CLINICAL APPLICATIONS OF CRYOTHERAPY AMONG SPORTS PHYSICAL THERAPISTS.

BACKGROUND: Therapeutic modalities (TM) are used by sports physical therapists (SPT) but how they are used is unknown. PURPOSE: To identify the current clinical use patterns for cryotherapy among SPT. STUDY DESIGN: Cross-sectional survey. METHODS: All members (7283) of the Sports Physical Therapy Section of the APTA were recruited. A scenario-based survey using pre-participation management of an acute or sub-acute ankle sprain was developed. A Select Survey link was distributed via email to participants. Respondents selected a treatment approach based upon options provided. Follow-up questions were asked. The survey was available for two weeks with a follow-up email sent after one week. Question answers were the main outcome measures. RESULTS: Reliability: Cronbach's alpha=>0.9. The SPT response rate = 6.9% (503); responses came from 48 states. Survey results indicated great variability in respondents' approaches to the treatment of an acute and sub-acute ankle sprain. CONCLUSIONS AND CLINICAL RELEVANCE: SPT applied cryotherapy with great variability and not always in accordance to the limited research on the TM. Continuing education, application of current research, and additional outcomes based research needs to remain a focus for clinicians. LEVEL OF EVIDENCE: 3.

Fate and Transport of 17ß-Estradiol beneath Animal Waste Holding Ponds.

Concentrated animal feeding operations typically store livestock waste in clay-lined ponds. Although these ponds are regulated to include a liner with a small hydraulic conductivity to limit leaching, previous studies have traced surface and groundwater contamination from such regulated animal waste ponds. This research examined the transport of 17ß-estradiol (E2) and its primary metabolite, estrone (E1), through soil liners using field- and laboratory-based studies. Additionally, a potential engineering solution to limit hormone transport-applying biochar to new pond liners to act as a retardant-was studied. Soil cores 80 cm in length were collected beneath a mature dairy waste pond and analyzed for moisture content and hormone concentrations. Unsaturated conditions and E2 concentrations of 4 to 250 ng g were detected beneath the waste pond. In the laboratory portion of the study, hand-packed columns of sand or clay were subjected to infiltration by a 2.3-m head of dairy waste. A subset of the hand-packed sand columns was amended with powdered biochar to test its ability to retard E2 and E1. For 3 mo, column leachate was analyzed for hormone concentrations, and at the conclusion of the study E2 and E1 concentrations in the soil were measured. In the 44 d after sealing, the clay, sand, sand with a thin layer of biochar, and sand mixed with a biochar amendment leached a total of 0.54, 1.3, 0.09, and 0.45 µg of E2, respectively. The biochar amendments to the hand-packed columns considerably minimized E2 in the leachate.

Molecular indicators of Nitrobacter spp. population and growth activity during an induced inhibition event in a bench scale nitrification reactor.

The Nitrobacter spp. ribosomal RNA gene (rDNA) and transcript (rRNAt) abundance were quantified in a bench scale nitrification reactor during baseline periods of high nitrification efficiency and an intervening staged inhibition event. The transcript to gene ratio (rRNAt/rDNA) was highly sensitive to changes in the reactor nitrite oxidation rate. During high nitrification efficiency, the rRNAt/rDNA metric displayed a range from 0.68 to 2.01 with one-sided (α=0.10) lower and upper prediction intervals of 0.70 and 1.78, respectively. When nitrification was inhibited by disabling the reactor pH control system, this activity metric declined an order of magnitude to ≈ 0.05, well below the lower prediction interval reflecting high nitrification efficiency. The decline was rapid (2h) and preceded a significant drop in reactor nitrification performance, which occurred as ammonia accumulated. The rRNAt/rDNA ratio remained low (≈ 0.05) for several days after the pH control system was re-enabled at a setpoint of 8.0, which otherwise induced rapid oxidation of accumulated ammonia and produced high free ammonia concentrations. The timing of a subsequent increase in the rRNAt/rDNA ratio, which transiently exceeded the upper prediction interval established during the baseline period of high nitrification efficiency, was not coincidental with resumption of pH control at 7.2 that lowered free ammonia concentrations to non-inhibitory levels. Rather, nitrite oxidation resumed and the rRNAt/rDNA ratio increased only after oxidation of accumulated ammonia was complete, which was coincidental with reduced reactor oxygen demand. In summary, the Nitrobacter rRNAt/rDNA activity metric reflected timely and easily recognizable changes in nitrite oxidation activity, illustrating that molecular data can be used to diagnose poor biological wastewater treatment performance.

Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter.

The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCM( lux ), was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.

Limited impact of free ammonia on Nitrobacter spp. inhibition assessed by chemical and molecular techniques.

Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10mg-N/L) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3mg-N/L/h), low nitrite oxidation rates (0.2mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration.

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1.

The genome of a fecal pollution indicator phage, Bacteroides fragilis ATCC 51477-B1, was sequenced and consisted of 44,929 bases with a G+C content of 38.7%. Forty-six putative open reading frames were identified and genes were organized into functional clusters for host specificity, lysis, replication and regulation, and packaging and structural proteins.

Response of Nitrobacter spp. ribosomal gene and transcript abundance following nitrite starvation and exposure to mechanistically distinct inhibitors.

The Nitrobacter spp. rRNA gene (rDNA) and relative rRNA transcript abundance (rRNAt/rDNA ratio) were evaluated in response to sudden changes in the nitrite oxidation rate. The rDNA abundance poorly indicated sudden transitions in the rate, whereas the relative rRNAt abundance usually varied quickly and significantly. In response to changes in nitrite concentration, 8 h were required for the rRNAt/rDNA ratio to transition from a minimum value at nitrite starvation (approximately 0.07) to a maximum value with excess nitrite present (approximately 4), and 5 h were required for this metric to return to the minimum value after nitrite starvation re-ensued. Generally, the relative rRNAt abundance dropped significantly after 4.5 h of exposure to three different inhibitors. A sharp decline in the rRNAt/rDNA ratio occurred during exposure to 3,5-DCP (from 4 down to 0.2) even as the fractional inhibition level remained low (< 0.10); the minimum ratio value was observed when nitrite oxidation was completely inhibited. The ratio decreased significantly during exposure to azide (from 4 to 0.5) and H+ (from 2 to 0.2), but only when the fractional inhibition levels were high (> 0.8). Interestingly, when the pH was suddenly changed to 4.5, inhibiting nitrite oxidation completely, the rRNAt/rDNA metric did not decline suggesting that rRNAt processing was inhibited. This effect was not observed during severe inhibition with 3,5-DCP and azide. Overall, the findings indicate the relative rRNAt abundance can be used to closely track in situ Nitrobacter spp. activity and in most instances will reveal inhibition events with the potential to impact treatment performance in reactors where Nitrobacter spp. are dominant.

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction.

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.

Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 +/- 2.0 x 10(11) for bacteria, 3.7 +/- 3.2 x 10(10) for Nitrospira, 1.2 +/- 0.9 x 10(10) for all AOB, and 7.5 +/- 6.0 x 10(9) for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 +/- 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 +/- 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.