Eszter Csoma
email: csomae@freemail.hu

Beáta Mészáros

László Asztalos

József Kónya

Lajos Gergely

Prevalence of WU and KI Polyomaviruses in Plasma, Urine and Respiratory Samples from 1 Renal Transplant Patients 2

Keywords: WU polyomavirus, KI polyomavirus, renal transplantation

15

WU and KI polyomaviruses (WUPyV, KIPyV) have been detected in respiratory, blood, stool 16 and lymphoid tissue, but not in urine samples. PCR based detection revealed higher frequency 17 in immunocompromised individuals. In this study the prevalence of WUPyV and KIPyV was 18 analyzed in respiratory, urine and blood samples from renal transplant patients compared with 19 healthy individuals. WUPyV and KIPyV were detected by nested PCR. The PCR products 20 were sequenced and viral DNA loads were determined by quantitative real-time PCR. 21

WUPyV and KIPyV were found in plasma (3.6 %; 7/195), urine (14 %; 7/50) and respiratory 22 samples (10 %; 9/90) of renal transplant patients, but not in plasma (0/200) and urine (0/36) 23 specimens from healthy blood donors. WUPyV and KIPyV were detected mainly early after 24 renal transplantation and the viral loads were low. A higher prevalence of WUPyV was found 25 in plasma and urine samples, KIPyV was found more frequently in respiratory samples from 26 renal transplant patients. It is hypothesized that immunosuppression due to the transplantation 27 may result in reactivation of these viruses or may establish greater susceptibility to infection 28 with KIPyV and WUPyV.

Serological studies suggest that KI and WU polyomaviruses (KIPyV, WUPyV) are 31 widespread. It is thought that primary infection may occur in childhood because the 32 seropositivity for both viruses is high in children and reaches 70-80 % in adults [Neske et al., 33 2010;[START_REF] Nguyen | Serologic evidence of frequent human infection with 204 WU and KI polyomaviruses[END_REF]. Both WUPyV and KIPyV have been identified from respiratory 34 specimens of patients with respiratory symptoms [START_REF] Allander | Identification of a third human polyomavirus[END_REF][START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF]. 35

Although the pathogenic roles of these viruses have not been clarified, PCR based detection 36 revealed 0.4-9 % prevalence in respiratory specimens of immunocompetent patients and 37 higher frequency in children and immunocompromised individuals [START_REF] Bialasiewicz | Detection of BK, JC, 172 WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory 173 samples[END_REF]38 Dalianis et al., 2009;[START_REF] Mourez | Polyomaviruses KI and WU in immunocompromised patients with respiratory 196 disease[END_REF]. Viral DNA was also detected in blood samples 39 from immunocompromised patients and children [START_REF] Miller | WU polyomavirus in patients 192 infected with HIV or hepatitis C virus, Connecticut, USA[END_REF][START_REF] Neske | Detection of WU 198 polyomavirus DNA by real-time PCR in nasopharyngeal aspirates, serum, and stool 199 samples[END_REF], in 40 stool samples of children with gastroenteritis [START_REF] Bialasiewicz | Detection of BK, JC, 172 WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory 173 samples[END_REF][START_REF] Neske | Detection of WU 198 polyomavirus DNA by real-time PCR in nasopharyngeal aspirates, serum, and stool 199 samples[END_REF], in 41 lymphoid tissues from immunocompromised patients [START_REF] Sharp | Reactivation and mutation of 206 newly discovered WU, KI, and Merkel cell carcinoma polyomaviruses in 207 immunosuppressed individuals[END_REF], but not in urine 42 samples from immunocompromised and immunocompetent patients [Bialasiewicz et al., 43 2009;[START_REF] Bofill-Mas | Newly 178 described human polyomaviruses Merkel cell, KI and WU are present in urban sewage 179 and may represent potential environmental contaminants[END_REF][START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF]. The higher prevalence in 44 immunocompromised patients suggest that these viruses may cause more severe problems in 45 these individuals in a manner similar to the effect of BK and JC virus (BKV, JCV) [Jiang et 46 al., 2009]. 47

The prevalence of WUPyV and KIPyV has been studied and examined in respiratory, urine 48 and blood samples from renal transplant patients. 49 (median 1188 days, range 3-7108). For control measurements, 200 blood samples from 200 53 healthy blood donors were taken (75 men, 125 women, median age 39 years, range 10-74 54 years). Fifty urine samples from 50 transplant patients were also collected after 55 transplantation (range 5-6230 days; median 141 days). Thirty six urine specimens from 56 healthy blood donors were used as controls. Ninety upper respiratory tract specimens using 57 throat swabs from 90 renal transplant individuals were obtained 18-6230 days after the 58 transplantation (median 1177 days). 59 Nucleic acids from 200 µL plasma, centrifuged for 10 min at 180×g at 4 ºC, 200 µl urine 60 specimen and throat swab sample washed in 200 µL buffer were isolated using High Pure 61 Viral Nucleic Acid Kit (Roche, Basel, Switzerland) according to the manufacturer's 62 instructions. Nucleic acid was eluted in 50 µl and stored at -20 ºC until use. 63

F
The Regional and Institutional Ethics Committee of University of Debrecen approved all of 64 the studies. All patients gave their written informed consent. 65

Qualitative and quantitative detection of KIPyV and WUPyV DNA. To detect WUPyV and 66

KIPyV DNA, the first round of WUKI nested-PCR was carried out with WUKI_OS and 67 WUKI_OAS primers as described previously [START_REF] Sharp | Reactivation and mutation of 206 newly discovered WU, KI, and Merkel cell carcinoma polyomaviruses in 207 immunosuppressed individuals[END_REF] in a final volume of 20 µL 68 containing 5 µL DNA solution, GenAmp Fast PCR Master Mix (Applied Bisystems, Foster 69 City, CA, USA) and 10-10 pmol of each primer. For the second round, 4 µL of the PCR 70 product from the first round was amplified in 20 µL final volume using GenAmp Fast PCR 71

Master Mix and 10-10 pmol WUKI_IS and WUKI_IAS primers [START_REF] Sharp | Reactivation and mutation of 206 newly discovered WU, KI, and Merkel cell carcinoma polyomaviruses in 207 immunosuppressed individuals[END_REF]. The 72 annealing temperature was 60 ºC in both rounds. Plasmids containing the genome of WUPyV 73 and KIPyV were used as positive controls [START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF]Lindau et al., 2009] were positive for KIPyV and five for WUPyV DNA. The level of DNA load in six plasma 88 samples was less then 250 GEq/mL urine, below the limit of detection, and 2.5 X 10 2 KIPyV 89 GEq/mL in one specimen. Significant difference was found between polyoma-positive and 90 negative samples regarding the time after renal transplantation (p=0.001) ( GEq/mL plasma) and six samples were WUPyV DNA positive by sequencing. The viral loads 95 of four samples were < 250 GEq/mL, and two samples had 5 X 10 2 and 1.1 X 10 3 GEq / mL 96 plasma. PCR positive samples were collected significantly earlier after transplantation then 97 the negative samples (p=0.001) (Table 2.). In the case of two patients whose urine samples 98 were WUPyV DNA positive (28.6 %), WUPyV viremia was also detected. 99

Prevalence of WUPyV and KIPyV by PCR in respiratory samples. Nine (10 %) of 90 100 respiratory samples were WUKI PCR positive (Table 1.). Six samples were KIPyV DNA 101 positive (66.7 %) with viral loads ranging from 2.8 X10 2 to 3.7 X 10 5 GEq/mL (median 4.2 X 102 10 4 ; in two samples the viral load was below the limit of detection). Only one out of the three 103

WUPyV positive samples had detectable viral load of 6.3 X 10 2 GEq/mL. Statistical analysis 104 revealed that the PCR positive samples were collected significantly earlier after 105 transplantation then the PCR negative samples (p=0.002; Table 2

.). The plasma sample of one 106

patient with a KIPyV positive respiratory specimen was positive for WUPyV DNA, the 107 plasma samples of the others were PCR negative. All of the patients with a positive 108 This study revealed that WUPyV and KIPyV can be detected in blood, urine and respiratory 113 samples from renal transplant patients, but these viruses were not found in blood and urine 114 specimens from healthy blood donors. 115 Viremia was detected in 3.6 % of the renal transplant patients, mostly early after 116 transplantation, but not in healthy blood donors. Other studies have found only WUPyV in 117 blood samples [START_REF] Bialasiewicz | Detection of BK, JC, 172 WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory 173 samples[END_REF][START_REF] Miller | WU polyomavirus in patients 192 infected with HIV or hepatitis C virus, Connecticut, USA[END_REF][START_REF] Neske | Detection of WU 198 polyomavirus DNA by real-time PCR in nasopharyngeal aspirates, serum, and stool 199 samples[END_REF], but apart 118 from WUPyV (2.6 %), KIPyV (1 %) was detected in the current study in renal transplant 119 patients. The viral loads were very low, ≤ 250 GEq /mL plasma. Previously, WU and KI 120 polyomaviruses were not found in urine samples from immunocompromised, renal transplant 121 patients, immunocompetent patients and pregnant women [START_REF] Bialasiewicz | Detection of BK, JC, 172 WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory 173 samples[END_REF]Bofill-122 Mas et al., 2010;[START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF]. In this study these viruses were not detected in urine 123 specimens from healthy blood donors, but 14 % of the samples from renal transplant patients 124 were positive for viral DNA. A higher prevalence of WUPyV was observed compared with 125 KIPyV (6/7 vs. 1/7). The viruses appeared early after renal transplantation, and all of the 126 positive samples were collected within two months after the transplantation. The viral loads in 127 these urine samples were low (≤1.1 X 10 3 GEq/mL), but in two patients WUPyV viremia was 128 also found at the same time. A different DNA isolation method in which samples were not 129 stored and DNA was isolated immediately after the collection of the urine samples, primers 130 and PCR conditions may be the reasons why these viruses were found while other 131 investigators did not detect these viruses in urine samples [START_REF] Bialasiewicz | Detection of BK, JC, 172 WU, or KI polyomaviruses in faecal, urine, blood, cerebrospinal fluid and respiratory 173 samples[END_REF]Bofill-132 Mas et al., 2010;[START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF]. 133

In immunocompetent individuals a slightly higher prevalence of WUPyV DNA was observed 134 in the respiratory samples [START_REF] Dalianis | KI, WU and Merkel cell 181 polyomaviruses: a new era for human polyomavirus research[END_REF] and also the seroprevalence of WUPyV was 135 found to be greater [START_REF] Neske | High 201 prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human 202 bocavirus in German blood donors[END_REF][START_REF] Nguyen | Serologic evidence of frequent human infection with 204 WU and KI polyomaviruses[END_REF] compared with KIPyV. In the 136 F o r P e e r R e v i e w 9 throat swab samples from renal transplant patients a higher prevalence of KIPyV (6/9; 6.7 %) 137 then WUPyV (3/9; 3.3 %) was observed. This is in accordance with the results of Murez et al. 138 [2009] who found a higher frequency of KIPyV in respiratory samples from 139 immunocompromised patients. A significant difference was found between the PCR positive 140 and the negative group of the patients regarding the date of samples collection. Viral DNA 141 was detected mostly early after renal transplantation. 142

The results of previous studies suggest that primary infection with WU and KI viruses occurs 143 during childhood with subclinical or mild illness [START_REF] Abedi Kiasari | Age-related 166 pattern of KI and WU polyomavirus infection[END_REF]Bialasiewicz et 144 al., 2007;[START_REF] Gaynor | Identification of a novel polyomavirus from patients 185 with acute respiratory tract infections[END_REF][START_REF] Neske | High 201 prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human 202 bocavirus in German blood donors[END_REF]. Transmission can be fecal-oral and/or via 145 the respiratory route [START_REF] Dalianis | KI, WU and Merkel cell 181 polyomaviruses: a new era for human polyomavirus research[END_REF]. Presumably, in a manner similar to BKV and 146 JCV, it may establish lifelong persistence [START_REF] Jiang | 188 time PCR assay for detection of the two newly characterized human KI and WU 190 polyomaviruses[END_REF]. This study revealed that viruria 147 with WUPyV and KIPyV can occur, so urine can also be a source of infection. The higher 148 prevalence of WUPyV and KIPyV in respiratory samples from immunocompromised 149 patients, and the findings that these viruses are present in blood and urine specimens from 150 renal transplant patients, but not in healthy blood donors, suggest that similar to BKV and 151 JCV, WUPyV and KIPyV might cause significant disease primarily in immunocompromised 152 individuals. At the same time, because of the high rates of coinfections with other respiratory 153 viruses, the pathological role and the clinical consequences of the KI and WU respiratory tract 154 infections are not clean. Based on the knowledge of other human pathogen polyomaviruses it 155 is hypothesized that immunosuppression due to transplantation may result in reactivation of 156 these viruses, or may establish greater susceptibility to KIPyV and WUPyV [Jiang et al., 157 2009]. Although viremia and viruria were found in the current study in renal transplant 158 patients, the viral loads were low. In the case of BKV the level of viruria correlates with the 159 degree of immunosuppression, and the higher viral loads in urine and blood samples can 160 result in more severe clinical consequences [START_REF] Ahsan | Polyomaviruses and human diseases[END_REF]

  acute upper respiratory tract infection, but none of these samples 109 were tested for any respiratory virus; a significantly higher frequency compared with the PCR 110 negative patients (9/9 vs. 47/81; p=0.01).

Table 1 .

 1 Prevalence of WU and KI polyomaviruses in different samples from renal transplant

	patients and healthy blood donors
			KI and WU polyomavirus
		DNA in samples, number (%)
						Total number
	Sample		KIPyV	WUPyV
		Sample			Negative	of samples
	source		positive	positive
						(patients)
	Renal
	transplant	plasma	2 (1)	5 (2.6)	188 (96.4)	195 (195)
	patients
	Renal
	transplant	urine	1 (2)	6 (12)	43 (86)	50 (50)
	patients
	Renal
	transplant	throat swab	6 (6.6)	3 (3.3)	81 (90)	90 (90)
	patients
	Healthy blood
		plasma	0 (0)	0 (0)	200 (100)	200 (200)
	donors
	Healthy blood
		urine	0 (0)	0 (0)	36 (100)	36 (36)
	donors

Table 2 .

 2 Detection of WU and KI polyomaviruses after renal transplantation

		Days after renal transplantation, range
		(median)
	Samples from renal	WUPyV and KIPyV
			Negative
	transplant patients	positive
	plasma	8-2122 (24) *	3-7108 (1271)
	urine	8-58 (30) *	7-6230 (745)
	throat swab	21-822 (101) #	18-6230 (1177)

prevalence of WUPyV and KIPyV in plasma samples.

  PCR was <100 genome equivalent/mL (GEq/mL). Since this PCR 75 method amplifies both WUPyV and KIPyV DNA and does not differentiate between them, 76 the PCR products were sequenced by using the ABI PRISM 3100 Genetic Analyzer (Applied 77 Biosystems). At the same time, real-time PCR for WUPyV and also for KIPyV was 78 performed with primers and probes described previously[Lindau et al., 2009] with 5 µL DNA 79 to confirm the result of the nested-PCR and to quantify the viral loads in the samples. 80 χ 2 and Fisher's exact test was used to assess the difference in frequency for categorical 81 variables. Mann-Whitney U test was applied for continuous variables. A difference was 82 considered significant if p value was less then 0.05. 83 Seven (3.6 %) of 195 plasma 85 samples from transplant patients and none from 200 healthy blood donors were positive by 86 WUKI PCR (p<0.01; Table 1.). Sequencing of the PCR products revealed that two samples 87

	84	F o r r o sensitivity of this nested-F RESULTS PCR
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
		w w
	74	. The
		5 6

Table 2 .

 2 ). 91

PCR prevalence of WUPyV and KIPyV in urine samples.

  Seven (14 %) urine specimens 92 from transplanted patients and none from 36 healthy blood donors were positive for WUKI by 93 PCR (p<0.05; Table 1.). One sample was KIPyV DNA positive (viral load was < 250 94

Table 1 .

 1 transplant patients may help to clarify whether the presence of WUPyV and 162KIPyV in urine and blood samples can result in severe disease as observed withBKV. 163 This work was supported by grants from the Hungarian Scientific Research Found (OTKA 213 73145) and from the Ministry of Health, Hungary (062-05), respectively. 214 Prevalence of WU and KI polyomaviruses in different samples from renal transplant 220

	. Further follow up

Table 2 .

 2 Detection of WU and KI polyomaviruses after renal transplantation 225

	226
		Days after renal transplantation, range
		(median)
	Samples from renal	WUPyV and KIPyV
			Negative
	transplant patients	positive
	plasma	8-2122 (24) *	3-7108 (1271)
	urine	8-58 (30) *	7-6230 (745)
	throat swab	21-822 (101) #	18-6230 (1177)

Acknowledgments 210

We thank Tobias Allander from Karolinska Institute (Sweeden) and David Wang from 211 Washington University (USA) for providing KIPyV and WUPyV plasmids, respectively. 212

Conflict of interest 216

The authors have no conflict of interest.


Asseray

M Lefebvre

C Jacqueline

G Amador

V Le Mabecque

A Miegeville

G Potel

J Caillon

N Asseray

Efficacy of daptomycin combined with rifampicin for the treatment of experimental meticillin-resistant Staphylococcus aureus (MRSA) acute osteomyelitis

Keywords:

Efficacy of daptomycin combined with rifampicin for the treatment of experimental meticillin-resistant (MRSA)

acute osteomyelitis

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Introduction

Orthopaedic infections are among the most difficult to treat, involving surgical procedures and prolonged antibiotherapy [START_REF] Lew | Osteomyelitis[END_REF]. Clinical guidelines are lacking in this area of infectious disease and most of the recommendations, such as the French recommendations [START_REF] Spilf) | Clinical practice recommendations. Osteoarticular infections on materials (prosthesis, implant, osteosynthesis) [[END_REF], are simply based on expert opinion as there are insufficient data to support a high level of evidence. Glycopeptides remain the first-line recommended therapy for the treatment of meticillin-resistant Staphylococcus aureus (MRSA) orthopaedic infections, in combination with rifampicin when the microorganism is susceptible [START_REF] Zimmerli | Prosthetic-joint infections[END_REF]. When MRSA is multiresistant, only a few therapeutic alternatives are available. Daptomycin is the first approved lipopeptide antibiotic and provides potent bactericidal activity against a broad range of Grampositive bacteria, including MRSA [START_REF] Critchley | Activity of daptomycin against susceptible and multidrug-resistant Gram-positive pathogens collected in the SECURE study (Europe) during 2000-2001[END_REF][START_REF] Sader | Antimicrobial susceptibility of Grampositive bacteria isolated from European medical centres: results of the Daptomycin Surveillance Programme (2002-2004)[END_REF][START_REF] Wootton | Comparative bactericidal activities of daptomycin and vancomycin against glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA isolates[END_REF]. The relevance of its combination with rifampicin has recently been demonstrated in animal models, particularly in foreignbody infection models [START_REF] John | Efficacy of daptomycin in implant-associated infection due to methicillin-resistant Staphylococcus aureus: importance of combination with rifampin[END_REF]. Daptomycin showed similar efficacy to vancomycin in previous experimental osteomyelitis studies but has not been evaluated at the early stage of treatment or in combination with rifampicin [START_REF] Norden | Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with LY146032[END_REF][START_REF] Mader | Comparative evaluation of daptomycin (LY146032) and vancomycin in the treatment of experimental methicillin-resistant Staphylococcus aureus osteomyelitis in rabbits[END_REF][START_REF] Luu | Treatment of chronic experimental Staphylococcus aureus osteomyelitis with LY146032 and vancomycin[END_REF]. Therefore, this new antibiotic needs to be appraised in combination with rifampicin in an acute osteomyelitis model.

Materials and methods

Bacterial strain

MRSA strain BCB8 isolated from blood culture was used in the study.

Antibiotics

Daptomycin powder was supplied by Cubist Pharmaceuticals (Lexington, MA).

Susceptibility testing

Minimum inhibitory concentrations (MICs) of daptomycin, vancomycin and rifampicin were determined by the microdilution method in cation-adjusted Mueller-Hinton broth. For daptomycin, the test medium was supplemented with 50 mg/L Ca 2+ according to Clinical and Laboratory Standards Institute (CLSI) guidelines [START_REF]Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement[END_REF].

Animals

Study animals were female New Zealand White rabbits. This study was approved by the animal research committee of the University of Nantes (France). Fentanyl analgesia (fentanyl transdermal patch, 12 g/h) was used for pain management.

Human pharmacokinetic simulation studies

A first step in the pharmacokinetic studies consisted of investigating the parameters allowing simulation of the kinetics of daptomycin in human serum. Blood samples were taken from three healthy rabbits at 0, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8

A c c e p t e d M a n u s c r i p t 5 and 24 h after administration of an intravenous (i.v.) bolus of daptomycin at 6 mg/kg body weight to determine spontaneous drug kinetics. Pharmacokinetic data were processed and were compared with those of humans [START_REF] Bugnon | In vivo simulation of human pharmacokinetics in the rabbit[END_REF]. A computer-controlled system was then used to obtain the human kinetic profiles for daptomycin in rabbits.

Pharmacokinetic parameters were intended to simulate those observed in healthy volunteers following administration of 6 mg/kg i.v. daptomycin every 24 h [START_REF] Dvorchik | Daptomycin pharmacokinetics and safety following administration of escalating doses once daily to healthy subjects[END_REF]. The infusion was delivered by a computer-controlled pump that allowed the flow to be adjusted to a profile mathematically defined in time. To validate the simulation, plasma concentrations were determined in five rabbits.

Vancomycin was given as a continuous infusion at a dose of 100 mg/kg body weight/day so that the steady-state serum level was equivalent to the usual target in humans (ca. 25 mg/L) [START_REF] Asseray | Activity of glycopeptides against Staphylococcus aureus infection in a rabbit endocarditis model: MICs do not predict in vivo efficacy[END_REF]. Rifampicin 20 mg/kg was injected intramuscularly every 12 h to simulate human 10 mg/kg twice-daily oral administration [START_REF] Saleh-Mghir | Combination of quinupristin-dalfopristin (Synercid) and rifampin is highly synergistic in experimental Staphylococcus aureus joint prosthesis infection[END_REF].

Osteomyelitis model

The experimental procedure used in this work has been published recently [START_REF] Amador | In vivo assessment of the antimicrobial activity of a calcium-deficient apatite vancomycin drug delivery system in a methicillin-resistant Staphylococcus aureus rabbit osteomyelitis experimental model[END_REF]. A transarticular aperture was drilled in the cortical bone between the condyles on the right femur of the rabbits under general anaesthesia (ketamine 20 mg/kg i.v. and xylazine 1 mg/kg i.v.). An 8 G needle was introduced into the medullary canal and was then removed. One millilitre of a bacterial suspension of MRSA adjusted to 10 8 colony-forming units (CFU)/mL was injected into the knee cavity. Three days after inoculation, debridement and irrigation of the infected joint with 50 mL of sterile saline was performed. Samples of infected joint fluid, bone marrow and cortical bone were removed, weighed and homogenised in 500 L of sterile saline and then serial A c c e p t e d M a n u s c r i p t 6 dilutions were plated on trypticase soy agar. Following overnight incubation at 37 C, the number of viable bacteria was determined. After lavage and debridement, rabbits were randomly assigned to five regimens: no treatment (controls); monotherapy arms (daptomycin or vancomycin); and combination arms with rifampicin (daptomycin or vancomycin). For each group, eight animals were required to validate the results. At the end of a 4-day course of antibiotics, animals were euthanized and infected joint fluid, epiphyseal bone sample and femoral bone marrow were obtained. Dilutions at 10 -1 , 10 -2 and 10 -4 were performed to avoid any carry-over effect. Bacterial counts were determined after 48 h of incubation at 37 C. The efficacy measurement was performed by comparing the bacterial load before (Day 3 after infection) and after (Day 7 after infection) antibacterial therapy. The lower limit of detection was 20 CFU/mL. The endpoint was expressed as the mean difference  standard deviation in log CFU/g of infected tissue between Day 3 and Day 7 (log CFU/g).

Emergence of resistance

To determine whether antibiotic regimens could induce the selection of in vivo resistant variants, undiluted samples were spread on agar plates containing daptomycin or rifampicin at concentrations corresponding to 4 MIC. Bacteria recovered after 48 h of incubation at 37 C were tested to determine the MIC of daptomycin or rifampicin by the broth microdilution method.

Histopathology

Samples of the distal half of the femur bone were fixed in neutral-buffered formalin solution, dehydrated in a graded alcohol solution and embedded in methyl

Statistical analysis

Mean loads from each group were compared by Newman-Keuls test after analysis of variance (ANOVA) using GraphPad Prism v4.0 (GraphPad Software Inc., San Diego, CA). A P-value <0.05 was considered significant.

Results

Susceptibility testing

MICs for MRSA strain BCB8 were 0.5 g/mL for daptomycin, 1 g/mL for vancomycin and 0.008 g/mL for rifampicin.

Pharmacokinetic data of daptomycin in serum

Pharmacokinetic parameters of daptomycin obtained with the human-simulated dose of 6 mg/kg were close to those observed in humans: mean half-life (T 1/2 ), 7.8 ± 1.0 h; peak concentration (C max ), 86.4 ± 7.1 mg/L; and area under the concentration-time curve (AUC), 705 ± 67 mg h/L. Following treatment, none of the animals in the daptomycin or vancomycin groups had sterile bone and the log CFU/g was not significantly different from that observed in the control group. In both antibiotic combination groups, the mean bacterial counts in the three osteoarticular samples were significantly lower than those in the control and in both monotherapy groups (P < 0.05). The combination of daptomycin plus rifampicin gave better results, with a greater reduction in the bacterial load (statistically significant only in the joint fluid, P < 0.01) and a greater number of sterile bone samples (Table 1).

Antibiotic resistance

No variant resistant to rifampicin was detected in the group treated with daptomycin plus rifampicin, but resistant mutants were detected in one animal in the group treated with vancomycin plus rifampicin. The rifampicin MIC of mutants was >32 mg/L compared with an initial MIC of 0.008 mg/L. Resistant mutants to daptomycin were detected in five samples from three animals treated with daptomycin alone; the MICs of mutants were 2 mg/L and 4 mg/L. In the inoculated femur, haematopoietic cells in the marrow spaces were preserved.

Minimal to mild acute inflammation with intramedullary abscess was observed in the medulla of the metaphysis. Occasional bone necrosis was noticeable (data not shown).

Discussion

In this acute osteomyelitis model, daptomycin and vancomycin alone were ineffective at reducing the bacterial count significantly. Both combinations were more effective than the corresponding monotherapy. Bacterial eradication was achieved more often with the combination of daptomycin and rifampicin compared with and the combination of vancomycin and rifampicin. No emergence of resistance to rifampicin or daptomycin was observed in the daptomycin/rifampicin group, whereas resistance to daptomycin was detected in the monotherapy group.

Since daptomycin is used to treat infections caused by Gram-positive cocci resistant to -lactams, its efficacy in the treatment of wound infections, especially in orthopaedic surgery, is of interest. This new antimicrobial could be a first step in the treatment of osteomyelitis and other orthopaedic infections. However, there are few data regarding its efficacy in treating such infections. Some experimental studies have compared daptomycin with vancomycin, showing a possible benefit of daptomycin [START_REF] Norden | Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with LY146032[END_REF][START_REF] Mader | Comparative evaluation of daptomycin (LY146032) and vancomycin in the treatment of experimental methicillin-resistant Staphylococcus aureus osteomyelitis in rabbits[END_REF][START_REF] Luu | Treatment of chronic experimental Staphylococcus aureus osteomyelitis with LY146032 and vancomycin[END_REF][START_REF] Rose | Evaluation of daptomycin pharmacodynamics and resistance at various dosage regimens against Staphylococcus aureus isolates with reduced susceptibilities to daptomycin in an in vitro pharmacodynamic model with simulated endocardial vegetations[END_REF]. The potential interest of daptomycin for the treatment of orthopaedic infections is supported by certain specific features of this antibiotic.

Indeed, daptomycin has recently been shown to demonstrate activity against

A c c e p t e d M a n u s c r i p t 10 intracellular meticillin-susceptible S. aureus and MRSA in monocyte-derived macrophages, to inhibit slime synthesis and to induce slime disruption in vitro [START_REF] Baltch | Activities of daptomycin and comparative antimicrobials, singly and in combination, against extracellular and intracellular Staphylococcus aureus and its stable small-colony variant in human monocyte-derived macrophages and in broth[END_REF][START_REF] Roveta | Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp[END_REF].

These properties could explain the potency of the association of daptomycin with rifampicin in such infections.

In this acute osteomyelitis model, no efficacy was achieved with either monotherapy regimen. Moreover, in another model, vancomycin and daptomycin monotherapy regimens were unable to cure any cage-associated infection [START_REF] John | Efficacy of daptomycin in implant-associated infection due to methicillin-resistant Staphylococcus aureus: importance of combination with rifampin[END_REF]. In this model, daptomycin at high dose in combination with rifampicin showed the highest activity against planktonic and adherent MRSA.

The rabbit model used here was similar to human osteoarticular infection because of the mechanism of inoculation, the high bacterial load in bone without spontaneous cure, the combination of medical and surgical treatment, and the simulation of human pharmacokinetics. This acute osteomyelitis model is of interest in assessing efficacy of antibiotics at an early stage of treatment. The other models were different owing to a more moderate bacterial count in bone tissues leading to a spontaneous cure rate, or mimicking of haematogenous osteomyelitis [START_REF] Norden | Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus with LY146032[END_REF][START_REF] Yin | Comparative evaluation of tigecycline and vancomycin, with and without rifampicin, in the treatment of methicillin-resistant Staphylococcus aureus experimental osteomyelitis in a rabbit model[END_REF].

These results and recent literature highlight the importance of adding rifampicin to daptomycin in difficult-to-treat staphylococcal bone infections. This combination could be a promising and easier treatment option for orthopaedic infections caused by MRSA.

Funding

A c c e p t e d M a n u s c r i p t 2 ABSTRACT

 2 Daptomycin exhibits rapid bactericidal activity against Gram-positive organisms, including meticillin-resistant Staphylococcus aureus (MRSA). Daptomycin in combination with rifampicin needs to be assessed in bone infection. An MRSA acute osteomyelitis model was used. Daptomycin and vancomycin were compared, alone or in combination with rifampicin, over 4 days. Surviving bacteria were counted in bone, bone marrow and joint fluid. Vancomycin and daptomycin as single therapies were ineffective, but both combinations were significantly more effective than the corresponding monotherapy. Combination of daptomycin and rifampicin could prevent S. aureus from developing resistance. This combination could be a useful alternative to treat MRSA osteomyelitis at an early stage.

  The present study aimed to assess the efficacy of daptomycin, compared with vancomycin, in combination with rifampicin in a rabbit model of experimental MRSA acute osteomyelitis.

  sections in a sagittal plane were cut at 5 mm and slices were stained with Masson-Goldner stain for histological analysis.

  modelAll of the animals infected with MRSA exhibited positive joint fluid, bone marrow and bone smear cultures, with mean bacterial counts 3 days after infection of 7.7  0.4, 7.9  0.3 and 8.4  0.5 log CFU/g, respectively.

  s c r i p t

Competing interests

None declared.

Ethical approval

In vivo studies were approved by the Animal Study Committee of the University of Nantes, France.

A c c e p t e d M a n u s c r i p t


Florian Astelbauer

Andreas Obwaller

Adriane Raninger

Brigitte Brem

Harald Greger

Michael Duchêne

Walther Wernsdorfer

Julia Walochnik
email: julia.walochnik@meduniwien.ac.at

High antitrypanosomal activity of plant-derived sulphur-containing amides

Keywords:

come

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Central and South America. The reported annual incidence is 41 200 cases, with 12 500 individuals dying every year [START_REF] Salvatella | Achievements in controlling Chagas disease in Latin America[END_REF], [START_REF]World Health Organization. Update of American Trypanosomiasis and Leishmaniasis Control and Research: Final Report (Rio de[END_REF].

Clinically, the pathogenesis of the disease can be divided into three phases, namely a short acute phase, a long lasting latent phase and a chronic phase, emerging in 10-30% of patients. Serious chronic symptoms such as cardiomyopathy, dementia and malformations of the intestines, e.g. megaoesophagus and megacolon, can be observed.

The drugs of choice are benznidazole and nifurtimox [START_REF] Coura | A critical review on Chagas disease chemotherapy[END_REF], however therapeutic outcome is phase-dependent and resistance against both drugs in T. cruzi is increasing. Both compounds are effective in the acute and initial chronic phases of the infection but not in the late chronic phase. Therefore, dose and the duration of treatment depend on the stage of disease and on the susceptibility of the respective T. cruzi strain. Moreover, severe side effects have been reported for both drugs.

Benznidazole is associated with hypersensibility, dermatitis with cutaneous eruptions, fever, lymphadenopathy, articular and muscular pain, functional insufficiency of bone marrow, thrombocytopenic purpura and agranulocytosis, polyneuropathy, paresthesia and polyneuritis of peripheral nerves. During treatment with nifurtimox, adverse effects including anorexia, weight loss, psychotic alterations, excitability or sleepiness, nausea, vomiting, intestinal colic and diarrhoea have been observed. An

A c c e p t e d M a n u s c r i p t alternative treatment option is amphotericin B (AmB) [START_REF] De-Castro | Differential effect of amphotericin B on the three evolutive stages of Trypanosoma cruzi and on the host cell-parasite interaction[END_REF], however it is prohibitively expensive when using its less toxic liposomal formulation.

The aim of this study was to find new drug candidates by screening compounds extracted and purified from tropical plants for their antitrypanosomal activity. It is estimated that 25% of all drugs prescribed today are derived from flowering plants [START_REF] Farnsworth | Higher plants-the sleeping giant of drug development[END_REF], [START_REF] Newman | Natural products as sources of new drugs over the last 25 years[END_REF]. Alkaloids, which form the biggest group of drugs in the modern pharmacopeia [START_REF] Raskin | Plants and human health in the twenty-first century[END_REF], are secondary metabolites on which plant growth is not dependent. Drugs extracted from plants, or their semisynthetic analogues, have been successfully used against other protozoan diseases, e.g. quinine [START_REF] Kyle | Discoverers of quinine[END_REF] and artemisinin [START_REF]Antimalaria studies on Qinghaosu[END_REF] in the treatment of malaria.

Standardised axenic cultivation of T. cruzi is a useful approach for yielding a defined number of parasites for in vitro drug testing and for comparison of novel compounds with standard drugs. In this study, we evaluated the in vitro antitrypanosomal activities of 15 plant-derived compounds, none of which have been studied

previously for antiprotozoal activity. The investigated compounds, extracted from tropical plants of the families Rutaceae, Meliaceae and Stemonaceae belong to eight different chemical groups, namely flavaglines, furoquinolines, quinolinones, acridones, coumarins, Stemona alkaloids, amides and sulphur-containing amides.

Materials and methods

Parasites

Epimastigote T. cruzi Y cultures (ATCC 50832) were cultured according to the American Type Culture Collection protocol 1029 in axenic liver infusion tryptose (LIT)

A c c e p t e d M a n u s c r i p t medium (pH 7.2) containing 10% fetal bovine serum at 26 C in 12.5 cm 2 tissue culture flasks (Falcon, Franklin Lakes, NJ) under aerobic conditions.

Compounds

Fifteen compounds, including the flavaglines aglafoline and rocaglamide, the furoquinolines dictamnine and iso--fagarine, the quinolinone zanthobungeanine, the acridones yukocitrine and 5-hydroxynoracronicine, the coumarins microminutine and methyllacarol, the Stemona alkaloid tuberostemonine, the amide dehydrothalebanin B, and the sulphur-containing amides methyldambullin, methylgerambullin, sakambullin and dehydroniranin A, were extracted from leaves, stem or root bark of tropical plants. Extraction and purification were achieved by preparative column and thin layer chromatography and purity was assessed by high-performance liquid chromatography and nuclear magnetic resonance. Stock solutions of the compounds were prepared in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St Louis, MO).

In vitro tests and evaluation

All compounds were tested in 96-well microtitre plates. Wells were inoculated with were determined by log-probit regression using SPSS 15.0 (SPSS Inc., Chicago, IL).

Results

Of the 15 compounds tested, only the 3 sulphur-containing amides (methyldambullin, methylgerambullin and sakambullin) (Fig. 1) showed antitrypanosomal activity. The results of growth inhibition assays after 48 h of exposure are shown in Fig. 2

Discussion

The novel compounds methyldambullin, methylgerambullin and sakambullin showed high antitrypanosomal activity in vitro. All three compounds were significantly more effective, with lower EC 50 and EC 90 values than the standard drug benznidazole.

Moreover, at least after 72 h treatment, all three compounds also had lower EC 90

A c c e p t e d M a n u s c r i p t values than AmB, although EC 50 values were higher. Methylgerambullin showed the lowest EC 90 (4.56 M) after 72 h compared with 16.82 M for AmB.

Methyldambullin, methylgerambullin and sakambullin are novel sulphur-containing amides. Currently, all three compounds could only be extracted from the genus Glycosmis, family Rutaceae, in contrast to other sulphur-containing amides that have been reported also to occur in the genus Raphanus, family Brassicaceae [START_REF] Moon | Total synthesis and biological evaluation of methylgerambullone[END_REF]. The sulphur-containing acid moieties are most likely derived from the amino acid cysteine, whereas the amine parts are mostly characterised by phenethyl or phenethenyl groups that additionally can be linked to various prenyloxy structures, e.g. methyldambullin and sakambullin, or geranyloxy groups, e.g. methylgerambullin.

These prenylated amides are mostly oxidised to sulfones or sulphoxides [START_REF] Hofer | Sulfur-containing amides from Glycosmis species (Rutaceae)[END_REF]. Apart from their chemotaxonomic importance, some sulphur-containing amides show pronounced antifungal or insecticidal activity [START_REF] Greger | Bioactive amides from Glycosmis species[END_REF]. Methylgerambullin, the most efficient compound in the present investigation, has been demonstrated to have anticancer activity in previous experiments against CEM-SS (T-lymphoblastic leukaemia), KU812F (chronic myelogenous leukaemia), HT29 (colon cancer) and UACC-62 (melanoma) cell lines, and has been shown to be non-toxic to peripheral blood mononuclear cells [START_REF] Mohamed | Apoptotic and necrotic cell death as manifestations in leukemic cells treated with methylgerambullin, a sulphone from Glycosmis calcicola[END_REF]. No antibacterial or antifungal activity has been observed for methylgerambullin and sakambullin [START_REF] Abdullah | Chemical constituents and biological activities of Glycosmis chlorosperma var. Elmeri (Merr.) Stone (Rutaceae)[END_REF]. Moreover, there is no further antimicrobial or cytotoxic activity known for methyldambullin, methylgerambullin and sakambullin or any other sulphur-containing amides.

Interestingly, methyldambullin, methylgerambullin and sakambullin also showed high

A

  c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t ABSTRACT Chagas disease, caused by Trypanosoma cruzi, represents an important public health problem in endemic geographic regions in Middle and South America, affecting 15 million infected people. Treatment options are still limited due to the toxicity of available drugs, parasite resistance and poor drug activity during the chronic phase of the disease. In this study, we investigated the in vitro antitrypanosomal activity of 15 tropical plant-derived compounds with the aim of finding new drug candidates. Three novel sulphur-containing amides (methyldambullin, methylgerambullin and sakambullin) showed promising high antitrypanosomal activities, with 50% effective concentrations (EC 50 values) after 72 h exposure of 1.7, 1.23 and 5.18 M, respectively, compared with EC 50 values for amphotericin B and benznidazole of 0.71 M and 30.89 M, respectively.

  Trypanosoma cruzi, transmitted by reduviid bugs (e.g. Triatoma infestans, Rhodnius prolixus), is the causative agent of Chagas disease. According to the World Health Organization (WHO), ca. 15 million people are infected in 18 countries throughout

10 5 T

 5 . cruzi Y cells/mL in LIT medium at 26 C. The applied compound concentrations were 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 M. AmB (Sigma-Aldrich) and benznidazole (Sigma-Aldrich) were used as control compounds. The final DMSO concentration never exceeded 0.2% (v/v).Viability of T. cruzi Y cells was determined after 48 h and 72 h treatment using aBuerker haemocytometer. All experiments were carried out in triplicate and were A c c e p t e d M a n u s c r i p t repeated twice. The 50% and 90% effective concentrations (EC 50 and EC 90 values)

  efficacy against Leishmania infantum and Plasmodium falciparum in preliminary experiments in our laboratory (Astelbauer et al., unpublished data). Furthermore, we A c c e p t e d M a n u s c r i p t found an efficient, high-yielding method to synthesise methyldambullin, methylgerambullin and sakambullin chemically (Obwaller et al., unpublished data). These results strongly encourage further studies, including assessment of in the vitro toxicity to various cell lines and primary cells as well as efficacy testing of the synthesised compounds in a murine model of T. cruzi infection.A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Fig. 1 .

 1 Fig. 1. Sulphur-containing amides showing antitrypanosomal activity, all derived from

Fig. 2 .

 2 Fig. 2. Growth inhibition of Trypanosoma cruzi Y after 48 h treatment with

Fig. 3 .

 3 Fig. 3. Growth inhibition of Trypanosoma cruzi Y after 72 h treatment with

A c c e p t e d M a n u s c r i p

  . The EC 50 values obtained for methyldambullin, methylgerambullin and sakambullin were 4.17, 2.83 and 4.50 M, respectively, and the EC 90 values were 24.01, 10.95 and 24.34 M. The EC 50 values for AmB and benznidazole were 0.46 M and 46.81 M, respectively, and the EC 90 values were 18.52 M and 412.20 M. M, respectively. The EC 90 values were 8.34, 4.56 and 12.93 M, respectively, compared with EC 90 values of the control compounds AmB and benznidazole of 16.82 M and 278.01 M, respectively.

	Results after exposure for 72 h are shown in Fig. 3. The EC 50 values for
	methyldambullin, methylgerambullin and sakambullin were 1.7, 1.23 and 5.18 M, ,
	respectively, compared with EC 50 values for AmB and benznidazole of 0.71 M and
	30.89

Funding

This study was supported by grant 814280 from the 'Österreichische Forschungsförderungsgesellschaft' (FFG).

Competing interests

None declared.

Ethical approval

Not required.


Carla M A Pinto

J A Tenreiro

J A Tenreiro Machado

Complex order van der Pol oscillator

Keywords: van der Pol oscillator, complex order derivative, dynamical behavior

HAL is

Introduction

The van der Pol (VDP) oscillator is an ordinary differential equation that has arisen as a model of electrical circuits containing vacuum tubes [START_REF] Van Der Pol | Forced oscillations in a circuit with nonlinear resistance (receptance with reactive triode)[END_REF] (re-edited [START_REF] Bellman | Selected Papers on Mathematical Trends in Control Theory[END_REF]). It produces self-sustaining oscillations in which energy is fed into small oscillations while is removed from large 1 NODY9886_source.tex; 3/11/2010; 13:48 p. 2 oscillations. This is the first relaxation oscillator appearing in the literature [START_REF] Van Der Pol | On relaxation-oscillations[END_REF][START_REF] Van Der Pol E | Frequency Demultiplication[END_REF]. It is given by the following second order differential equation ẍ + μ x 2 -1 ẋ + x = 0 (1)

Parameter μ controls the way the voltage flows through the system. For μ = 0 this is just a simple linear oscillator. For large values of μ, that is for μ 1, the system exhibits a relaxation oscillation. This means that the oscillator has two distinct phases: a slow recovery phase and a fast release phase (vaccuum tubes quickly release or relax their voltage after slowly building up tension).

This equation has been used in the design of various systems, from biology, with the modeling of the heartbeat [START_REF] Fitzhugh | Motion picture of nerve impulse propagation using computer animation[END_REF][START_REF] Nagumo | An active pulse transmission line simulating nerve axon[END_REF][START_REF] Glass | Theory of Heart[END_REF], the generation of action potentials [START_REF] Fitzhugh | Mathematical models of threshold phenomena in the nerve membrane[END_REF][START_REF] Fitzhugh | Impulses and physiological states in theoretical models of nerve membrane[END_REF], up to acoustic systems [START_REF] Altmann | Acoustic and seismic signals of heavy military vehicles for co-operative verification[END_REF] and electrical circuits [START_REF] Appelbe | Hopf bifurcation in a van der Pol type oscillator with magnetic hysteresis[END_REF][START_REF] Elabbasy | Synchronization of van der Pol oscillator and Chen chaotic dynamical system[END_REF]. The VDP oscillator has also been used in the context of chaos theory [START_REF] Chen | Chaotic dynamics of the fractionally damped van der Pol equation[END_REF][START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF][START_REF] Ge | Chaos excited chaos synchronizations of integral and fractional order generalized van der Pol systems[END_REF][START_REF] Ge | Chaos in a modified van der Pol system and in its fractional order systems[END_REF][START_REF] Elabbasy | Synchronization of van der Pol oscillator and Chen chaotic dynamical system[END_REF].

Fractional calculus (FC) has been an important research issue in the last few decades. FC is a generalization of the ordinary integer differentiation and integration to an arbitrary, real or complex, order [START_REF] Oldham | The fractional calculus: theory and application of differentiation and integration to arbitrary order[END_REF][START_REF] Samko | Fractional integrals and derivatives: theory and applications[END_REF][START_REF] Miller | An introduction to the fractional calculus and fractional differential equations[END_REF]. Application of FC have been emerging in different and important areas of physics and engineering [START_REF] Oustaloup | La commande CRONE: commande robuste d'ordre non entier[END_REF][START_REF] Mainardi | Fractional relaxation-oscillation and fractional diffusion-wave phenomena[END_REF][START_REF] Machado | Analysis and design of fractional-order digital control systems[END_REF][START_REF] Nigmatullin | The statistics of the fractional moments: Is there any chance to "read quantitatively" any randomness?[END_REF][START_REF] Podlubny | Fractional-order systems and PID-controllers[END_REF][START_REF] Machado | Discrete-time fractional-order controllers[END_REF][START_REF] Chen | Discretization schemes for fractionalorder differentiators and integrators[END_REF][START_REF] Baleanu | About fractional quantization and fractional variational principles[END_REF][START_REF] Machado | Fractional Derivatives: Probability Interpretation and Frequency Response of Rational Approximations[END_REF][START_REF] Nigmatullin | Is It Possible to Derive Newtonian Equations of Motion with Memory?[END_REF]. Fractional order behavior has been found in areas such as fluid mechanics [START_REF] Momani | Analytical approach to linear fractional partial differential equations arising in fluid mechanics[END_REF], mechanical systems [START_REF] Ge | Chaos, control and synchronization of a fractional order rotational mechanical system with a centrifugal governor[END_REF], electrochemistry [START_REF] Oldham | Fractional differential equations in electrochemistry[END_REF], and biology [START_REF] Darwish | On quadratic integral equation of fractional orders[END_REF][START_REF] Ahmed | On fractional order differential equations model for nonlocal epidemics[END_REF], namely in the modeling of the central pattern generators for animal locomotor rhythms [START_REF] Pinto | Complex order biped rhythms[END_REF][START_REF] Pinto | Fractional Central Pattern Generators for Bipedal Locomotion[END_REF].

There are several definitions of fractional derivatives of order α ∈ R being three of the most important the Riemann -Liouville, the Grünwald -Letnikov, and the Caputo given by:

D α t f (t) = 1 Γ(n -α) d n dt n t a f (τ ) (t -τ ) α-n+1 dτ, n -1 < α < n (2) D α t f (t) = lim h→0 1 h α [ t-a h ] h=0 (-1) k α k f (t -kh) ( 3
)
D α t f (t) = 1 Γ(α -n) t a f (n) (τ ) (t -τ ) α-n+1 dτ, n -1 < α < n (4)
where Γ() is the Euler's gamma function, [ ] means the integer part of x, and h represents the step time increment. It is also possible to generalize results based on transforms, yielding:

L{D α t f (t)} = s α L{f (t)} - n-1 k=0 s k D α-k-1 t f (0 + ) ( 5 )
where s and L represent the Laplace variable and operator, respectively.

The definitions demonstrate that fractional derivatives capture the history of the variable, or, by other words, that have memory, contrary to integer derivatives, that are local operators. The Grünwald -Letnikov formulation inspires the numerical calculation of the fractional derivative based on the approximation of the time increment h through the sampling period T and the series truncation at the r th term. This method is often denoted as Power Series Expansion (PSE) yielding the equation in the z -domain:

Z{D α x(t)} ≈ 1 T α r k=0 (-1) k Γ(α + 1) k!Γ(α -k + 1) z -k X(z) ( 6 )
where X(z) = Z{x(t)} and z and Z represent the z-transform variable and operator, respectively. In fact, expression (3) represents the Euler (or first backward difference) approximation in the s → z discretization scheme, being the Tustin approximation another possibility. The most often adopted generalization of the generalized derivative operator consists in α ∈ R. The case of having fractional derivative of complex-order α ± jβ ∈ C leads to complex output valued results and imposes some restrictions before a practical application. To overcome this problem, it was proposed recently [START_REF] Hartley | Complex-order distributions[END_REF][START_REF] Hartley | Conjugatedorder differintegrals[END_REF][START_REF] Barbosa | Discretization of Complex-order Algorithms for Control Applications[END_REF] the association of two complex-order derivatives. In fact, there are several arrangements that produce real valued results. For example, with the real part of two complex conjugate derivatives D α±jβ we get:

Z 1 2 D α-jβ x(t) + D α+jβ x(t) ≈ 1 T α sin β ln( 1 T ) βz -1 + 1 2 β(1 -2α)z -2 + • • • + + cos β ln( 1 T ) -1 + αz -1 -1 2 β(α 2 -α -β 2 + • • • ) X(z) (7)
Other combinations and the adoption of a Padé fraction, instead of the series for the approximation, are also possible. Nevertheless, in the sequel it is explored the case of expression [START_REF] Barbosa | Analysis of the Van der Pol Oscillator Containing Derivatives of Fractional Order[END_REF].

We must remark that it is adopted a scheme of discretized fractional derivative commonly accepted in engineering, but not yet fully investigated from a mathematical view point. Let us recall the basic definitions of Riemann-Liouville (R-L) and Caputo (C) derivatives, in equations ( 2) and ( 4), based on integral expressions, and of Grünwald-Letnikov (G-L), in equation ( 3), based on a series expression suitable for discretization. The C derivative is a regularization of the R-L derivative and their G-L derivative is the series representation of the R-L derivative. In equation ( 5) the Laplace transform of the R-L deriva-tives differs from that of the C derivative, as shown by Gorenflo and Mainardi [START_REF] Gorenflo | Fractional calculus, integral and differential equations of fractional order[END_REF][START_REF] Gorenflo | Timefractional diffusion: a discrete random walk approach[END_REF], subsequently pointed out in the well known treatises on Fractional Calculus by Podlubny [START_REF] Podlubny | Fractional Differential Equations. An Introduction to Fractional Derivatives, Fractional Differential Equations, Some Methods of Their Solution and Some of Their Applications[END_REF] and Kilbas-Srivastava and Trujillo [START_REF] Kilbas | Theory and Applications of Fractional Differential Equations[END_REF]. So, in practice it is generalized to complex orders the classical R-L derivative of real order. In case of adopting the Caputo derivative (in view of standard initial conditions), it should be adopted a different Laplace transform (see Gorenflo-Mainardi [START_REF] Gorenflo | Fractional calculus, integral and differential equations of fractional order[END_REF][START_REF] Gorenflo | Timefractional diffusion: a discrete random walk approach[END_REF]), and also a modified G-L series representation, because the C derivative is a regularization of the R-L derivative in the time origin. For real order less than one the G-L representation of the C derivative has been adopted in the paper by Gorenflo et al. [START_REF] Gorenflo | Timefractional diffusion: a discrete random walk approach[END_REF]. As a consequence, the Z representation in equation ( 6) in the complex domain should be modified accordingly for the Caputo derivative. While tackling these matters is not straightforward, we must note that the simulations carried in the sequel correspond to steady state responses and that the initial conditions have a minor impact in the resulting charts.

Having these ideas in mind, this paper is organized as follows. In Section 2, we introduce the two approximations of the complex order van der Pol oscillator (CVDP) and we present results from numerical simulations. In Section 3 we outline the main conclusions of this study.

Complex order van der Pol system

Fractional VDP systems have been studied by many authors [START_REF] Chen | Chaotic dynamics of the fractionally damped van der Pol equation[END_REF][START_REF] Barbosa | Analysis of the Van der Pol Oscillator Containing Derivatives of Fractional Order[END_REF][START_REF] Tavazoei | More Details on Analysis of Fractional-order Van der Pol Oscillator[END_REF][START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF][START_REF] Attari | Analysis of a fractional order Van der Pol-like oscilattor via describing function method[END_REF][START_REF] Ge | Chaos excited chaos synchronizations of integral and fractional order generalized van der Pol systems[END_REF][START_REF] Ge | Chaos in a modified van der Pol system and in its fractional order systems[END_REF]. Their work differ in the approaches considered to express the fractional derivative. Chen et al [START_REF] Chen | Chaotic dynamics of the fractionally damped van der Pol equation[END_REF] considered a forced van der Pol equation with fractional damping of the form:

ẍ + μ(x 2 -1)D α x(t) + x(t) = a sin(ωt) ( 8
)
where μ is an endogenous damping parameter, a denotes the amplitude of a periodic forcing, and ω is the forcing frequency. Barbosa et al [START_REF] Barbosa | Analysis of the Van der Pol Oscillator Containing Derivatives of Fractional Order[END_REF] considered the following modified version of the van der Pol equation:

x (1+λ) + α(x 2 -1)x (λ) + x = 0 ( 9
)
with 0 < λ < 1.

Tavazoei et al [START_REF] Tavazoei | More Details on Analysis of Fractional-order Van der Pol Oscillator[END_REF] determined the parametric range for which the fractional VDP system studied by Barbosa et al [START_REF] Barbosa | Analysis of the Van der Pol Oscillator Containing Derivatives of Fractional Order[END_REF] can perform as an undamped oscillator. They also showed that, contrary to the integer order VDP, trajectories in a fractional VDP oscillator do not converge to a unique cycle.

Ge et al [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF] studied the autonomous and non-autonomous fractional van der Pol oscillator. The non-autonomous system in statespace oscillator model is given by:

d α x 1 dt α = x 2 d β x 2 dt β = -x 1 -μ(1 -x 2 1 )(c -ax 2 1 )x 2 + b sin t ( 10
)
where α and β are fractional numbers.

To the best knowledge of the authors, little attention has been given to CVDP oscillators. In this paper, we consider the following two complex order state-space models of the VDP oscillator:

1 2 D α+jβ + D α-jβ x 1 ẋ2 = 0 1 -1 -μ(x 2 1 -1) x 1 x 2 (11) ẋ1 1 2 D α+jβ + D α-jβ x 2 = 0 1 -1 -μ(x 2 1 -1) x 1 x 2 (12)
where D α±jβ , α, β ∈ R + , is a generalization of the concept of the integer derivative, that corresponds to α = 1 and β = 0. We adopt the PSE method for the approximation of the complexorder derivative in the discrete time numerical integration. Several experiments demonstrated that it is required a slight adaption to the standard approach based on a simple truncation of the series. In fact, since our objective is to generate limit cycles, the truncation corresponds to a diminishing of the gain [START_REF] Machado | Fractional Derivatives: Probability Interpretation and Frequency Response of Rational Approximations[END_REF] and, consequently, leads to difficulties in the promotion of periodic orbits. Therefore, in order to overcome this limitation, we decided to include a gain adjustment factor corresponding to the sum of the missing truncated series coefficients.

The discretisation of the CVDP oscillators [START_REF] Darwish | On quadratic integral equation of fractional orders[END_REF] and ( 12) leads to, respectively: In Table 1 we depict periodic solutions of systems ( 13)-( 14) for α = 0.4, β = 0.8 and μ = 0.5. One can observe the appearance of the relaxation oscillation phenomena as α increases (first row of the table). Note that in the case of system [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF] this phenomenon is already present for α = 0.4 and is emphasized for α = 0.8 (second row of the table ).

x 1 (k + 1) = 1 ψ(β,Δt) H(x 1 (k)) + (Δt) α x 2 (k) x 2 (k + 1) = x 2 (k) + Δt -x 1 (k) -μ(x 2 1 (k) -1)x 2 (k) ( 13
)
x 1 (k + 1) = x 1 (k) + Δt x 2 (k) x 2 (k + 1) = 1 ψ(β,Δt) H(x 2 (k)) + (Δt) α -x 1 (k) -μ(x 2 1 (k) -1)x 2 (k) (14
In Figures 12, we show the phase portraits of solutions (x 1 (t), x 2 (t)) of systems ( 13)-( 14) for β = 0.8, μ ∈ {0.5, 2} and different values of α. As expected, the larger the value of μ, the more nonlinear the oscillation becomes. We verify also that we can control the period of the oscillation by varying α. Table 1: Periodic solutions of the CVDP systems ( 13)-( 14) for β = 0.8, μ = 0.5 and α ∈ {0.4, 0.8}.

We now simulate the ordinary differential systems given by expressions ( 13)-( 14) for β = 0.8, α ∈ {0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0}, μ = {0.5, 1.0, 1.5, 2}, and we measure the amplitude and the period of the solutions. Values of α ∈ [0.0, 0.2[ were also considered in the simulations. For system [START_REF] Ge | Chaos, control and synchronization of a fractional order rotational mechanical system with a centrifugal governor[END_REF] we found stable periodic solutions nevertheless for system [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF] simulation results led to unstable solutions, so we have decided to omit these values. Are adopted the initial conditions x 1 (1) = 0.0, x 1 (2) = 0.005,

x 1 (3) = 0.010, x 1 (4) = 0.015, x 1 (5) = 0.02, x 2 (1) = 1.0, x 2 (2) = 1.005, x 2 (3) = 1.010, x 2 (4) = 1.015, x 2 (5) = 1.02.
Each simulation is executed until a stable periodic solution is found. The amplitude and the period of the solutions versus α are depicted in Figures 34. We find that the period increases as α goes from 0.2 to 1.0, in both systems ( 13)-( 14). On the other hand, the amplitude is almost constant. To be precise, the amplitude shows a very tiny increase in system (13) and a very tiny decrease in system [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF], as α increases to one.

We now compute the Fourier transforms |F{x 1 (t)}| of the periodic solutions of systems ( 13) and ( 14), for β = 0.8 and α = {0.3, 0.5, 0.7, 1.0}. Figures 56depict the amplitude of the Fourier transforms vs the fre-quency ω.

The charts demonstrate that the main part of the 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2.5 14) (line) for β = 0.8, α ∈ {0.2, 0.3, 0.4, 0.5, 06, 0.7, 0.8, 0.9, 1.0} and μ = 0.5 (left) and μ = 2.0 (right).

signal energy is concentrated in the fundamental frequency ω 0 . The remaining energy, located in the higher harmonics increases with μ. Furthermore, it is also observed that the fundamental frequency of the oscillations ω 0 varies with α and μ. For the range of tested values, the numerical fitting lead to exponential and rational fraction approximations, for systems ( 13) and ( 14), respectively, given by: ω 0 ≈ 23.032 exp (-3.0686α) [START_REF] Ge | Chaos excited chaos synchronizations of integral and fractional order generalized van der Pol systems[END_REF] and We verify that in the first case the frequency of oscillation is independent of μ, while in the second case it is related both with α and μ.

ω 0 ≈ 1 0.0214 + 0.29μ + (0.7437 -0.1202μ) α 2.3158+0.8522μ (16)

Conclusions

In this paper two complex-order approximations to the well-known van der Pol oscillator were proposed. The amplitude and the period of solutions produced by these two approximations were then measured. The imaginary part was fixed while the real component was varied, for two distinct values of parameter μ. It was observed that the waveform period increases as α varies between 0.2 and one. On the other hand, the amplitude values are almost constant as α varies. Moreover, it seems there is a tiny increase in the amplitude of solutions for system [START_REF] Ge | Chaos, control and synchronization of a fractional order rotational mechanical system with a centrifugal governor[END_REF] and a tiny decrease for system [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF] as α approaches one.

It was also calculated the Fourier transform |F{x 1 (t)}| for systems ( 13) and [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF]. It was verified that the main part of the signal energy is concentrated in the fundamental frequency ω 0 . The remaining energy, located in the higher harmonics increases with μ. It is also observed that the fundamental frequency of the oscillations ω 0 varies with α and μ. For the range of tested values, the numerical fitting lead to exponential and rational fraction approximations for systems [START_REF] Ge | Chaos, control and synchronization of a fractional order rotational mechanical system with a centrifugal governor[END_REF] and [START_REF] Ge | Chaos in a generalized van der Pol system and in its fractional order system[END_REF], respectively.

  ) where Δt = 0.0005 is the time increment, ψ(β, Δt) = cos b log 1 Δt and function H(x i ), i = 1, 2, results from the Taylor series expansion truncation.

Figure 1 :

 1 Figure 1: Phase-space solutions (x 1 (t), x 2 (t)) of the CVDP system (13) for α ∈ {0.4, 0.6, 0.8}, β = 0.8 and μ = 0.5 (left) and μ = 2.0 (right).

Figure 2 :

 2 Figure 2: Phase-space solutions (x 1 (t), x 2 (t)) of the CVDP system (14) for α ∈ {0.4, 0.6, 0.8}, β = 0.8 and μ = 0.5 (left) and μ = 2.0 (right).

Figure 3 :

 3 Figure3: Amplitude of the periodic solutions x 1 (t) produced by the CVDP oscillators (13) (dashed) and (14) (line) for β = 0.8, α ∈ {0.2, 0.3, 0.4, 0.5, 06, 0.7, 0.8, 0.9, 1.0} and μ = 0.5 (left) and μ = 2.0 (right).

Figure 4 :

 4 Figure4: Period of the solutions x 1 (t) produced by the CVDP oscillators (13) (dashed) and (14) (line) for β = 0.8, α ∈ {0.2, 0.3, 0.4, 0.5, 06, 0.7, 0.8, 0.9, 1.0} and μ = 0.5 (left) and μ = 2.0 (right).

Figure 5 :

 5 Figure 5: Amplitude of the Fourier transform |F {x 1 (t)}| vs ω for the CVDP oscilators (13) (left) and (14) (right) with β = 0.8, α ∈ {0.3, 0.5, 0.7, 1.0} and μ = 0.5.

Figure 6 :

 6 Figure 6: Amplitude of the Fourier transform |F {x 1 (t)}| vs ω for the CVDP oscilators (13) (left) and (14) (right) with β = 0.8, α ∈ {0.3, 0.5, 0.7, 1.0} and μ = 2.0.

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Acknowledgments

CP was supported by Fundação para a Ciência e a Tecnologia (FCT) through the Centro de Matemática da Universidade do Porto www.fc.up.pt/cmup.


Keywords: screening, public heath, epidemiology, cancer, quality

teaching and research institutions in France or abroad, or from public or private research centers.

Cancer screening: inequalities ...

  Cancer screening: inequalities … in the data. Journal: International Journal of Epidemiology Manuscript ID: IJE-2010-07-0698.R1 Manuscript Type: Letter to the Editor Date Submitted by the Author: 23-Aug-2010

  Last but not least, the publication in 2010 of raw data from 2002 underlines the urgent need for an European Cancer Observatory with adequate funding.(http://eu-cancer.iarc.fr/7-abouteco.html) The large inequalities in cancer care which exist among European countries are not acceptable. Comparison of recent data is mandatory for benchmarking. The results of the healthcare systems must be compared to allow for the disclosure of irrelevant policies and for identification of the best practices. Epidemiology is the key factor for improvement of quality and security of care.

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	5 6	Letter to the Editor
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	8	Cancer screening: inequalities … in the data.
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	10 11 12 13	Alain Braillon MD PhD Public Health, 80000 Amiens, France braillon.alain@gmail.com
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	16 17	Keywords: Cancer screening, inequality, benchmarking
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	20 21 22 23 24 25	Palencia et al were rightly concerned by inequalities in the use of cancer screening in 2002.(1) The discussion rightly began by an analysis of the strengths and weakness of the study but I F am concerned by two points. o r
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	48 49 50 51	1 Palència L, Espelt A, Rodríguez-Sanz M et al Socio-economic inequalities in breast and cervical cancer screening practices in Europe: influence of the type of screening program. Int. J. Epidemiol. 2010;39: 757-765.
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	53	No conflict of interest
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The validity of the data could have been discussed, at least for France. The prevalence of breast cancer screening can hardly be found in peer-reviewed journals but data exists.(http://www.invs.sante.fr/presse/2010/communiques/depistage_cancer_sein_060410/in dex.html) The French organized breast cancer screening programme achieved national coverage in 2004. Acceptance rate for this organized program increased from 40.2 % in 2004 to 49.3 % in 2006 and 52.5 % in 2008 which are far lower than the 79.4 cited by Palencia et al. Even in 2008 and 2009 only one territory, out of a hundred, reached the 70% level (Haute-Vienne), whereas at the opposite end Paris has the lowest rate (28.2 %).

The second point is the relevance of the definition of screening prevalence (ie. "mammography use in the previous 3years."). The benefit of screening programs is evidenced from randomized trials with durations reaching a decade. Evaluating the prevalence of screening by a cross-sectional analysis is misleading ; it overestimates the quality of the programs and therefore the potential benefits.


Martin Volmer
email: martin.volmer@uni-bielefeld.de

Stefan Northoff

Sebastian Scholz

Tobias Thüte

Heino Büntemeyer

Thomas Noll

Fast filtration for metabolome sampling of suspended animal cells

Keywords: Animal cells, Fast filtration, Metabolome, Microstructure heat exchanger, Quenching, Rapid sampling

come

Introduction

Recent years have seen an increase in systems biological approaches for the investigation of cellular processes. In this respect the "omics" technologies have not only been developed to investigate the processes within cells but also to use the obtained knowledge to improve cultivation processes. From the classical functional genomics/systems biology tools, metabolomics is the most recent technique. Unlike for bacteria and yeast [START_REF] Bolten | Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts[END_REF][START_REF] Faijes | Comparison of quenching and extraction methodologies for metabolome analysis of lactobacillus plantarum[END_REF][START_REF] Schaub | Integrated sampling procedure for metabolome analysis[END_REF][START_REF] Villas-Bôas | Global metabolite analysis of yeast: evaluation of sample preparation methods[END_REF], there are no standard protocols for sampling and extraction of suspended animal cells. A number of different rapid sampling methods are currently used [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF][START_REF] Wiendahl | A microstructure heat exchanger for quenching the metabolism of mammalian cells[END_REF][START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF]), but have not been compared to other methods in depth.

It is most important for metabolome sampling to stop the organism's metabolic activity quantitatively and rapidly. This is to avoid loss of metabolites caused by residual enzyme activity (de Koning and van Dam 1992) or quenching conditions. A standard quenching method for bacteria and yeast is cold methanol, though other protocols using a microstructure heat exchanger or fast filtration are also applied [START_REF] Wellerdiek | Metabolic quenching of corynebacterium glutamicum: efficiency of methods and impact of cold shock[END_REF][START_REF] Bolten | Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts[END_REF]. [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] showed that using methanol, as proposed by [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF], causes increased cell permeabilization and leakage of intracellular components. Thus, alternative methods for the quenching of suspended animal cells are required. Furthermore, it is of utmost importance to separate the cells from the supernatant before metabolite extraction, as residual extracellular components will interfere with the measurement of low concentrated intracellular metabolites, making filtration or pelleting by centrifugation the most suitable techniques. Both methods require a washing step to remove residual medium from the cells. Phosphate buffered saline (PBS) is widely used as washing solution [START_REF] Teng | A direct cell quenching method for cell-culture based metabolomics[END_REF]), but the high phosphate concentration in the sample after the washing step is problematic. Phosphate is a target for metabolome analysis and in large quantities interferes with analytical methods for metabolite quantification, e.g. GC-MS. [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] use an iso-osmotic saline solution, which is also applied in this work.

Another important step of the sample preparation process is the extraction of metabolites from cells. A number of different extraction methods are currently employed for bacteria and yeast as well as for animal cells using boiling methanol, chloroform/methanol and acidic extractions with acidic acetonitrile/methanol or acetic acid. For suspended animal cells methanol is most commonly used as extraction agent [START_REF] Ritter | Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design[END_REF][START_REF] Bennett | Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach[END_REF][START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF][START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF].

Fast filtration as a sampling method for suspended animal cells has already been described by [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] but, due to problems with the metabolite recovery they did not consider it a suitable technique. In this study we describe new method for sampling suspended animal cells using fast filtration and subsequent extraction of metabolites. Furthermore, this method was used for taking metabolite samples during a cultivation of CHO cells. Samples were compared to those taken by a microstructure heat exchanger method derived from [START_REF] Wiendahl | A microstructure heat exchanger for quenching the metabolism of mammalian cells[END_REF] as well as to sampling without quenching.

Material and methods

Cultivation of cells

CHO DP12 cells (ATCC, CRL-12445) were cultivated using the chemically defined medium TC-42 (TeutoCell AG, Germany).

Sampling using fast filtration

The fast filtration set-up contained a custom build two part PTFE filtration device. The lower part accommodated a 50 mm stainless steel filter support frit (Sartorius Stedim Biotechnology, Germany) and a connection for the vacuum pump and controller (CVC3000, Vacuubrand, Germany). The upper part contained a 15 cm polystyrene pipe of 8 mm diam. for application of the sample and washing solution.

For sampling with the fast filtration device a volume of cell suspension correlating to 5 × 10 7 cells total was drawn from the bioreactor using a luer-lock coupled syringe. The cell suspension was applied to the custom build PTFE filtration device and drawn through a 47 mm depth filter disk of 3 µm nominal pore size (Type A/D, Pall Corporation, USA) using a vacuum of 40 mbar. Subsequently, 30 ml of a 0 °C cold iso-osmotic NaCl solution (290 mOsm kg -1 ) was drawn through the filter to remove residual culture medium. The filter was taken from the filtration device and placed inside a 50 ml tube pre-cooled to 0 °C. Tube and filter were frozen in liquid N 2 , immediately.

Sampling using a microstructure heat exchanger A microstructure heat exchanger (Forschungszentrum Karlsruhe, Germany) was connected to a cooling unit (Julabo, Germany) maintained at -4 °C. Cell suspension from the bioreactor was drawn through the heat-exchanger with a vacuum of 180 mbar. The cooled cell suspension was collected in a glass bottle on ice and a volume correlating to 5 × 10 7 cells was transferred to pre-5 cooled 50 ml tubes. The tubes were centrifuged for 1 min at -2 °C and 3000 g in a fixed-angle centrifuge. The supernatant was discarded and the cell pellets were washed with 30 ml ice-cold NaCl solution. The washed cell pellets were frozen in liquid N 2 and stored at -80 °C until extraction of metabolites.

Reference sampling

The cell suspension was directly drawn into 50 ml tubes. Further treatment was according to the microstructure heat exchanger sampling protocol.

Extraction of metabolites

Pelleted cells and cells on filters were extracted using a similar protocol: 4 ml 85 % (v/v) aqueous methanol at -20 °C was added to the washed filter and cell pellets. As the filter still contains a certain volume of washing solution, 1 ml ice-cold water was added to the pellets. Internal standards for metabolite analysis were added and incubated for 24 h at -20 °C. The filter was removed from the extraction solution by drawing the solution through the porous frit of an empty PD10 column (GE-Healthcare, Sweden). Subsequently, 400 µl chloroform (20 °C) were added and the mixture was vortexed thoroughly for 20 s and spun down for 10 min at 2000 rcf and 20 °C.

The supernatant was transferred to a new 15 ml tube and evaporated at 10 °C. Extracts were resuspended in 500 µl water and 500 µl chloroform was added. The suspension was vortexed thoroughly for 20 s and re-centrifuged for 15 min at 2000 g and 20 °C. Three 100 µl aliquots of the aqueous phase were transfered to new 1.5 ml tubes. The extracts were stored at -80 °C until analysis.

Analytical methods

A lactate dehydrogenase (LDH) test kit (Roche Diagnostics, Germany) was used to measure cell disruption given as percentage of total cellular LDH activity.

Adenosine triphosphate (ATP) was quantified using a commercial luminescence assay (Biaffin, Germany) or as described below for metabolites from the central energy metabolism. Amino acids and pyruvate were measured using standard protocols on by HPLC [START_REF] Büntemeyer | Off-Line Analysis in Animal Cell Culture[END_REF].

The concentrations of intracellular metabolites from the central energy metabolism were determined by HILIC-mass spectrometry (Hydrophilic Interaction Liquid Chromatography) on a MonoChrom diol column (Varian, Germany) using acetonitrile with 0.1 % formic acid (A) and 5 mM ammonium acetate with 0.1 % formic acid (pH 3.5, adjusted with ammonia) (B), at 0.2 ml min -1 .

For GC-MS analysis, the keto-and aldehyde-functions of the metabolites were converted into their oxime derivatives using methoxyamine hydrochloride. All other relevant functions like amines, carboxylic acids or hydroxyls were masked with trimethylsilyl groups resulting in volatile derivatives. Statistical errors were reduced by using ribitol as internal standard, while systematic bias were avoided by randomization. The derivatives were separated in a GC 3800 chromatograph and detected by a MS4000 mass spectrometer (both Varian, Germany). A FactorFour VF-5ms column (30 m × 0.25 µm × 0.25 µm) consisting of 95 % methyl-and 5 % phenyl-groups was used (Varian, Germany).

Results and discussion

Validation of fast filtration as means of metabolome sampling

A microstructure heat exchanger method proposed by [START_REF] Wiendahl | A microstructure heat exchanger for quenching the metabolism of mammalian cells[END_REF] and the use of methanol/ammonium bicarbonate as proposed by [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF] have been used for quenching animal cell suspensions to 0 °C in the sub-second scale, but residual metabolic activity still occurs below 0 °C [START_REF] Sauter | Die bakterielle Signalverarbeitung am Beispiel des Sucrose Phosphotransferasesystems in Escherichia coli -Modellierung und experimentelle Überprüfung[END_REF]. Therefore it is key to authentic metabolome sampling of animal cells to not only cool the cells down as fast as possible but also to keep overall sampling time to a minimum. Sampling using centrifugation has certain drawbacks in this respect. We found that a centrifugation time of 1 min at 3000 g is advisable for reliable results. This results in a total sampling time of about 3 min after quenching when using a washing step. Fast filtration allows sampling within 25 s.

The shear forces caused by drawing cell suspension and washing solution through the filter are a major concern, as they can result in high rates of cell disruption during sampling. Data on LDH release during sampling with different vacuums are shown in Fig. 1.

The release of LDH from the cells is only slightly higher during filtration compared to sampling using centrifugation. Sampling at 40 mbar vacuum results in 5.43 % ± 0.57 % LDH measured in filtrate and wash compared to 2.56 % ± 0.66 % LDH measured in supernatant and wash of reference centrifugation experiments.

An increase in vacuum results in higher amounts of LDH in the filtrate and wash due to higher shear forces caused by the increased transmembrane pressure. Lower vacuums increase the sampling time, as illustrated in Fig. 2, making 40 mbar an optimal vacuum allowing short sampling time with minimal cell damage. Higher vacuums account for increased cell disruption which leads to the loss of intracellular metabolites from the sample.

To reduce residual medium components in the extrac,t a rapid washing step is implemented in the sampling procedure. Fig. 3 shows the cellular content of four exemplary amino acids extracted from filters loaded with 10 7 cells to 6 × 10 7 cells. For comparison data of reference experiments with centrifugation of 5 × 10 7 cells are shown.

Cellular amino acid concentrations were independent from the number of cells on the filter, indicating that residual extracellular components are negligible. High amounts of residual amino acids would lead to significantly higher amino acid concentrations at lower cell counts. Data of other amino acids support these findings further. The average amount of residual amino acid in the filter was calculated to be 0.19 % ± 0.01 % of the total amino acid amount in the cell suspension used with a maximum for glycine of 0.22 % ± 0.04 %. These values are in accordance to those presented by [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF] for direct quenching in methanol with an additional washing step.

Additionally, the slightly increased cell disruption measured when using fast filtration for sampling has no significant effect on the metabolite content. Both, sampling using fast filtration and centrifugation yielded the same results on intracellular amino acid amounts. Potential differences caused by cell disruption seem to be smaller than the error from sampling and measurement.

Fig. 4 shows the results of experiments on leakage of ATP from the cells during sampling. ATP was chosen as a model for larger polar metabolites and to compare results to data from [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF], who use solutions of methanol and substances like ammonium bicarbonate for quenching and washing. No detectable leakage was observed when using an iso-osmotic solution of sodium chloride in water and only minor leakage when using pure water as washing solution. In contrast, [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF] describe ATP leakage of more than 50 % in a washing step. The main reason for this is the disintegration of the cell membrane due to the methanol in the quenching solution, as assessed by [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF].

Intracellular ATP was measured in this study to be about 1.5 fmol cell -1 . While [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] determined the intracellular ATP amount of a recombinant CHO cell line (clone C2.8 SPF) to be 4.6 fmol cell -1 . Differences to this study might be caused by a multitude of reasons including differences in the cell line or culture conditions. A comparison with results to data for CHO cells from [START_REF] Sellick | Effective quenching processes for physiologically valid metabolite profiling of suspension cultured mammalian cells[END_REF] is difficult as they measured intracellular concentrations instead of the cellular amount. Assuming perfectly circular cells of 12 µm diam. the intracellular concentration of 6 mM would translate into an amount of approx. 5.4 fmol cell -1 . All these intracellular ATP amounts are well within the range [START_REF] Cordell | Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry[END_REF] states as normal for CHO cells.

Pyruvate leakage from cells during fast filtration is shown in Fig. 5. Pyruvate was chosen as model for smaller polar metabolites, especially monocarboxylates, which are prone to 'bleed' during sampling. In experiments with water as washing solution' nearly all pyruvate bled from the cells.

When using iso-osmotic NaCl solution for washing about 600 nmol or 50 % of the total pyruvate bled from the cells. The efflux of monocarboxylates, especially lactate, from cells is caused by concentration driven transport of proton-linked monocarboxylate transporters (MCT) rather than by diffusion across the membrane [START_REF] Sharpe | Lactate efflux from sarcolemmal vesicles isolated from rainbow trout oncorhynchus mykiss white muscle is via simple diffusion[END_REF]. Therefore, it can be assumed that the much higher release of pyruvate from the cells compared to LDH release during sampling is caused by transporters and, possibly, ion-channels rather than cell disruption. This is supported by the negligible ATP leakage as there are no ATP transporters in the cell membrane.

Application of fast filtration for metabolome sampling

To evaluate the applicability of metabolome sampling of animal cells using fast filtration samples were drawn from a bioreactor cultivation of CHO DP12 cells (Fig. 6) using different sampling protocols. Samples for metabolome analysis were taken on days three, four, and five using fast filtration and microstructure heat exchanger sampling as well as centrifugation as reference. Fig. 7 illustrates the adenylate energy charge (AEC) measured for each sampling protocol at each sampling point. The AEC was calculated as described by [START_REF] Ataullakhanov | What determines the intracellular atp concentration[END_REF].

The AEC for the samples taken using the fast filtration method is higher for all sampling points. This indicates a higher efficiency of stopping the metabolic activity, as the turnover rates of ATP to ADP and AMP are rather high [START_REF] Ataullakhanov | What determines the intracellular atp concentration[END_REF].

The intracellular glucose concentration for the three sampling points is shown in Fig. 8. The glucose concentration from the samples of day five of the cultivation was below the limit of detection. A higher amount of intracellular glucose is observed for the samples taken by fast filtration. This can be due to a higher quenching efficiency of the fast filtration method or to residual extracellular glucose on the filter. The latter can be ruled out by the fact, that fast filtration samples do not exhibit higher amino acid concentrations than samples taken with the other methods (data not shown).

Large differences were also found for ribose/ribulose 5-phosphate concentrations (Fig. 9). On cultivation day 5 concentrations of up to 0.243 fmol cell -1 were measured in fast filtration extracts and concentrations below 0.038 fmol cell -1 and 0.037 fmol cell -1 were measured in extracts from samples taken using the microstructure heat exchanger and centrifugation, respectively. This strongly supports the higher quenching efficiency of the fast filtration method. By using fast filtration the metabolism of the cells was more efficiently stopped and concentrations of metabolites with high turnover rates were maintained at higher concentrations, whereas the metabolism of cells sampled with the microstructure heat exchanger or just centrifugation was stopped less efficiently. [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] describe considerably lower amounts of extracted metabolites from cells on filters compared to extracts from cell pellets. This was not observed in this study (see also Fig. 10).

Conclusions

Fast filtration is a valuable and reliable tool for metabolome sampling of suspended animal cells.

Even metabolites with high turnover rates were detected in higher concentrations compared to other sampling methods. Furthermore, leakage of metabolites from the cells during sampling is low, as is cell damage.

There is no obvious advantage using the microstructure heat exchanger method in comparison to centrifugation at -2 °C. This is most likely due to the extended period of time in which the cells are handled at about 0 °C, which is not sufficient to completely stop the metabolic activity.

Depending on the nature and the turnover rates of the target metabolites, a simple sampling method like centrifugation might be sufficient to yield some results. But to generate a snapshot of the metabolic state of a cell at any given moment, the fast filtration method presented in this study is a reliable tool superior to other sampling techniques like microstructure heat exchanger or rapid centrifugation.

The cold methanol extraction described in this work is more capable of releasing metabolites from cells on filters than the method used by [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF]. This may be due to the longer exposure of the cells to the extraction reagent (i.e. 24 h as opposed to 10 min).

Furthermore, inert borosilicate glass-fiber filter material was used in this study to prevent interactions of metabolites with the filter material. The composite PFTE and PE filters used by [START_REF] Dietmair | Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol[END_REF] might cause adherence of metabolites to the filter material.

Thus, the presented combination of fast filtration and cold methanol extraction can be considered as well applicable for sampling of intracellular metabolites from suspended animal cells.

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10

 12345678910 Fig. 1 Dependence of LDH release and vacuum applied to the filtration device. Reference experiments with centrifugation of cells at 3000 rcf for 1 min exhibited a release of LDH during sampling of 2.56 % ± 0.66 % compared to the total LDH amount in the sample cell solution. For each cell count three individual experiments were done. LDH measurements were done in triplicates