Dry Bath Research Articles

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Effect of Waon therapy on functional mitral valve regurgitation

It has been reported that Waon therapy improves cardiac and vasucular function in patient with chronic heart failure using a far infrared-ray dry sauna bath of 60 degrees Celsius. The acute effect of Waon therapy is brought by peripheral artery expansion and increasing cardiac output. Furthermore, the reduction of the preload with the expansion of pulmonary and systematic vein brings the decrease in mitral regurgitation (MR) and pulmonary capillary wedge pressure. In addition, chronic effect of Waon therapy is the improvement in cardiac function, hemodynamics, vascular function and the neurohumor-related factors.

Effects of dry and mist saunas on circulatory and thermoregulatory functions in humans

To test the hypothesis that mist sauna is a safer way of bathing than dry sauna, we compared changes in circulatory and thermoregulatory functions during 10 min sauna bathing in mist sauna at 40℃ with relative humidity of 100%, and in dry sauna by infrared ray at 70℃ with relative humidity of 15%. Subjects were seven healthy young men aged 29 ± 6 yrs (mean ± SD). We measured blood pressure, heart rate, skin temperatures at chest, forearm, thigh, and leg, tympanic temperature (Tty) by thermistors, skin blood flow at forearm by laser Doppler flowmetry, and sweat rate by ventilated capsule method at 1 min intervals throughout the experiment. Total sweating and change of hematocrit were also measured for dehydration analysis. Blood pressure was elevated more and changes in heart rate and total sweating were larger in dry sauna than mist. A significant hematocrit increase was observed in dry sauna bathing only. Mean skin temperature and Tty in dry sauna were elevated higher than those in mist. Heat stress of the dry sauna may be stronger than that of the mist, leading to dehydration and hypovolemia by sweating. Percent plasma volume loss was significantly larger in the dry than mist sauna. Changes in skin blood flow and sweat rate/Tty during mist sauna were significantly larger than those during dry sauna bathing despite heat stress of the mist sauna. The mist sauna bathing may thus be safer physiologically, and provide more effective vascular dilatation and sweating than the dry sauna bathing.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

The presence of water influences reproductive function in the song sparrow (Melospiza melodia morphna)

Cumulative evidence indicates that song sparrows, Melospiza melodia morphna, of western Washington State prefer territories close to water. Densities of territories were higher within 50m of open water and analysis of stomach contents revealed aquatic organisms. An unusually dry period (less open water) in May and June 1997 had no effect on latency of territorial males to respond to a simulated territorial intrusion (STI), but the number of songs and closest approach to the decoy were lower than those recorded in June 1988 (no major dry periods). A laboratory experiment was conducted in which males and females were exposed to long days to stimulate reproductive development. One group received a bath with water daily and the other group received a dry bath (both groups had ad libitum access to drinking water through a glass tube). Birds with a dry bath tended to show reduced photoperiodically-induced gonadal growth compared with birds that had access to water in the bath. Plasma levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased following photostimulation in all groups. However, LH levels were higher in females with access to water. There were no differences in hormone levels in any of the other groups. Taken together these novel data indicate that presence and access to open water could be an important environmental cue for song sparrows in western Washington State. Implications for global climate change and droughts are suggested.

Functionality of egg white proteins as affected by high intensity ultrasound

The goal of this contribution was to determine the impact of HIUS on the thermal aggregation, gelation, foaming and emulsifying properties of egg white (EW) proteins.EW solutions were sonicated for 20 min using an ultrasonic processor Vibra Cell Sonics, model VCX 750 (frequency: 20 kHz; amplitude: 20%). The following properties were determined: particle size distribution by light scattering, the dynamics of gelation upon time and temperature (70, 75, 80 and 85 °C), surface hydrophobicity, concentration of sulfhydryl (SH) groups, denaturation temperatures (Tpeak), bulk viscosity, foaming by a whipping method and emulsifying properties by the use of a vertical scan analyzer and droplet size determinations. In order to study aggregation, EW solutions were heated in a dry bath at 70, 75, 80 and 85 °C for different periods of time from 0 to 30 min and analyzed by static light scattering and confocal laser scanning microscopy.Surface hydrophobicity increased after sonication, but total SH content was not affected. The apparent viscosity decreased, which seemed to affect the stability of foams prepared with sonicated protein. Emulsions from sonicated samples resulted more stable to creaming and flocculation. The gelation temperature of EW did not vary substantially after sonication as well as the gelation properties studied. The rate of formation of aggregates upon heating was accelerated by sonication. This fact could be attributed to the increase in hydrophobicity of the protein. Thus, HIUS could allow improving some functional properties of EW.

Repeated sauna therapy improves myocardial perfusion in patients with chronically occluded coronary artery-related ischemia

BackgroundRepeated low-temperature sauna (Waon) therapy relieves ischemic symptoms in patients with peripheral arterial disease. We investigated whether Waon therapy could improve myocardial perfusion in patients with ischemia related to chronic total occlusion (CTO) of coronary arteries. MethodsTwenty-four patients who had ischemia in the CTO-related area were examined. The severity of ischemia was quantified by thallium-201 myocardial perfusion scintigraphy with adenosine. The Waon group (n=16) was treated daily for three weeks with a 60°C far infrared-ray dry sauna bath for 15min and then kept in a bed covered with blankets for 30min. The control group (n=8) underwent myocardial perfusion scintigraphy twice with a three-week interval. ResultsIn the control group, neither summed stress score (SSS) nor summed difference score (SDS) of myocardial scintigraphy changed. However, Waon therapy improved both SSS (16±7 to 9±6, p<0.01) and SDS (7±4 to 3±2, p<0.01), and the improvement was greater in patients with higher SSS and SDS scores at the baseline. Waon therapy extended treadmill exercise time (430±185 to 511±192s, p<0.01) and improved flow-mediated dilation of the brachial artery (4.1±1.3 to 5.9±1.8%, p<0.05), but tended to decrease the number of circulating CD34-positive bone marrow-derived cells. ConclusionsWaon therapy improves CTO-related myocardial ischemia in association with improvement of vascular endothelial function. This therapy could be a complementary and alternative tool in patients with severe coronary lesions not suitable for coronary intervention.

Genetic Analysis of the Heparan Modification Network in Caenorhabditis elegans

Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.

BioSpotlight

BioTechniquesVol. 50, No. 1 BioSpotlightOpen AccessBioSpotlightNathan S. Blow & Patrick C.H. LoNathan S. BlowSearch for more papers by this author & Patrick C.H. LoSearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113580AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Automatic for the PeopleWhen it comes to studying gene or microRNA expression patterns, quantitative PCR (qPCR) is still the method of choice. While in a low-throughput format the equipment to perform qPCR and the downstream analysis tools are readily available, when it comes to higher-throughput applications and studies, automating the assay can be challenging. One of the major stumbling blocks comes from the rigid robotic scripts that are often supplied with liquid handling robots, making optimization for complicated applications such as qPCR intricate and time-consuming. In the current issue of BioTechniques, S. Callejas and colleagues from the Centro Nacional de Investigaciones Cardiovasculares in Madrid describe a new program they developed called Automatic Genomics which can be used for designing medium-throughput qPCR experiments and load 96- and 384-well plates. The software is based on Microsoft Excel with a Visual Basic interface and requires only a limited programming background to execute. Subapplications give users the opportunity to automate steps such as sample transfer, reverse transcription, and volume calculations according to their defined parameters without the need for specific vendor-supplied scripts. To validate their software, the authors tested technical reproducibility for three genes with 88 cDNA samples, with each sample-gene combination loaded in triplicate. The final data showed that 96% of the sample-gene combinations passed the technical quality control metric cutoff of 0.5 cycles (the maximum allowable difference in cycles between the technical replicates) and 61.5% passed a cutoff of 0.2 cycles. The authors note that this level of reproducibility is higher than what could have been achieved by hand. Although designed for use specifically with the Freedom EVO series of liquid handling robots, this software will enable researchers using those platforms to more easily perform larger scale qPCR experiments, with high reproducibility, without the need for complicated modifications to robotics scripts for each experiment.See “Automatic Genomics: a user-friendly program for the automatic designing and plate loading of medium-throughput qPCR experiments”Convection, ‘Vection, What's Your Function?Continuous improvements in PCR thermocycler designs have significantly reduced instrument size, cost, complexity, and amplification times, facilitating the development of simplified platforms suitable for point-of-care applications. One alternative to traditional PCR is convective PCR, where the sample is repeatedly circulated by convection through different temperature zones corresponding to the denaturation, annealing, and extension steps. Previous convective PCR systems, however, have relied on sophisticated temperature controllers and complex designs requiring tubing and chambers to circulate samples, which has limited their widespread usage. In this issue, P.J. Chen and colleagues at the National Taiwan University (Taipei, Taiwan) describe a greatly simplified convective PCR platform that dispenses with these requirements by carrying out amplification in a single capillary tube placed on a dry heat bath set at 95°C. In their capillary convective PCR (CCPCR) approach, the heated base acts to both denature the DNA sample at the bottom of the tube and also cause it to rise by convection. As this portion of the sample rises, it is cooled by the air surrounding the upper part of the tube and undergoes the annealing and extension steps of PCR, after which it sinks down to begin another cycle of convective PCR. Complete amplification can be achieved in as little as 30–40 minutes and does not require any of the complicated manipulations necessary with previous convective PCR systems. The authors demonstrate that CCPCR can amplify fragments up to 500 bp starting from as few as 30 copies, although proper design of amplicons and primers is required such that their Td and Tm, respectively, are optimized for convective PCR. The hardware requirements of the CCPCR platform can be further reduced by using a basic water bath or the heating plate contained in a portable scent-based mosquito repellent. The low cost and ease of operation of CCPCR makes it ideally suited for use in less-developed countries for the rapid detection of disease outbreaks and point-of-care applications.Schematic diagram of CCPCR illustrating the locations of the different steps of PCR along the temperature gradient of the capillary tube heated at 95°C from the bottom.See “Rapid amplification in a capillary tube by natural convection with a single isothermal heater”FiguresReferencesRelatedDetails Vol. 50, No. 1 Follow us on social media for the latest updates Metrics History Published online 28 June 2018 Published in print January 2011 Information© 2011 Author(s)PDF download

Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater

Herein we describe a simple platform for rapid DNA amplification using convection. Capillary convective PCR (CCPCR) heats the bottom of a capillary tube using a dry bath maintained at a fixed temperature of 95°C. The tube is then cooled by the surrounding air, creating a temperature gradient in which a sample can undergo PCR amplification by natural convection through reagent circulation. We demonstrate that altering the melting temperature of the primers relative to the lowest temperature in the tube affects amplification efficiency; adjusting the denaturation temperature of the amplicon relative to the highest temperature in the tube affects maximum amplicon size, with amplicon lengths of ≤500 bp possible. Based on these criteria, we successfully amplified DNA sequences from three different viral genomes in 30 min using CCPCR, with a sensitivity of ~30 copies per reaction.

Combined application of interference therapy and "dry" carbon dioxide baths for the treatment of hemodynamic disorders in patients with implantation failure due to inflammation

This work was designed to evaluate the influence of different physiotherapeutic modalities on the possibility of implantation, development and outcome of pregnancy in patients with chronic endometriosis and previous failures of implantation. The study confirmed initial sonographic changes and deficit of uterine blood supply as well as gradual improvement of these parameters under effect of adequate physiotherapy. Inclusion of dry carbon dioxide baths in the combined treatment of hemodynamic disorders made it possible to "prepare" endometrium for implantation and optimize blood circulation in the uterine vasculature in order to enhance the probability of implantation, development of pregnancy, and its success.

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-beta-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.

Rapid identification of blaKPC-possessing Enterobacteriaceae by PCR/electrospray ionization-mass spectrometry

Rapid identification of blaKPC-possessing Enterobacteriaceae by PCR/electrospray ionization-mass spectrometry

An Improved Method for Determining Medium‐ and Long‐Chain FAMEs Using Gas Chromatography

The existing protocols for analyzing fatty acid methyl esters (FAMEs) using a one-step acetyl chloride (AC) catalyzed transesterification and extraction procedure cannot accurately determine the medium- and long-chain fatty acids simultaneously in clinical (enteral, parenteral) formulations. For example: (1) addition of AC at room temperature generates an exothermic reaction that often results in loss of sample and possible injury to the analyst; (2) certain polyunsaturated fatty acids (PUFAs) are less stable at elevated temperatures during the transesterification and contribute to the over-estimation of the C16:0 and C18:1 fatty acids; and (3) the flame-ionization detector (FID) response varies depending on the carbon chain length of the fatty acids, that consequently impacts the underestimation of medium-chain fatty acid (C6-C10) recoveries. To overcome these deficiencies and accurately determine FAMEs, we have developed an improved one-step transesterification method that employs the addition of AC in tubes kept on a dry ice bath, the transesterification at room temperature, and the data analysis using relative response factors. Using this modified protocol, we determined the fatty acid composition of lipid emulsions (Omegaven and Lipidem) on a Shimadzu GC2010 gas chromatography (GC) system using a capillary GC column (Zebron ZB-WAX plus, 30 m, 0.25 mm ID, 0.25 microm). Our data suggest that the improved method can be easily used to accurately determine fatty acids (C6-C24) in functional foods and lipid emulsions.

The influence of "dry" bi-carbonate baths on the circadian profile of arterial pressure in patients who suffered acute myocardial infarction

The objective of this study was to evaluate effect of "dry" bi-carbonate baths (DBB) on the circadian profile of arterial pressure (AP) in patients who suffered acute myocardial infarction (AMI). Diurnal AP profiles were analysed in all patients after AMI. The patients comprising group 1 were treated with the use of DBB while the remaining ones received a sham treatment. 24 hour AP monitoring was performed before and after therapy. The study revealed a reduction in the frequency of hypotensive diastolic values at the daytime and in the duration of night-time hypertensive episodes under the influence of therapy with the use of DBB. Variability of systolic AP throughout 24 hours and of diastolic AP at daytime also decreased. Generally speaking, patients with elevated AP showed a more pronounced effect of DBB on the night AP profile. It is concluded that therapy with the use of DBB after AMI has beneficial effect on the clinical state of the patients and reduces the risk of cardiovascular complications.

SYNTHESIS OF O-SUBSTITUTED BENZOPHENONES BY GRIGNARD REACTION OF 3-SUBSTITUTED ISOCOUMARINS

ABSTRACT A series of o-substituted benzophenones was prepared by efficient method involving the 3-substituted isocoumarins and phenylmagnesium bromide. The reaction was completed in presence of anhydrous ether under dry ice-solvent bath in few hours with modest to good yields.Key words: Isocoumarins, diketones, aryl, phenyl magnesium bromide, ether, dry ice.e-mail: nawaz_f@yahoo.co.in. INTRODUCTION Synthetic and naturally occurring isocoumarins possess important biological properties. They are useful intermediates in the synthesis of a variety of carbocyclic and heterocyclic compounds 1-6 .In continuous of our interest in carbon-carbon bond formation reaction 7-12 ,the present paper presents an efficient method to synthesize o-substituted benzophenones (2) from 3-substitued isocoumarins (1) and phenyl magnesium bromide in presence of anhydrous ether under dry ice-solvent bath. Benzophenone and its derivatives are important intermediates in dyes, pharmaceutical and other chemical industries

Radon concentration levels in dry CO2 emanations from Harghita Băi, Romania, used for curative purposes

The aim of this work is to study the exposure due to the radon of the patients from the dry carbon dioxide baths (mofettas) from Harghita Băi, Romania. These mofettas consist of emanated gas with high carbon dioxide content, and proved curative effects. The vertical and horizontal distribution of the radon activity concentration was monitored indoor, the seasonal variation, as well as the soil radon and thoron activity concentration outdoor, in the vicinity of the bath to determine the origin of the radon and the location of the fault through which it propagates. The equilibrium factor of the baths and the effective dose received by the patients was also calculated.

Cwc24p, a Novel Saccharomyces cerevisiae Nuclear Ring Finger Protein, Affects Pre-snoRNA U3 Splicing

U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 co-transcriptionally, thereby affecting splicing and 3'-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING finger protein that had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistent with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35 S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35 S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

Contamination à Sphingomonas paucimobilis : à propos de sept cas isolés sur des milieux de conservation et de transport de greffons cornéens

From September to December 2004, contaminations were found in fifteen conservation and transport mediums of corneal grafts at the tissue bank of Brest, including seven by Sphingomonas paucimobilis. The pulsed-field gel electrophoresis made it possible to establish the genotypic profiles of each strain and to compare them. Similarities were found between certain strains of the contaminated mediums and those of the thermostated double boiler of the tissue bank. The link between the contamination and the defrosting of the mediums in the double boiler was thus established. Measures of prevention are currently proposed to defrost the bottles like the use of a dry bath to replace the current one.