Effect of Arsenic on EPS Synthesis, Biofilm Formation, and Plant Growth-Promoting Abilities of the Endophytes Pseudomonas PD9R and Rahnella laticis PD12R.

Phytoremediation, a cost-effective, eco-friendly alternative to conventional remediation, could expand efforts to remediate arsenic-contaminated soils. As with other pollutants, the plant microbiome may improve phytoremediation outcomes for arsenic-contaminated sites. We used in vitro and in silico methods to compare the arsenic resistance mechanisms, synthesis of extracellular polymeric substances (EPS), biofilm formation, and plant growth-promoting abilities of the endophytes Pseudomonas sp. PD9R and Rahnella laticis PD12R. PD12R, which tolerates arsenate (As(V)) and arsenite (As(III)) to concentrations fivefold greater than PD9R, synthesizes high volumes of EPS in response to arsenic, and sequesters arsenic in the capsular EPS and cells. While arsenic exposure induced EPS synthesis in both strains, only PD12R continued to form biofilms at high As(III) and As(V) concentrations. The effects of endophyte inoculation on Arabidopsis growth varied by strain and As(V) concentration, and PD9R had positive effect on plants exposed to low levels of arsenic. Comparative genomic analyses exploring the EPS synthesis and arsenic resistance mechanisms against other Pseudomonas and Rahnella strains suggest that both strains possess atypical arsenic resistance mechanisms from other plant-associated strains, while the configuration of the EPS synthesis systems appeared to be more broadly distributed among plant- and non-plant-associated strains.

Potential Biocontrol Activities of Populus Endophytes against Several Plant Pathogens Using Different Inhibitory Mechanisms.

The plant microbiome can be used to bolster plant defense against abiotic and biotic stresses. Some strains of endophytes, the microorganisms within plants, can directly inhibit the growth of plant fungal pathogens. A previously isolated endophyte from wild Populus (poplar), WPB of the species Burkholderia vietnamiensis, had robust in vitro antifungal activity against pathogen strains that are highly virulent and of concern to Pacific Northwest agriculture: Rhizoctonia solani AG-8, Fusarium culmorum 70110023, and Gaemannomyces graminis var. tritici (Ggt) ARS-A1, as well as activity against the oomycete, Pythium ultimum 217. A direct screening method was developed for isolation of additional anti-fungal endophytes from wild poplar extracts. By challenging pathogens directly with dilute extracts, eleven isolates were found to be inhibitory to at least two plant pathogen strains and were therefore chosen for further characterization. Genomic analysis was conducted to determine if these endophyte strains harbored genes known to be involved in antimicrobial activities. The newly isolated Bacillus strains had gene clusters for production of bacillomycin, fengicyn, and bacillibactin, while the gene cluster for the synthesis of sessilin, viscosin and tolaasin were found in the Pseudomonas strains. The biosynthesis gene cluster for occidiofungin (ocf) was present in the Burkholderia vietnamiensis WPB genome, and an ocf deletion mutant lost inhibitory activity against 3 of the 4 pathogens. The new isolates lacked the gene cluster for occidiofungin implying they employ different modes of action. Other symbiotic traits including nitrogen fixation, phosphate solubilization, and the production of auxins and siderophores were investigated. Although it will be necessary to conduct in vivo tests of the candidates with pathogen-infected agricultural crops, the wild poplar tree microbiome may be a rich source of beneficial endophyte strains with potential for biocontrol applications against a variety of pathogens and utilizing varying modes of action.

Gonadotropin Releasing Hormone Agonists or Antagonists for Preimplantation Genetic Diagnosis (PGD)? A Prospective Randomised Trial.

BACKGROUND: The use of GnRH analogue medication is essential in reproductive medicine to avoid premature ovulation by pituitary suppression for the duration of ovarian stimulation by gonadotrophins. The type of pituitary suppression by either GnRH agonist analogues versus GnRH antagonist analogues may result in different embryological hence clinical results. Preimplantation genetic diagnosis is a subtype of IVF in which embryos are created for genetic diagnosis of hereditary disorders in order to avoid genetically affected children. Embryological quality hence ovarian stimulation in preimplantation genetic diagnosis is crucial as genetic selection will reduce the number of available embryos to a fraction of the total. OBJECTIVE: The aim of this study was to assess the efficiency of GnRH antagonist versus GnRH agonist treatment for pituitary suppression in ovarian stimulation for PGD, by proxy of number and quality of embryos at cleavage stage available for biopsy. METHOD: We conducted a prospective randomised controlled trial comparing pituitary suppression by GnRH antagonist versus GnRH agonist in ovarian stimulation for PGD. The primary outcome measure was the number of embryos of sufficient quality for biopsy at cleavage stage. Secondary outcome parameters were the number of blastocysts available of top quality, and clinical pregnancy rate. RESULTS: There was no difference in number of oocytes retrieved, embryos at cleavage stage available for biopsy or embryo quality. The clinical pregnancy rate was higher in the GnRH agonist group; however the sample size was insufficient to allow conclusions. CONCLUSION: The use of GnRH agonist versus antagonist treatment does not result in differences in a number of oocytes, embryos or embryo quality in ovarian stimulation for preimplantation genetic diagnosis.

Forward-looking infrared imaging predicts ultimate burn depth in a porcine vertical injury progression model.

INTRODUCTION: Current methods of assessing burn depth are limited and are primarily based on visual assessments by burn surgeons. This technique has been shown to have only 60% accuracy and a more accurate, simple, noninvasive method is needed to determine burn wound depth. Forward-looking infrared (FLIR) thermography is both noninvasive and user-friendly with the potential to rapidly assess burn depth. The purpose of this paper is to determine if early changes in burn temperature (first 3 days) can be a predictor of burn depth as assessed by vertical scarring 28 days after injury. METHODS: While under general anesthesia, 20 burns were created on the backs of two female Yorkshire swine using a 2.5cm×2.5cm×7.5cm, 150g aluminum bar, for a total of 40 burns. FLIR imaging was performed at both early (1, 2 and 3 days) and late (7, 10, 14, 17, 21, 24 and 28 days) time points. Burns were imaged from a height of 12 inches from the skin surface. FLIR ExaminIR(©) software was used to examine the infrared thermographs. One hundred temperature points from burn edge to edge across the center of the burn were collected for each burn at all time points and were exported as a comma-separated values (CSV) file. The CSV file was processed and analyzed using a MATLAB program. The temperature profiles through the center of the burns generated parabola-like curves. The lowest temperature (temperature minimum) and a line midway between the temperature minimum and ambient skin temperature at the burn edges was defined and the area of the curve calculated (the "temperature half-area"). RESULTS: Half-area values 2 days after burn had higher correlations with scar depth than did the minimum temperatures. However, burns that became warmer from 1 day to 2 days after injury had a lower scar depth then burns that became cooler and this trend was best predicted by temperature minima. When data were analyzed as a diagnostic test for sensitivity and specificity using >3mm scarring, i.e. a full-thickness burn, as a clinically relevant criterion standard, temperature minima at 2 days after burn was found to be the most sensitive and specific test. CONCLUSIONS: FLIR imaging is a fast and simple tool that has been shown to predict burn wound outcome in a porcine vertical injury progression model. Data showed that more severe burn wounds get cooler between 1 and 2 days after burn. We found four analytic methods of FLIR images that were predictive of burn progression at 1 and 2 days after burn; however, temperature minima 2 days after burn appeared to be the best predictive test for injury progression to a full-thickness burn. Although these results must be validated in clinical studies, FLIR imaging has the potential to aid clinicians in assessing burn severity and thereby assisting in burn wound management.

Development and evaluation of a novel mucus diffusion test system approved by self-nanoemulsifying drug delivery systems.

The aim of this study was the development of a novel mucus diffusion model and the approval thereof by self-nanoemulsifying drug delivery systems (SNEDDSs). For diffusion experiments, various SNEDD formulations were developed, spiked with fluorescein diacetate, and evaluated for their mucus diffusion behavior through an intestinal mucus layer within the novel setup. In brief, SNEDD formulations resulting in particle sizes of 12.0 nm produced 70.3% of diffused model drug through the mucus layer. In comparison, SNEDDSs with particle sizes of 455.5 nm led to a permeation of 8.3% only. Apart from this size dependence, two SNEDDS excipients namely Cremophor RH 40 and triacetin were identified to strongly affect the permeation through mucus. Hence, it could be demonstrated that particle size and single excipients can positively influence mucus diffusion of SNEDDSs. Furthermore, it could be shown that the developed mucus diffusion model is a promising tool for pharmaceutical research in comparison with already established systems as it allows an easy handling coupled with the possibility to test different kinds of mucus in parallel within one setup.

Are the enzymatic methods currently being used to measure bronchoalveolar lavage bile salt levels fit for purpose?

BACKGROUND: Microaspiration after gastroesophageal reflux has been implicated in the chronic loss of allograft function in lung transplant patients. Bronchoalveolar lavage fluid (BALF) assessment for pepsin and bile salts is a common method to document reflux and aspiration. Clinically used methods for bile salt analysis include tandem mass spectrometry and diagnostic enzymatic kits designed to measure bile salts in serum. In clinical research, the enzymatic kits have been commonly used for BALF assays in lung transplant recipients, with reports of detection limits of 0.2 µmol/liter, and the levels used to inform clinical decisions. This study assessed the sensitivity of detection by 2 enzymatic assay kits compared with tandem mass spectrometry. METHODS: These 2 kits were used to measure (1) the absorbance changes for 0 to 50 µmol/liter bile salts, (2) levels in gastric juice (10-10,010 µmol/liter), and (3) bile salt levels of 40 BALF samples that were also measured using tandem mass spectrometry (0.01-1.19 µmol/liter). Measurements of pH/impedance were done in 14 of 15 patients. RESULTS: Neither kit had detection limits as low as claimed in previous BALF studies. The kits could be made more sensitive with a longer incubation time, (5 µmol/liter). All patients had detectable lavage bile acids using mass spectroscopy, 71% had pathologic distal gastroesophageal reflux, and 43% had pathologic proximal reflux. CONCLUSIONS: The enzymatic kits are not sensitive enough for use in situations where bile salt levels are much below 5 µmol/liter, which is the case in BALF. In addition, reports in the literature of levels significantly below 5 µmol/liter need reassessing. Tandem mass spectrometry with a lower limit of detection of 0.01 µmol/liter should be the method of choice.

Bile acids in laryngopharyngeal refluxate: will they enhance or attenuate the action of pepsin?

OBJECTIVES/HYPOTHESIS: To assess if, as previously reported in the literature, bile acids inhibit pepsin activity, resulting in pepsin having a less important role in laryngopharyngeal damage in reflux disease. STUDY DESIGN: Prospective translational research study. METHODS: A total of 78 patient's fasting gastric juice samples were obtained from routine endoscopy. The total bile acid (TBA) content and pepsin activity were measured using a 3α-hydroxysteroid dehydrogenase-based kit for bile acids; and pepsin activity was measured using succinyl albumin/dimethylhaemoglobin as a substrate and the development of new N-terminals. The ability of bile acids to effect pepsin activity was assessed with three primary bile acids, two unconjugated and one taurine conjugated, using the above N-terminal assay. RESULTS: Gastric juice contained median TBA of 40 (range 10-10010) µM and pepsin activity of 408 (range 27-3892)ug/ml. We used this data to inform the relative levels of pepsin and bile acids that might occur in a reflux event, and we used concentrations of bile acids between 10-100µM. Pepsin activity was pH dependent, but 28% of the activity was retained at pH 5.5. None of the bile acids showed any significant effect on pepsin activity across the pH range 2.0-6.0. CONCLUSIONS: At the levels and pH that pepsin and bile acids might occur in an LPR event, bile acids do not attenuate pepsin activity. Pepsin could be considered a damaging factor even at high pH, and it will aggravate further any damaging effects of bile acids in the refluxate. Laryngoscope, 2012.