Digital gene expression analysis might aid in the diagnosis of thyroid cancer.

Background: Thyroid cancer represents approximately 90% of endocrine cancers. Difficulties in diagnosis and low inter-observer agreement are sometimes encountered, especially in the distinction between the follicular variant of papillary thyroid carcinoma (fvptc) and other follicular-patterned lesions, and can present significant challenges. In the present proof-of-concept study, we report a gene-expression assay using NanoString nCounter technology (NanoString Technologies, Seattle, WA, U.S.A.) that might aid in the differential diagnosis of thyroid neoplasms based on gene-expression signatures. Methods: Our cohort included 29 patients with classical papillary thyroid carcinoma (ptc), 13 patients with fvptc, 14 patients with follicular thyroid carcinoma (ftc), 14 patients with follicular adenoma (fa), and 14 patients without any abnormality. We developed a 3-step classifier that shows good correlation with the pathologic diagnosis of various thyroid neoplasms. Step 1 differentiates normal from abnormal thyroid tissue; step 2 differentiates benign from malignant lesions; and step 3 differentiates the common malignant entities ptc, ftc, and fvptc. Results: Using our 3-step classifier approach based on selected genes, we developed an algorithm that attempts to differentiate thyroid lesions with varying levels of sensitivity and specificity. Three genes-namely SDC4, PLCD3, and NECTIN4/PVRL4-were the most informative in distinguishing normal from abnormal tissue with a sensitivity and a specificity of 100%. One gene, SDC4, was important for differentiating benign from malignant lesions with a sensitivity of 89% and a specificity of 92%. Various combinations of genes were required to classify specific thyroid neoplasms. Conclusions: This preliminary proof-of-concept study suggests a role for nCounter technology, a digital gene expression analysis technique, as an adjunct assay for the molecular diagnosis of thyroid neoplasms.

Brain activity sustaining the modulation of pain by empathetic comments.

Empathetic verbal feedback from others has been shown to alleviate the intensity of experimental pain. To investigate the brain changes associated with this effect, we conducted 3T-fMRI measurements in 30 healthy subjects who received painful thermal stimuli on their left hand while overhearing empathetic, neutral or unempathetic comments, supposedly made by experimenters, via headsets. Only the empathetic comments significantly reduced pain intensity ratings. A whole-brain BOLD analysis revealed that both Empathetic and Unempathetic conditions significantly increased the activation of the right anterior insular and posterior parietal cortices to pain stimuli, while activations in the posterior cingulate cortex and precuneus (PCC/Prec) were significantly stronger during Empathetic compared to Unempathetic condition. BOLD activity increased in the DLPFC in the Empathetic condition and decreased in the PCC/Prec and vmPFC in the Unempathetic condition. In the Empathetic condition only, functional connectivity increased significantly between the vmPFC and the insular cortex. These results suggest that modulation of pain perception by empathetic feedback involves a set of high-order brain regions associated with autobiographical memories and self-awareness, and relies on interactions between such supra-modal structures and key nodes of the pain system.

First outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit.

AIM: To describe an outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit. METHODS: Isolates of linezolid-resistant E. faecium were typed by pulsed-field gel electrophoresis (PFGE), and examined by polymerase chain reaction (PCR) and sequencing for the transmissible cfr gene that confers resistance to linezolid. Enhanced environmental cleaning, initial and weekly screening of all patients, and monitoring of adherence to standard infection control precautions were implemented. FINDINGS: Five patients with pre-existing renal disease were found to have rectal colonization with linezolid-resistant E. faecium over a two-week period. The index case was a 57-year-old male from India who had travelled to the UK. One patient also had a linezolid-resistant E. faecium of a different PFGE profile isolated from a heel wound. All isolates were confirmed to harbour the cfr gene by PCR and Sanger sequencing, and all were resistant to glycopeptides (VanA phenotype). CONCLUSIONS: This article describes the first UK outbreak with a single strain of linezolid- and glycopeptide-resistant E. faecium harbouring the cfr gene, affecting five patients in a nephrology unit. Following the implementation of aggressive infection control measures, no further cases were detected beyond a two-week period.

Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections. AIM: To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem. METHODS: Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10µg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis. FINDINGS: Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-ß-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C. freundii, two NDM-producing E. cloacae complex, and four out of five GES-5-producing E. cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K. oxytoca overall shared 79% similarity. CONCLUSION: CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals.

Isolation and detection of extended spectrum ß-lactamase (ESBL)-producing enterobacteriaceae from meat using chromogenic agars and isothermal loop-mediated amplification (LAMP) assays.

The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity.

Evaluation of the NucliSENS EasyQ KPC assay for detection of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae.

The NucliSENS EasyQ KPC assay (bioMérieux SA, Marcy l'Etoile, France) was compared with a routinely used phenotypic method for detection of Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases, using 806 stool samples and rectal swabs. Compared with the phenotypic method, the EasyQ KPC assay had a sensitivity and specificity of 93.3% and 99.0%, respectively, in this setting, with diverse KPC producers not limited to ST258 Klebsiella pneumoniae.

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae in a UK district hospital; an observational study.

BACKGROUND: Extended-spectrum beta-lactamases (ESBLs) are an increasingly important cause of resistance in Gram-negative bacteria throughout the world. AIM: We investigated the clinical and molecular epidemiology of infections caused by ESBL-producing Enterobacteriaceae in a UK hospital, to identify the types of ESBL produced and risk factors for acquisition. METHODS: Between July 2008 and June 2009, all patients yielding ESBL-producing Enterobacteriaceae from any clinical specimen were prospectively investigated using a questionnaire. API20E was used for bacterial identification; susceptibility testing and ESBL production were assessed by BSAC disc diffusion and cefpodoxime-clavulanate synergy tests, respectively. Polymerase chain reaction was used to screen a subset of isolates for bla(CTX-M) genes, to assign Escherichia coli isolates to their phylogenetic groups, and to identify members of the uropathogenic ST131 lineage. RESULTS: The overall prevalence of ESBL producers among clinical samples yielding Enterobacteriaceae was 1%; ESBL producers, obtained from 124 patients, were E. coli (N = 105), Klebsiella pneumoniae (N = 12), and others (N = 7). The main risk factors identified include recent antibiotic use (93%) and presence of a urinary catheter (24%). CTX-M group 1 ESBLs dominated (in 59 of 78, 76%, isolates studied). Most E. coli (35 of 56 tested) were phylogroup B2; of these, 23 belonged to the ST131 clone, 12 were phylogroup D, and four each belonged to phylogroups A and B1. CONCLUSION: ESBLs are an uncommon but significant problem in north-west Cambridgeshire. CTX-M-type enzymes were found in 75% of ESBL-positive isolates. All but two patients had at least one recognized risk factor. This study supports the requirement for interventions to reduce inappropriate urinary catheterization and antibiotic prescribing.

Development of high temperature comprehensive two-dimensional liquid chromatography hyphenated with infrared and light scattering detectors for characterization of chemical composition and molecular weight heterogeneities in polyolefin copolymers.

The application of high temperature comprehensive two-dimensional (2D) liquid chromatography for quantitative characterization of chemical composition and molecular weight (MW) heterogeneities in polyolefins is demonstrated in this study by separating a physical blend of isotactic-polypropylene, ethylene-random-propylene copolymer, and high density polyethylene. The first dimension separation is based on adsorption liquid chromatography that fractionates the blend from low to high ethylene content. The second dimension is size-exclusion chromatography connected with light scattering (LS) and infrared (IR) detectors. The IR detector shows desired sensitivity and linearity for monitoring analyte concentrations in the eluent after 2D separations. In addition, the compositions of the analytes are also determined from the ratio of two IR absorbances at the specified wavelength regions, an absorbance for measuring the level of methyl groups in polyolefins and another absorbance for measuring concentration. The LS detector is used to determine absolute molecular weight of the analytes from the ratio of the light scattering signal to the IR concentration signal. The ability to obtain concentration, chemical composition, and MW of polyolefins after 2D separation provides new opportunities to discover structure-property relationships for polyolefins with complex structures/architectures.

Identifying antimicrobial multiresistance patterns of Escherichia coli sampled from diarrhoeic calves by cluster analysis techniques: a way to guide research on multiresistance mechanisms.

Multiresistance is a critical issue. This study points out the usefulness of cluster analysis techniques to describe concisely the antimicrobial susceptibility patterns of bacterial isolates in a way that could effectively help in generating hypotheses on multiresistance mechanisms. Data were selected from the French antimicrobial resistance survey network on veterinary pathogens (Resapath). They were related to 1545 Escherichia coli isolates, which were isolated from faecal samples of diarrhoeic calves in France between 2002 and 2006. Ten clusters of isolates displaying similar features in terms of resistance profile to 13 relevant antimicrobials were computed. The presence of two to ten simultaneous resistances was detected in nine out of the ten clusters. Looking at potential mechanistic interpretations, results may suggest genetic links between some resistance mechanisms, but this should be confirmed by molecular investigation of the corresponding isolates. Looking at therapeutical potential implications, the high level of resistance and multiresistance to several antimicrobials observed in E. coli makes a critical reassessment of empiric oral antimicrobial therapy in calves highly desirable.

[Antibiotics susceptibility of Streptococcus and Enterococcus: data of Onerba network]. / Sensibilité aux antibiotiques chez les streptocoques (hors pneumocoque) et les entérocoques: données Onerba.

This work was aimed to analyze trends in susceptibility to antibiotics among the main species of beta-hemolytic streptococci involved in community-acquired infections in human (Streptococcus pyogenes and Streptococcus agalactiae), or in animals (Streptococcus suis and Streptococcus uberis) and also among the main enterocci species, Enterococcus faecalis and Enterococcus faecium. Data were recorded since 1996 through the Onerba networks. S. pyogenes, as the other beta-hemolytic streptococci studied remained fully susceptible to beta-lactam antibiotics. However, susceptibility to macrolides is clearly decreasing in S. pyogenes. In 2002, only 62 to 65% of the strains according to the network considered, were susceptible to erythromycin. A similar trend was observed for S. agalactiae with only 75% of erythromycin susceptibility in 2002, and for both species isolated from animals S. suis and S. uberis, with respectively 35 and 76% of strains susceptible to erythromycin. In enterococci, susceptibility to beta-lactams remained stable between 2000 and 2004. Indeed, the susceptibility to aminopenicillins remained high in E. faecalis (about 98%), whereas the proportion of E. faecium isolates susceptible to these antibiotics were lower than 60%. From 1999 to 2004, various studies conducted in French hospitals showed that the vancomycin resistance among enterococci accounted for less than 2%. However, the recent emergence of glycopeptide resistant enterococci clusters in French hospitals is a matter of concern and emphasizes the need for an ongoing surveillance. Such trend in macrolide resistance among S. pyogenes or S. agalactiae should consequently lead to propose other alternatives in case of beta-lactam allergy, and for pharyngitis, to rethink the place of the culture for susceptibility testing.

Branched-polymer separations using comprehensive two-dimensional molecular-topology fractionation x size-exclusion chromatography.

Branching has a strong influence on the processability and properties of polymers. However, the accurate characterization of branched polymers is genuinely difficult. Branched molecules of a certain molecular weight exhibit the same hydrodynamic volumes as linear molecules of substantially lower weights. Therefore, separation by size-exclusion chromatography (SEC), will result in the co-elution of molecules with different molecular weights and branching characteristics. Chromatographic separation of the polymer molecules in sub-microm channels, known as molecular-topology fractionation (MTF), may provide a better separation based on topological differences among sample molecules. MTF elution volumes depend on both the topology and molar mass. Therefore co-elution of branched molecules with linear molecules of lower molar mass may also occur in this separation. Because SEC and MTF exhibit significantly different selectivity, the best and clearest separations can be achieved by combining the two techniques in a comprehensive two-dimensional (MTFxSEC) separation system. In this work such a system has been used to demonstrate branching-selective separations of star branched polymers and of randomly long-chain-branched polymers. Star-shaped polymers were separated from linear polymers above a column-dependent molecular weight or size.

[Impact of nociceptive stimuli on cervical kinesthesia]. / Influence de stimulations nociceptives sur le sens de repositionnement céphalique.

UNLABELLED: The goal of this study was to evaluate the impact of nociceptive stimuli upon the cervical proprioception ability. METHOD: Thirty healthy young subjects performed a cervicocephalic relocation test (CRT) in two random conditions: the first one was based on a nociceptive electric stimulation called condition "pain", whereas the second one was targeting a painless electric condition called condition "control". The CRT consisted of repositioning the head on the trunk, after an active transversal movement of the head in the transverse field with closed eyes. The pointing was recorded at the beginning and at the end of each rotation using a custom video acquisition system. RESULTS: The average mean of error repositioning was worth 3.98+/-0.99 degrees (average mean, standard deviation) in the condition "pain", and 1.75+/-0.37 degrees in the condition "control" (p<0.01). CONCLUSION: Acute pain provokes a disturbance of the cervical proprioception ability without damaging the anatomic structure. This observation suggests the interest of an early follow-up of the pain to avoid sensory disturbances, as well as the establishment of a cervical proprioceptive rehabilitation program after an algic event.

GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis.

The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.

A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets.

This study, Vetoquinol S.A. epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains from infected pets originated from six European countries. Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S. intermedius, P. aeruginosa). The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995. In E. coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously. Over this period, there was no significant evolution of MIC(90) for any bacterial species studied and no development of resistance was observed. Marbofloxacin was the most active antibiotic against P. multocida isolates and had the lowest MIC. No difference in MIC distribution was seen between the S. intermedius (unimodal distribution) isolated from dermatological infections and those from otitis. This was also true for P. aeruginosa. The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria.

Seven years survey of susceptibility to marbofloxacin of bovine pathogenic strains from eight European countries.

This study was conducted from 1994 to 2001 to determine the susceptibility of bovine pathogenic bacteria to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains originated in bovine diseases from eight European countries. They were isolated from gut infections (Escherichia coli, salmonellae), mastitis (E. coli, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. dysgalactiae) and respiratory diseases (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus). There was no change in the MIC distributions for each species after the launch of marbofloxacin in 1997. In E. coli, a resistant population was present before the use of marbofloxacin having been induced by co- or cross-resistance to other antibiotics used previously. Over this period the only a significant change seen was an increase in MIC(90) of E. coli from the gut (1.275 microg/ml in 1994/1995 to 5.098 microg/ml in 2001). All the salmonellae were susceptible to marbofloxacin with a MIC(90) = 0.073 microg/ml in 2001 without development of high level resistance. The use of marbofloxacin seems not to have favoured a significant increase and spreading of resistant bacteria.

Apparent redundancy of electron transfer pathways via bc(1) complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.

Solvent extraction of organic molecules of exobiological interest for in situ analysis of the Martian soil.

A solid-liquid extraction method able to perform in situ extraction of organic compounds on Mars is proposed. The extraction efficiency of various organic solvents was tested and compared to that of water. The selected key compounds are molecules of exobiological interest: glycine, alanine, serine, glutamic acid, oxalic acid, benzoic acid, phthalic acid, isophthalic acid, terephthalic acid and 1,3,5-benzenetricarboxylic acid. Among the organic solvents, propanol gives the highest yield of extraction for all the targeted compounds except for benzoic acid. A mixture of propanol and ethyl acetate increases significantly the extraction yield of benzoic acid. The extraction time was considerably reduced (140 h to 15 min) by using sonication. The method is discussed for an easy automation with coupling to an in situ GC-MS space instrument.

[About the rational use of intrathoracic transfers. Anatomical and surgical bases for the selection of twelve different muscular and omental flaps]. / De l'utilisation raisonnée des transferts intrathoraciques. Bases anatomiques et chirurgicales pour la sélection de 12 différents lambeaux musculaires et omentaux.

Because of their rich blood supply and of their original detersive and filling properties, the muscular pedicled flaps harvested from the trunk or the omental flap elevated from the abdomen may be very usefull to treat large defects or major septic problems in pleural, pericardic or mediastinal cavities. We here describe the main principles to be followed in such intrathoracic reconstructions performed in order to control severe mediastinites, aortic prosthetic infections, pleural empyemas and broncho-pleural, tracheo-esophageal or broncho-esophageal fistulas. In all these circumstances, the muscular or omental flaps which are transferred into the chest are selected according to the recipient field and to their respective access to the upper, middle and lower portions of the pleural space or mediastinum. Twelve different flaps so appear available to achieve the adequate reconstruction, filling or coverage of nine distinct topographic sites. Their rational use, based on various anatomical guidelines, allows to prevent or to cure efficiently 90% of the infectious or fistular complications frequently observed in the postoperative course of aggressive, functional or oncological, intrathoracic surgical procedures.

Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae.

Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.