduction in the radiated breast could be safely performed without interfering with mammography and cancer surveillance. Our experience using different techniques in three patients demonstrates that such reductions can be effectively and safely done if certain principles are followed. Pedicles should be designed to be broader and shorter than usual, and breast flaps should be undermined or elevated either minimally or not at all.