Ice nucleation active bacteria and their potential role in precipitation

Certain bacteria that are commonly found on plants have the capacity to catalyze the freezing of supercooled water at temperatures as warm as -1$^{\circ}$C. This is conferred by a protein in the outer membrane of the bacterial cell. Because of the abundance of these bacteria and the warm temperature at which they function as ice nuclei, they are considered to be among the most active of the naturally-occurring ice nuclei. As plant pathogens, antagonists of plant pathogens and as causal agents of frost damage, these bacteria have well-studied interactions with plants. Here we propose that these bacteria also play a role in atmospheric processes leading to rain, given that they are readily disseminated into the atmosphere and have been found in clouds at altitudes of several kilometers. That they participate in a sort of biological cycle of precipitation - whereby they are transported into clouds from plant canopies and incite rain thereby causing favorable conditions for their growth on plant surfaces - was proposed about 20 years ago. Today, sufficient evidence and meteorological tools have emerged to re-ignite interest in bioprecipitation and in the ways in which plants play a role as cloud seeders

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin

The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes' urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 °C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes' urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation. © 2010 Springer-Verlag

Stabilization of human urine doping control samples: II. Microbial degradation of steroids

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 °C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 °C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 °C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 °C. © 2009 Elsevier Inc. All rights reserved

Stabilization of human urine doping control samples: A current opinion

Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples' integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested. © 2011 Springer-Verlag

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin

Background: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. Methods: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. Results: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 °C or storage at - 20 °C. Conclusions: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples. © 2009 Elsevier B.V. All rights reserved

Doping control container for urine stabilization: a pilot study

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd