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29 mars 2011 2 29 /03 /mars /2011 08:42

Nephrol Ther. 2010 Feb;6(1):35-9. Epub 2009 Oct 2.

Traditional medicine in kidney diseases in Burkina Faso.

Lengani ALompo LFGuissou IPNikiema JB.

Service de néphrologie et hémodialyse, CHU Yalgado Ouédraogo, Ouagadougou 01, Burkina Faso. alengani2002@yahoo.fr

Kidney diseases are becoming public health problem worldwide, particularly in Africa. Kidney symptoms are frequent in African population witch have recourse to traditional medicine for primary cares.

For a better knowledge of medicinal plants used for the treatment of kidney symptoms, we conducted a prospective study from January to March 2007 to collect the opinion of traditional healers in the area of three towns (Ouagadougou, Ziniare, Fada NGourma) of Burkina Faso.

Thirty traditional healers were interrogated and results showed that they had limited knowledge on the kidney, urine origin (70% do not know it) and kidney diseases symptoms.

Eighty-three percent of traditional healers found that the origin of kidney symptoms was related to food supplementation.

We identify 61 medicinal plants related to 30 families and 51 genders.

Combretum micranthum, Chrysantellum americanum and Tamarindus indica were the most frequent species plants under use.

This traditional use is justified par bibliographic data on few plants. In conclusion, although traditional healers have limited knowledge on kidney, they continue to provide medicinal plants to treat kidney symptoms.

There is a great need, in Burkina Faso, for studies to define the medicinal plants inventory, their evaluation use, and their effects on the kidneys diseases.

PMID: 19800856 [PubMed - indexed for MEDLINE]

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25 mars 2011 5 25 /03 /mars /2011 19:33



Chronic Fatigue Syndrome (CFS) by the numbers


  • Number of Americans estimated to have CFS: 
  •                                           1 to 4 million

  • Annual productivity lost to the U.S. economy attributable to CFS: 
  •                                           $9.1 billion

  • Annual CFS-related medical costs for an individual with the condition:                                               $3,286

  • Recovery rate:  31% during the first 5 years of illness, 
  • 48% during the first 10 years of illness

  • CFS is most common in women (522 cases per 100,000) and minorities, especially Latinos (726 cases per 100,000)



Source: Centers for Disease Control and Prevention




Diagnostic Criteria for Chronic Fatigue Syndrome

The cause or causes of CFS have not been identified, and no specific diagnostic tests are available for the condition. In order to be diagnosed with chronic fatigue syndrome, a patient must satisfy two criteria:

1. Severe chronic fatigue that lasts for at least six months and cannot be explained by other known medical conditions whose manifestations include fatigue.

2. A patient also must have at least four of the following symptoms: 

* malaise after physical exertion 
* impaired memory or concentration 
* unrefreshing sleep 
* muscle pain 
* multi-joint pain without redness or swelling 
* tender cervical or axillary lymph nodes 
* sore throat 
* headache

The symptoms must have persisted or recurred during six or more consecutive months of illness and must not have predated the fatigue.

Source: Centers for Disease Control and Prevention



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25 mars 2011 5 25 /03 /mars /2011 18:23

Plaidoyer pour la valorisation de la médecine traditionnelle africaine

18 mars 2011

APA-Dakar (Sénégal) Le Président de l’ONG internationale de Promotion des médecines traditionnelles (PROMETRA), le Docteur Erick Gbodossou, a plaidé pour l’inscription dans les cursus scolaires et universitaires de la médecine traditionnelle africaine en vue de la valoriser.


Dans un entretien exclusif avec APA, il a soutenu que le moment est plus qu’opportun de promouvoir la valorisation de la médecine traditionnelle africaine, avant de relever : ‘’on voit que pour un petit rien, les médicaments manquent dans les pharmacies. Alors il ne faut pas exposer les populations à des dangers. Il faut valoriser la médecine traditionnelle en produisant des médicaments locaux pour répondre aux problèmes de santé locaux’’.


Dr Gbodossou, interpellé en marge de la Conférence sur savoirs traditionnels et innovation scientifique organisé jeudi à Dakar par le Groupe des amis de la francophonie, a précisé qu’’’il faut formaliser ce type de médecine en l’inscrivant dans les cycles scolaires et universitaires et en lui traçant un cadre administratif et légal’’.


Selon le président de PROMETRA, ‘’les Etats perdent beaucoup d’argent pour importer des médicaments toxiques, inefficaces et sans effet qui ont d’ailleurs montré leurs limites et lacunes’’.


Soulignant l’urgence de chercher des pistes aptes à de réconcilier les deux formes de médecines, il a déclaré : ‘’il faut des études collaboratives entre médecine moderne et médecine traditionnelle. Cela peut permettre d’avoir des résultats extrêmement importants et positifs pour l’Humanité’’.


A en croire, Dr Erick Gbodossou, 80% de la population au sud du Sahara s’adressent aux guérisseurs traditionnels pour leur santé, mais aussi pour des besoins d’éducation à la santé.


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25 mars 2011 5 25 /03 /mars /2011 18:19

Unlocking Chronic Fatigue Syndrome


As scientists race to find a biological cause for chronic fatigue syndrome, long considered by many doctors to exist in patients' heads, the National Institutes of Health could shed new light on the debate at a major scientific workshop on the disorder.

A Short History of XMRV

Researchers at the University of Utah and elsewhere are working to create diagnostic tests, based partly on proteins or other markers that appear to show up in greater quantities in patients with chronic fatigue syndrome. Diagnosing the disorder is difficult, in part because symptoms vary among patients.

Other scientists are trying to understand why other infections, such as mononucleosis, appear to prompt chronic fatigue syndrome in some patients. And in a program at New York's Columbia University, researchers are seeking to identify pathogens that may appear prominently in patients with the disorder. Researchers will be testing "for all those agents that we know affect vertebrates on this globe," says Mady Hornig, who heads the Columbia program.

Chronic fatigue syndrome affects between one million and four million Americans. They suffer from memory and concentration problems, debilitating pain and severe fatigue. Unable to identify a cause, doctors often dismissed these patients as complainers.

Currently, diagnosing chronic fatigue syndrome is largely a process of elimination. Molly J. Billings, 22 years old, first showed symptoms of the disorder in 2004, including headaches, muscle aches, fatigue and weakness. A year later, she could only get around by wheelchair and was bed-bound most of the time. She endured years of tests to rule out other medical explanations for her condition

"It was horrible to go and not find anything," says Ms. Billings, who lives in Kendall, N.Y. "I want a test that will give me a finite result." Today, her symptoms have shown gradual improvement. She attends classes twice a week at a local community college and is able occasionally to go out with friends.

CFS, also known as ME for myalgic encephalomyelitis, got a boost of attention in 2009 when the journal Science published a study that found the retrovirus XMRV was present in most members of a group of chronic fatigue syndrome patients. The 2009 study divided scientists and led to intense debate about whether the XMRV link is a breakthrough or a result of lab contamination. The study launched a wave of new research.

Anthony Komaroff, a doctor at Harvard Medical School who treats chronic fatigue syndrome patients, was involved in a study that found viruses in the same family as XMRV in his patients. Meanwhile, Brigitte Huber, professor of pathology at Tufts University, was involved in separate work that failed to find XMRV. They are now collaborating on a project that received funding last year to study two viruses in patients with the syndrome


Because symptoms of the syndrome tend to wax and wane, the researchers are investigating whether the viruses may be easier to detect when the symptoms are flaring. Dr. Komaroff is taking blood samples from patients when they are feeling relatively well and when their symptoms are pronounced. Dr. Huber will then analyze the blood to see if she can detect higher amounts of these viruses during times when people feel worse.

Jose G. Montoya, a researcher at Stanford University, is searching for possible infectious agents in chronic fatigue syndrome. "If we can find the infectious triggers, we can provide intervention," he says. Dr. Montoya's team enrolled 30 patients with elevated levels of antibodies against Epstein-Barr virus and HHV-6, a herpes virus, in a trial and treated them with valganciclovir, an anti-viral medicine. Dr. Montoya says patients on the drug showed improvement in cognition and fatigue. Researchers are now analyzing the patients' immune proteins to see if there are patterns that will help doctors figure out in advance who will and won't respond to therapy.

A number of efforts are underway to try to develop diagnostic tests. Researchers at the University of Medicine and Dentistry of New Jersey and Pacific Northwest National Laboratory reported recently that they found proteins in the spinal fluid of people with chronic fatigue syndrome that distinguished them from people with Lyme disease, which has some similar symptoms, and healthy controls. The next step is to narrow down the list of proteins to find "the best biomarkers for what is going wrong in the central nervous system," says Steven E. Schutzer of the University of Medicine and Dentistry of New Jersey who helped lead the study.



Nearly 25 years after the "Lyndonville outbreak" of chronic fatigue syndrome, a controversy is brewing among scientists over what causes the disease. A small-town doctor hopes his patients will help provide the answer. WSJ's Jason Bellini reports.

At the University of Utah, researchers are working on what they hope might ultimately lead to a test for chronic fatigue syndrome. Forty-eight patients with the disorder and healthy controls are involved in a trial in which they undergo a 30-minute exercise challenge. Even after moderate exercise, there were increases in gene expression markers in the blood for two days that allowed researchers to distinguish chronic fatigue syndrome patients from healthy controls.

More than 100 scientists, researchers and advocates are expected to gather at the NIH workshop in Bethesda, Md., attending sessions focused on such medical topics as infectious diseases, systems biology, immunology and neurology. By contrast, the last NIH scientific workshop, in 2003, had more emphasis on the psychological aspects of the disease, including stress, insomnia and depression.

Medical history has other examples of diseases that were not taken seriously but later turned out to have biological causes. Multiple sclerosis was once misdiagnosed as hysteria or chronic alcoholism. Today multiple sclerosis is suspected to be an auto-immune disorder. Stomach ulcers were thought to be caused by stress until two Australian scientists proved the bacteria Helicobacter pylori was the cause, work that won the Nobel Prize in 2005.

"The door has been opened by the retrovirus,'' says Mary Schweitzer, a former history professor who has chronic fatigue syndrome and was tapped to serve on the steering committee planning the NIH conference. "Now we want to bring in all the scientific research that is being done."

Write to Amy Dockser Marcus at amy.marcus@wsj.com


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25 mars 2011 5 25 /03 /mars /2011 17:15

Paris, le vendredi 25 mars 2011 –


Nous connaissons tous le fronton désuet des écoles communales précisant qu’il s’agit d’une « école de filles » ou de « garçons » voir un établissement mixte.

Un collectif de parents d’enfants souffrant d’autisme publie aujourd’hui dans la presse un encart où la célèbre inscription est détournée pour indiquer : « Ecole de garçons et de filles mais pas d’autistes ».

Par ce message choc, ils souhaitent sensibiliser les Français mais surtout les autorités au défaut de scolarisation des enfants souffrant d’autisme : selon eux, ils seraient plus de 80 % à être exclus de l’école.

« Quinze pour cent des enfants autistes seulement ont accès à l’école. Trente pourcent sont accueillis en instituts médico-pédagogiques ou en hôpitaux de jour. Plus de la moitié n’est donc accueillie nulle part » affirme ce collectif.

Si ce groupement est conscient que tous ces jeunes patients ne peuvent être admis en milieu scolaire, il considère qu’au moins la moitié pourrait avoir accès à l’école.

Les freins restent cependant nombreux alors que la scolarisation des enfants handicapés physiques n’a elle cessé de progresser.


Le manque d’assistant de vie scolaire est souvent décrié mais n’apparaît pas comme l’unique difficulté. La réticence des enseignants semble représenter également un obstacle majeur.


Un sondage réalisé par Opinion Way commandé par le collectif révèle ainsi que seuls 18 % des professeurs interrogés jugent que l’école ordinaire est « le meilleur environnement » pour les jeunes autistes, tandis que plus de la moitié redoutent de ne pouvoir enseigner dans de bonnes conditions s’ils accueillaient un enfant autiste.




Publié le 25/03/2011

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25 mars 2011 5 25 /03 /mars /2011 15:31

Congrès UEGW

Des résultats prometteurs de phase II avec l'antibiotique rifaximine dans le traitement de la maladie de Crohn ont été présentés le 27 octobre.

« Une réponse immunitaire altérée de la microflore entérique semble jouer un rôle clef dans le développement et l'entretien de l'inflammation intestinale dans la maladie de Crohn », a indiqué le Pr Herbert en présentant ses résultats au congrès de l’United European Gastroenterology Week (UEGW). « Un nombre limité d'études » a évalué certains antibiotiques dans le traitement de la maladie de Crohn active mais leur efficacité est « controversée », ajoutent les chercheurs.

Selon les résultats de cette étude de phase II menée avec de la rifaximine à libération intestinale prolongée. « Il s'agit du plus large essai montrant l'efficacité d'un antibiotique sur l'induction de la rémission de la maladie de Crohn », estiment les investigateurs. La rifaximine est un antibiotique à large spectre ciblant les voies gastro-intestinales. Déjà homologué aux Etats-Unis pour le traitement de la diarrhée du voyageur et la réduction du risque de récurrence de l'encéphalopathie hépatique manifeste, il a fait l'objet de deux études pivots dans le syndrome de l'intestin irritable avec diarrhée.

L'étude de phase II présentée à l'UEGW a porté sur 402 patients atteints d'une maladie de Crohn, avec un indice d'activité compris entre 220 et 400. Les patients ont reçu pendant 12 semaines soit un placebo soit la nouvelle formulation de rifaximine à raison de 400, 800 ou 1200 mg deux fois par jour. Les traitements tels que l'acide 5-aminosalicylique (5-ASA, mésalazine) ou les immunosuppresseurs pouvaient être poursuivis à des doses stables pendant l'étude. Le taux de rémission clinique, définie par un indice d'activité de moins de 150, était significativement supérieur avec la rifaximine et surtout à la dose de 800 mg qu'avec le placebo. Les effets de la rifaximine à 800 mg ont été maintenus pendant les 12 semaines suivantes de suivi sans traitement.

Virginie BAGOUET

Impact-santé.fr 2010 

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23 mars 2011 3 23 /03 /mars /2011 10:35



Parmi les bactéries à l'origine des arthrites réactionnelles  ; (ARe), Chiamydia trachoinatis est la mieux étudiée

(rappelons  ici que  CHIamydiae pneumoniae, à l'origine d'infections respiratoires, peut aussi être à l'origine d'une ARe). Il est estimé   que, dans au moins 30 % des cas, les ARe ont une évolution  |avec des poussées récidivantes ou une évolution chronique, avec dans certains cas développement d'une authentique SPA [1].



Au cours des dernières années, les techniques de PCR   ; ont permis de montrer que C. frachomatis persiste de manière  viable au sein des articulations. Dans une étude effectuée chez  |26 patients ayant une Spa (critères de l'ESSG) indifférenciée évoluant depuis au moins 6 mois, la recherche d'ADN de C. frachomafis et de C. pneumoniae dans le tissu de biop sies synoviales était positive dans 62 % des cas (10 fois pour C. frachomafis, 4 fois pour C. pneumoniae, 2 fois pour les deux) [2]. Cette recherche était positive chez 12 % de 167 sujets témoins atteints d'arthrose. Des travaux expérimentaux ont montré que Chiamydia peut survivre (bactérie viable mais non cultivable) longtemps au sein d'articulations inflamma toires, dans une forme intracellulaire aberrante caractérisée par une modification de l'expression de ses gènes [3]. On a mis en évidence différents mécanismes par lesquelles Chiamydia pouvait échapper aux défenses immunitaires de l'hôte, par exemple en inhibant l'apoptose des cellules infectées. Des données expérimentales in vitro montrent que, contrairement  à une monothérapie, une association d'antibiotiques comportant de la rifampicine (azithromycine et rifampicine) permet de faire disparaître les formes viables de Chiamydia.

Carter et al. [3] ont effectué un essai thérapeutique randomisé, en double insu, de deux associations d'antibiotiques contre placebo chez 42 patients ayant une Spa, répondant aux critères de l'ESSG et évoluant depuis au moins 6 mois, et chez lesquels les techniques de PCR avaient mis en évi dence de l'ADN de C. trachomafis ou de C. pneumoniae dans le tissu synovial ou dans les cellules mononucléées du sang périphérique. L'ancienneté moyenne de la Spa dépassait 10 ans. Le traitement était donné 6 mois et l'étude durait 9 mois.

Les deux bras supposés actifs étaient : association rifampicine 300 mg/j et doxycycline 100 mg/j ; association  rifampicine 300 mg/j et azithromycine (500 mg/j pendant 5 jours, puis 500 mg deux fois par semaine).

L'évaluation était faite avec un index composite et le critère principal d'évaluation était l'amélioration d'au moins 20 % de 4/6 paramètres à 6 mois.

Ceci a été obtenu chez 63 % traités par une association d'antibiotique (17/27) et 20 % des patients traités par placebo (3/15) (p = 0,01).

Pour une amélioration d'au moins 50 % de 4/6 critères, le taux de réponse était de 41 % sous antibiotiques et de 7% sous placebo.

Les recherches d'ADN de Chiamydia par PCR à 6 mois étaient devenues plus souvent négatives sous antibiotiques que sous placebo.


Cette étude montre que l'efficacité possible d'une association d'antibiotiques comportant de la rifampicine, aussi bien sur la persistance de Chiamydia que sur les signes cliniques de la Spa associée.

Si les résultats de cette étude nécessiteraient d'être confirmés, ils représentent une étape particulièreme  encourageante vers une guérison potentielle.



 1. Rihl M, Kuipers JG, Kôhler L et al. Combination antibiotics for ChIamydia-Induced arthritis: breakthrough to a cure? Arthritis Rheum 2010 ; 62:1203-7.

 2. Carter JD, Gérard HC, Espinoza LR et al. Chiamidiae as etiologic agents in chronic undifferentiated spondyloarthritis, Arthritis Rheum 2009 ; 60:1311-6.

3. Carter JD, Espinoza LR, Inman RD et al. Combination antibiotics as a treatment for chronic ChIamydia-Induced Reactive Arthritis. Arhritis Rheum 2010; 62:1298-307.                                                                                                                              '

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23 mars 2011 3 23 /03 /mars /2011 10:10


Symptômes datant de plus de 3 mois



(parmi les 19 suivants) . Les symptômes soulignés sont les plus spécifiques


A :                         CRITERES MUSCULAIRES (5) : très évocateurs 

1-             Crampes nocturnes ou au repos

2-             Myoclonies ou sursauts d’endormissement (impression de tomber)

3-             « Impatience » des jambes, diurne, en position assise

4-             Fasciculation d’une paupière (clonies)

5-             Sensation de manquer d’air (besoin de souffler ou d’inspirer profondément)


B :                         CRITERES CASCULAIRES (9)

6-             Ecchymoses spontanées ou au moindre choc

7-             Sueurs excessives (notamment nocturnes) ou flushs ou rashs du visage ou du torse

8-             Troubles visuels atypiques intermittents (flou visuel, ombres visuelles latérales fugaces, pseudo hallucinations, phosphènes, traits, photophobie excessive)

9-             Palpitations

10-         Sensations lipothymiques (positionnelles)

11-         Extrémités froides ou syndrome de Raynaud ou frilosité générale.

12-         Dysesthésies des extrémités (positionnelles ou non)

13-         Acouphènes

14-         Jambes lourdes vespérales


C :                         CRITERES « IRRITATIFS » (5)

15-         Prurit cutané (même localisé)

16-         Arthralgies ou myalgies (fugaces, mobiles), ou tendinites multiples

17-         Irritations oculaires (paupière inférieure, «yeux secs »)

18-         Irritation pharyngée ou rhinite per-annuelle non allergique

19-         Gastralgies ou dyspepsie ou trouble du transit ou troubles fonctionnels intestinaux.


II.                        FATIGUE

                        (non réactionnelle à un vécu psychique conflictuel douloureux)


Elle peut être soit

a-             Physique : « coups de fatigue » (notamment post prandiaux ), besoin de siestes, fatigue sportive

b-             Psychique : Anxiété généralisée, perte d’élan vital , ou syndrome dépressif.

c-              Intellectuelle : difficultés de concentration (« brain fog ») , ralentissement de l’idéation, baisse des performances scolaires, troubles de l’attention et de la mémoire.

d-             Troubles du sommeil : réveils nocturnes, sommeil non réparateur, sueurs nocturnes et somnolence diurne.


Dr Ph. Raymond

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16 mars 2011 3 16 /03 /mars /2011 15:22

Journal of Bacteriology, February 2011, p. 1018-1020, Vol. 193, No. 4
0021-9193/11/$12.00+0     doi:10.1128/JB.01158-10
Copyright © 2011American Society for Microbiology. All Rights Reserved.


Whole-Genome Sequences of Thirteen Isolates ofBorrelia burgdorferi{triangledown},{dagger}

Steven E. Schutzer,1* Claire M. Fraser-Liggett,2 Sherwood R. Casjens,3* Wei-Gang Qiu,4John J. Dunn,5 Emmanuel F. Mongodin,2 and Benjamin J. Luft6

Department of Medicine, University of Medicine and Dentistry of New Jersey—New Jersey Medical School, Newark, New Jersey 07103,1Institute for Genome Sciences, University of Maryland, School of Medicine, Department of Microbiology and Immunology, Baltimore, Maryland 21201,2Department of Pathology, Division of Microbiology and Immunology, University of Utah Medical School, Salt Lake City, Utah 84112,3Department of Biological Sciences, Hunter College of the City University of New York, New York, New York 10021,4Biology Department, Brookhaven National Laboratory, Upton, New York 11793,5Department of Medicine, Health Science Center, Stony Brook University, Stony Brook, New York 117946

Received 28 September 2010/ Accepted 6 October 2010


Borrelia burgdorferiis a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence ofB. burgdorferistrain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation anddisease phenotype, we determined the whole-genome sequences of 13 additionalB. burgdorferiisolates that span the range of natural variation. These sequences should allow improvedunderstanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.

Lyme disease is the most frequent tick-borne disease in North America and Europe (3,16,17). There are multiple variants ofB. burgdorferi(1,7,15,20,21), the causative agent, but questions remain about how their variation correlates with different clinical manifestations. Whole-genome sequencing (WGS) can orient approaches to diagnostics and vaccines and help avoid potential host cross-reactivity. Improved diagnostics are needed because the best clinical sign, the erythema migrans skin rash, does not always occur. Diagnostic assays and vaccines (18) have been less than satisfactory. However, these were developed before WGS of microbes and the human genome. This project was stimulated by the initial finding of genotypes ofB. burgdorferiassociated with invasiveness/dissemination (15). This has been substantiated (7,21).

The sequencing of strain B31 (6,8) has accelerated progress in Lyme disease research. We sequenced 13 additional isolates, chosen to cover a large fraction of the genetic and geographic diversity and obtained from humans and other natural hosts (Table 1).


View this table:
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TABLE 1. B. burgdorferi isolates used in this study


These genomes were sequenced by the random shotgun method as described previously, using Sanger DNA sequencing to an estimated8-fold coverage (12). Approximately 10,000 and 6,000 successful reads for the small and medium insert plasmid libraries, respectively,were sequenced, representing a total of about 14 Mbp of sequencing data for each. All plasmids were sequenced to closure unless noted otherwise (see Table S1 in the supplemental material). Genome annotation was performed using the JCVI Prokaryotic AnnotationPipeline (www.jcvi.org/cms/research/projects/prokaryotic-annotation-pipeline/overview/).

The B31 sequence showed thatB. burgdorferihas many more replicons (DNA molecules) than other bacteria. Besides its 910-kbp linearchromosome, strain B31 has been shown to have 12 linear and 10 circular plasmids (5), expanding observations (2,10) indicating thatBorreliabacteria universally harbor numerous plasmids, many essential for survival of the bacteria in mice and/or ticks (4). The newly sequenced genomes contain a total of 17,084,900 bp, averaging 1,314,223 bp/genome. Each strain carried between 13 and 21 plasmids (239 plasmids were sequenced, about half predicted to be linear replicons). At least 9 new plasmid types not in B31 were identified. Many plasmids underwent substantial rearrangements in different lineages. The linear chromosomes are very stable, with little variation among isolates. With the exception of a few differences at their right ends, the gene content of the chromosomes is essentially identical. Contrary to previous assumptions that genetic changes occurred only by slower point mutations, our initial WGS comparison of 4 strains showed that closely relatedB. burgdorferistrains frequently and more rapidly than by point mutation undergo horizontal exchange of genetic information (14). Evidence of this is also found in the newer genomes sequenced in this work.

The genetic diversity ofB. burgdorferiappears to be maintained in part by neutral and adaptive processes, such as resistance to host immune defense mechanisms and host preferences (4,9). Key questions remain on the genomic basis of these intra- and interspecific variations, particularly those associated with host resistance, high-frequency proliferation in wildlife populations, and invasiveness in humans.

Our long-range objectives are to develop a pangenomic picture ofB. burgdorferidiversity (13) and to understand how the variationsinfluence pathogenicity. We believe solutions for many of the problems associated with Lyme disease will come from scientificinformation, beginning with comparative genomics of this organism. Sequencing is a superb discovery tool whose greatest impact is realized when additional biology can implemented. Information from WGS of these well-characterized strains should provide a foundation for new hypotheses on the pathogenesis of Lyme disease and rational diagnostics and vaccines.

Nucleotide sequence accession numbers.

These sequences have been deposited in GenBank, and their Genome Project ID numbers and accession numbers are listed in Table 1and in Table S1 in the supplemental material, respectively.


This research was supported by the following grants from the National Institutes of Health: AI49003, AI37256, AI30071, GM083722, and RR03037. Additional funding was provided by the Lyme Disease Association and the Tami Fund.


* Corresponding author. Mailing address for Steven E. Schutzer: Department of Medicine, University of Medicine and Dentistry of New Jersey—New Jersey Medical School, Newark, NJ 07103. E-mail: schutzer@umdnj.edu. Mailing address for Sherwood R. Casjens: Department of Pathology, University of Utah Medical School, Room 2200 EEJMRB, 15 North Medical Dr. East, Salt Lake City, UT 84112. E-mail: sherwood.casjens@path.utah.edu Back


{triangledown}Published ahead of print on 8 October 2010. Back

{dagger}Supplemental material for this article may be found athttp://jb.asm.org/. Back


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Journal of Bacteriology, February 2011, p. 1018-1020, Vol. 193, No. 4
0021-9193/11/$12.00+0     doi:10.1128/JB.01158-10
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  • Banik, S., Terekhova, D., Iyer, R., Pappas, C. J., Caimano, M. J., Radolf, J. D., Schwartz, I. (2011). BB0844, an RpoS-Regulated Protein, Is Dispensable for Borrelia burgdorferi Infectivity and Maintenance in the Mouse-Tick Infectious Cycle.Infect. Immun.79: 1208-1217[Abstract] [Full Text]  
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16 mars 2011 3 16 /03 /mars /2011 15:18

Distinct Cerebrospinal Fluid Proteomes Differentiate Post-Treatment Lyme Disease from Chronic Fatigue Syndrome.

1 Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America, 2 Department of Neurology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America, 3 Division of Biostatistics and Epidemiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey, United States of America, 4 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America, 5 Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden, 6 Department of Neurology, State University of New York-Stony Brook, Stony Brook, New York, United States of America, 7 Department of Psychiatry, Columbia University Medical Center, New York, New York, United States of America, 8 Department of Pain Medicine and Palliative Care and Beth Israel Medical Center, Albert Einstein School of Medicine, Bronx, New York, United States of America

 Steven E. Schutzer1#*, Thomas E. Angel4#, Tao Liu4#,Athena A. Schepmoes4, Therese R. Clauss4, Joshua N. Adkins4, David G. Camp II4, Bart K. Holland3, Jonas Bergquist5, Patricia K. Coyle6, Richard D. Smith4, Brian A. Fallon7, Benjamin H. Natelson2,8


Neurologic Post Treatment Lyme disease (nPTLS) and Chronic Fatigue (CFS) are syndromes of unknown etiology. They share features of fatigue and cognitive dysfunction, making it difficult to differentiate them. Unresolved is whether nPTLS is a subset of CFS.

Methods and Principal Findings

Pooled cerebrospinal fluid (CSF) samples from nPTLS patientsCFS patients, and healthy volunteers were comprehensively analyzed using high-resolution mass spectrometry (MS), coupled with immunoaffinity depletion methods to reduce protein-masking by abundant proteins. Individual patient and healthy control CSF samples were analyzed directly employing a MS-based label-free quantitative proteomics approach. We found that both groups, and individuals within the groups, could be distinguished from each other and normals based on their specific CSF proteins (p<0.01). CFS (n = 43) had 2,783 non-redundant proteins, nPTLS (n = 25) contained 2,768 proteins, and healthy normals had 2,630 proteins. Preliminary pathway analysis demonstrated that the data could be useful for hypothesis generation on the pathogenetic mechanisms underlying these two related syndromes.


nPTLS and CFS have distinguishing CSF protein complements. Each condition has a number of CSF proteins that can be useful in providing candidates for future validation studies and insights on the respective mechanisms of pathogenesis. Distinguishing nPTLS and CFS permits more focused study of each condition, and can lead to novel diagnostics and therapeutic interventions.

Introduction Top

Prime objectives in studying neurologic and psychiatric disorders are to develop discriminating markers and generate data that can provide insight into disease pathogenesis. This can lead to novel treatment strategies. Chronic Fatigue Syndrome (CFS) and Lyme disease, particularly Neurologic Post Treatment Lyme disease syndrome (nPTLS), represent two conditions that share common symptoms of fatigue and cognitive dysfunction [1][7]. Despite extensive research CFS and nPTLS remain medically unexplained. There are no biological markers to distinguish these syndromes, creating diagnostic dilemmas and impeding research into understanding each individual syndrome.

Cerebrospinal fluid (CSF) is an ideal body fluid to examine for signature protein profiles informative for diagnosis or etiology of central nervous system (CNS)-related symptoms and dysfunction. Not only is the CSF an accessible liquid extension of the brain, but recent data suggests CSF may provide more relevant data than brain parenchyma itself in certain neurologic diseases [8]. Specific abnormalities found in CSF relating to CFS and nPTLS would suggest CNS involvement, and could facilitate their mechanistic understanding.

Liquid chromatography coupled to mass spectrometry (LC-MS) is becoming the method of choice for examining complex biological specimens, that contain hundreds to thousands of proteins [9], such as CSF[10]. This is particularly the case in the initial discovery phase. This discovery phase may be viewed as casting a wide net to maximize identification of as many proteins as possible in a sample. This initial list of identified proteins has value by itself for qualitative or semi-quantitative comparisons between diseases. Recent studies demonstrated the reliability and reproducibility of LC-MS results, with different mass spectrometers across different laboratories, when performed by experienced individuals [9][11]. In a discovery phase investigation, the MS technique is unbiased and does not require prior knowledge of what proteins may be in a sample. This is in contrast to subsequent validation studies where targeted approaches are used and which do require knowledge of target proteins. In searching for a disease biomarker, the discovery phase should provide a list of proteins and serve as a precursor phase for targeted approaches. These subsequent targeted approaches, whether they use other MS techniques or are immuno-based, are designed to validate the use of the biomarker protein(s) to distinguish one disease from another.

In practice tailored strategies are often needed to achieve a balance between ideal and real world constraints – especially where sample volumes and numbers are limited such as with CSF. In an ideal situation it is desirable to have numerous samples from individuals with a particular disease. It is further desirable to have sufficient total protein content in each sample so that a variety of protein separation and fractionation methods can be used prior to MS analysis. This will minimize abundant proteins from masking the detection of less abundant ones, and will permit full qualitative and quantitative analyses. Limited sample numbers and quantities do not preclude employment of tailored strategies to get meaningful results. It should be remembered that in the example of a biomarker search, the protein(s) will be confirmed or dismissed in future targeted validation studies, but failure to identify them in the broad discovery list would preclude them from examination for validation.

Until recently, technical hurdles impeded the use of CSF to distinguish conditions such as CFS and nPTLS. Advances in sample preparation, separations and MS platform capabilities enabled us to recently establish a comprehensive reference normal CSF proteome [10]. This provides the basis for comparative proteome analyses with other diseases, which should provide greater insight into their underlying pathogenesis.

To address the possibility that CFS and nPTLS could be distinguished from one another and healthy subjects, we searched for distinguishing protein marker profiles by applying our advanced proteomics strategy [10] to characterize the CSF proteomes from well described CFS and nPTLS patients (detailed in Methods). We performed comparative whole CSF proteome analyses between CFSnPTLS, and healthy normal controls, and complemented these findings with label-free quantitative analysis of individual subject samples. In addition, we performed a preliminary pathway analysis [12] using these data, to examine the feasibility of this type of tool for future investigations to probe for clues to the pathogenetic mechanisms behind these diseases.

Materials and Methods Top

Ethics Statement

Approval for the conduct of this study was obtained from the Institutional Review Board of New Jersey Medical School and the Institutional Review Board of Pacific Northwest National Laboratory (Exempt status and consent not required, using previously banked de-identified samples in accordance with federal regulations).

Overview and Rationale

We performed analysis of pooled CSF samples allowing for a broad and deep view as well as qualitative comparison of each disease-related and control CSF proteome. To determine if these two syndromes could be quantitatively differentiated we performed a label-free quantitative analysis of protein abundances for individual subject CSF samples. Pooling samples provided sufficient protein mass for effective downstream proteomics analysis following immunoaffinity depletion of the 14 most abundant proteins present (representing approximately 95% of the total protein mass in CSF), reducing the dynamic range of protein concentrations present in CSF, where proteins with highest concentrations mask proteins at lower concentrations from detection. Coupling immunoaffinity depletion with strong cation exchange (SCX) fractionation further reduces sample complexity, and allowed for the in-depth analysis of the CSF proteomes. These comprehensive CSF proteomics datasets were then used to create an accurate mass and time (AMT) tag database for subsequent label-free quantitative analysis of individual subject CSF samples. Due to the limit in sample volume, the CSF samples used in individual LC-MS analyses were not immunoaffinity depleted and fractionated, and therefore had much lower proteome coverage compared to the pooled samples. Nevertheless, the label-free quantitative analysis of single subject samples provided a means for statistical evaluation of the quantified protein abundances for many subjects suffering from CFS and nPTLSas well as normal healthy volunteers. Together these analyses represent the discovery phase of our studies on CFS and nPTLS, generating targets for follow up verification and validation in the later stages of the biomarker discovery workflow [13].

Cerebrospinal Fluid (CSF) specimens

CFS Subjects.

Both pooled and individuals CSF samples were analyzed. Equal aliquots from individual CSF samples were pooled to provide sufficient volume for extensive fractionation and two-dimensional LC coupled to tandem MS (2D-LC-MS/MS) analysis with immunoaffinity depletion from 30 women and 13 men (n = 43) who fulfilled the 1994 case definition for CFS [1]. All subjects were 18–54 years old (median = 43) and underwent a careful history and physical examination by an expert experienced in evaluating patients with medically unexplained fatigue and pain. Patients had blood tests to rule out common causes of severe fatigue such as anemia, liver disease, hypothyroidism, systemic lupus erythematosus, and Lyme disease[14]. All subjects then underwent a psychiatric diagnostic interview designed to identify major psychiatric diagnoses for exclusion in this study. Eleven of the patients were not taking medicines. Subjects then underwent lumbar puncture. CSF was sent to the laboratory for white blood cell (wbc) count and total protein [10]. A majority of CFS patients had normal CSF protein and cell counts (protein less than 45 mg/dl and wbc less than or equal to 5/mm3). Ten of the patients had increased protein values ranging from 46–93 mg/dl (with a median of 59 mg/dl) and 3 patients had minimally elevated wbc counts of 6, 7, and 9 respectively. Individual CSF samples from 14 of the 43 CFS subjects (aged 33–48 years with a median age of 43 years, 7 female and 7 male) were also used in direct LC-MS analysis (i.e., no MS/MS was performed) without immunoaffinity depletion. Twelve of the 14 patients had normal CSF protein levels and all had normal cell counts. All subjects provided written informed consent approved by the Institutional Review Board.

nPTLS Subjects.

Both pooled and individuals CSF samples were analyzed. Equal aliquots from individual CSF samples were pooled to provide sufficient volume for extensive fractionation and 2D-LC-MS/MS analysis with immunoaffinity depletion from 15 females and 10 males (n = 25) with nPTLS. All were documented to have had prior Lyme disease which met CDC surveillance case definition criteria [15], persistent neurologic features, including cognitive impairment and fatigue, despite appropriate antibiotic treatment [16][17]. Subjects were 17–64 years old (median = 48). All were seropositive for antibodies to B. burgdorferi (the etiologic agent of Lyme disease). Patients, enrolled in an NIH funded study, met the following criteria [17]: (1) current positive IgG Western blot using CDC surveillance criteria assessed using a single reference laboratory (University Hospital of Stony Brook); (2) treatment for Lyme disease with at least 3 weeks of intravenous ceftriaxone or cefotaxime that was completed at least 4 months before study entry; and (3) objective evidence of memory impairment as documented by the Wechsler Memory Scale-III compared to age-, sex-and education-adjusted population norms. nPTLS subjects were excluded if history or testing revealed a medical condition that could cause cognitive impairment or confound neuropsychological assessment (e.g., neurological disease, autoimmune disease, unstable thyroid disease, learning disability, substance abuse, B12 deficiency). Patients with cephalosporin allergy or a history of significant psychiatric disorder prior to onset of Lyme disease were also excluded. All patients had a comprehensive battery of neurocognitive testing and a full-physical exam with detailed rheumatologic and neurologic assessments.nPTLS patients then had a lumbar puncture and CSF was evaluated for cell count, total protein, glucose, total gammaglobulin, oligoclonal bands and evidence of B. burgdorferi (ELISA, Bb DNA by PCR, and culture using BSKII medium). None had evidence of another active tick-borne disease. A majority of nPTLS patients included in the pooled sample had normal CSF protein and cell counts (protein less than 45 mg/dl and wbc less than or equal to 5/mm3), except for 3 patients who had elevated protein values of 58, 69, and 71 mg/dl respectively and 1 patient with elevated wbc count of 6. Individual CSF samples from a group of 14 of the 25 nPTLS subjects (aged 25–58 years with a median age of 48 years, 6 female and 8 male) were also used in direct LC-MS analysis without immunoaffinity depletion. Two of the 14 patients had increased CSF protein levels of 69 and 71 mg/dl and 1 had a slightly elevated wbc of 6. All subjects provided written informed consent approved by the Institutional Review Board.

Normal Controls.

We used the 2D-LC-MS/MS data obtained previously from pooled CSF of 11 healthy control subjects [10]. Briefly, there were 8 women and 3 men, aged 24–55 years with a median age of 28 years. Individual CSF samples from another set of 10 healthy volunteers, age 37–44 years (median = 40) and 5 women and 5 men, were analyzed by LC-MS analysis without immunoaffinity depletion.

Immunoaffinity depletion of 14 high abundance CSF proteins

We had previously shown that this technique could increase our protein identification yield by 70% [10]. Pooled CSF samples from CFS or nPTLS patients (total volume of 18 mL each), were fractionated using a 12.7×79.0 mm Seppro® IgY14 LC10 affinity LC column (Sigma, St Louis, MO) as previously described [18]. Pooling was done to compensate for lack of sufficient volume (and consequent protein content) available for immunoaffinity depletion of individual patient samples. Both the flow-through (lower abundance proteins) and bound fractions from both pooled CSF samples were collected and processed identically until LC-MS/MS analysis. These analyses resulted in an in-depth characterization of the CSF proteome and the combined results of abundant protein and less abundant protein fractions allowed the creation of an AMT tag database[19] for high-throughput analysis of a larger number of individual subject samples using LC-MS.

Protein digestion

CSF proteins (from the immunoaffinity depletion processed pooled samples and the individual samples without immunoaffinity depletion processing) were digested with trypsin and cleaned up with SPE C18 columns as previously described [10]. Final peptide concentration was determined by BCA assay (Pierce, Rockford, IL). All tryptic digests were snap frozen in liquid nitrogen and stored at −80°C until further processing and analysis.

Strong cation exchange (SCX) fractionation

A total of 300 µg of tryptic peptides from both the IgY14 bound and flow-through fractions from the pooledCFS and nPTLS CSF samples were fractionated by SCX chromatography as described [20]. Thirty SCX fractions were collected for each sample and 20% of each fraction was injected for reversed-phase LC-MS/MS analysis.

Reversed-phase capillary LC-MS/MS for CSF pooled fraction analysis

SCX fractions of the IgY14 bound fraction samples were analyzed on an LTQ (ThermoFisher, San Jose, CA) linear ion trap, and SCX fractions of the IgY14 flow-through fraction samples were analyzed on an LTQ-Orbitrap Velos (ThermoFisher) instrument, operated in data-dependent mode with the same LC conditions as previously described [10].

Reversed-phase capillary LC-MS for label-free quantification of unfractionated CSF samples

For label-free quantification analyzing unfractionated CSF samples (individual patient samples with insufficient volume (protein content) for immunoaffinity depletion and SCX fractionation), the LTQ-Orbitrap Velos mass spectrometer was operated in the data-dependent mode with full scan MS spectra (m/z 400–2000) acquired in the LTQ-Orbitrap Velos with resolution of 60,000 at m/z 400 (accumulation target: 1,000,000). MS/MS data acquired here were not used for the quantitative analysis.

Data analysis

The LTQ raw data from the pooled samples was extracted using Extract_MSn (version 3.0; ThermoFisher) and analyzed with the SEQUEST algorithm (V27 revision 12; ThermoFisher) searching the MS/MS data against the human IPI database (Version 3.40). Mass tolerances of 3 Daltons for precursor ions and 1 Dalton for fragment ions without an enzyme defined, as well as static carboxyamidomethylation of cysteine and dynamic oxidation of methionine were used for the database search. The LTQ-Orbitrap Velos MS/MS data were first processed by in-house software DeconMSn [21] accurately determining the monoisotopic mass and charge state of parent ions, followed by SEQUEST search against the IPI database in the same fashion as described above, with the exception that a 0.1-Dalton mass tolerance for precursor ions and 1-Dalton mass tolerance for fragment ions were used. Data filtering criteria based on the cross correlation score (Xcorr) and delta correlation (ΔCn) values along with tryptic cleavage and charge states were developed using the decoy database approach and applied for filtering the raw data to limit false positive identifications to <1% at the peptide level [22][24]. For the LTQ-Orbitrap Velos data, the distribution of mass deviation (from the theoretical masses) was first determined as having a standard deviation (σ) of 2.05 part per million (ppm), and a mass error of smaller than 3σ was used in combination with Xcorr and ΔCn to determine the filtering criteria that resulted in <1% false positive peptide identifications.

The AMT tag strategy [19] was used for label-free quantification of MS features observed in the LTQ-Orbitrap Velos analysis of the individual CSF samples from normal, CFS and nPTLS conditions. The filtered MS/MS peptide identifications obtained from the 2D-LC-MS/MS analyses of all pooled CSF samples were included in an AMT tag database with their theoretical mass and normalized elution time (NET; from 0 to 1) recorded. LC-MS datasets were then analyzed by in-house software VIPER [25] that detects features in mass–NET space and assigned them to peptides in the AMT tag database [26]. The data was further filtered by requiring that all peptides must be detected in at least 30% of the datasets in each of the three conditions. The false discovery rate of the AMT tag analysis was estimated using an 11-Da shift strategy as previously described [27]. A false positive rate of <4% was estimated for each of the LC-MS data sets. The resulting lists of peptides from 2D-LC-MS/MS or direct LC-MS analysis were further processed by ProteinProphet software [28] to remove redundancy in protein identification.

Data normalization and quantification of the changes in protein abundance between the normal, CFS andnPTLS CSF samples were performed and visualized using in-house software DAnTE [29]. Briefly, peptide intensities from the LC-MS analyses of the individual samples (volume limited) were log2 transformed and normalized using a mean central tendency procedure. Peptide abundances from the individual samples were then “rolled up” to the protein level employing the R-rollup method (based on trends at peptide level) implemented in DAnTE. ANOVA, principal component analysis (PCA) and clustering analyses were also performed using DAnTE.

Pathway Analysis of the data was performed with Ingenuity Pathways Analysis (Ingenuity Systems,www.ingenuity.com). Canonical pathway analysis identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the CFS and nPTLS proteins identified. The significance of the associations were assessed with the Fisher's exact test.

Results Top

We first performed pooled sample analysis, then individual sample analysis, and then pathway analysis using the observed proteins. These analyses represent a discovery phase of our studies on CFS and nPTLS, generating targets which can be followed up in future verification and validation stages studies [13].

Proteomic analysis of pooled CSF samples

In the pooled analysis, we examined individual sets of CSF samples from CFS patients (n = 43) and nPTLSpatients (n = 25), respectively. We used the proteomic strategy described in Methods to assure that the maximum number of proteins would be analyzed and the more abundant proteins did not obscure the less abundant ones having biomarker potential. The bound fraction of abundant proteins from the immunoaffinity depleted flow through fraction was analyzed separately and included in the subsequent analysis. Combining immunoaffinity-based partitioning, SCX fractionation and LC-MS/MS, we identified approximately 30,000 peptides for each pooled sample corresponding to 2,783 nonredundant proteins inCFS patient samples and 2,768 proteins in nPTLS patient samples, compared to the 2,630 proteins present in the CSF of healthy normal control subjects. These can be graphically seen in Figure 1 which shows the number of proteins identified solely in each group, and shared or not shared between the groups (see Table S1). Figure 1 also shows that the nPTLS and CFS groups shared significantly more proteins (n = 305) than each disease group shared with healthy controls (n's = 135 and 166, respectively). (Note that, as with any assay, when we indicate that a protein was “not found” or “not identified” that is defined as within the limits of detection).


Figure 1. Characterization of the proteome from pooled and individual CSF samples.

A) Venn diagram of the qualitative distribution of proteins identified in the pooled, immunodepleted, and fractionated cerebrospinal fluid (CSF) from normal healthy control subjects, Chronic Fatigue Syndrome (CFS), and Neurologic Post Treatment Lyme Syndrome (nPTLS). The numbers of proteins for each of these three categories separately is shown outside the circles below the category (2,630 for true normal controls, 2,783 forCFS, and 2,768 for nPTLS). The subsets of intersections between these categories are shown within the circles. There were 1) 738 proteins that were identified in CFS, but not in either healthy normal controls or nPTLS; 2) 1,582 proteins that were not identified in CFS, but were in either nPTLS disease or healthy normal controls; 3) 692 proteins that were identified in the nPTLS patients, but not in healthy normal controls or CFS; and 4) 1,597 proteins that were not identified in nPTLS, but were identified in either healthy normal controls or CFS. This figure also shows that the nPTLS and CFS groups shared significantly more proteins (n = 305) than each disease group shared with controls (n's = 135 and 166). The specific lists of these subsets are presented in additional Table S1.


Proteomic analysis of individual CSF samples

Quantitative analyses were performed on individual CSF samples from 14 CFS patients and 14 nPTLSpatients. They were compared to 10 normal healthy volunteers (samples chosen at random) to provide insights on the variation among individuals within and between different groups. Limited volumes of the individual samples reduced the sample preparation options (i.e., immunoaffinity depletion and SCX fractionation), and hence resulted in less depth of proteome coverage than possible with the pooled samples, where approximately 20 ml were available for depletion and fractionation. Nevertheless, we identified 4,522 peptides across all individual samples, representative of 474 non-redundant proteins identified and quantified in the individual sample analysis (Table S2).

Unsupervised hierarchical clustering and PCA were employed to determine if the observed quantitative differences in protein abundances were sufficient to distinguish these two patient groups (this was de factoblinded – as samples were run in a random order and uncoded as to disease group afterwards). The proteins considered in the unsupervised hierarchical clustering analysis were quantified in individual samples and found to be significantly different in abundance by analysis of variance (ANOVA p ≤ 0.01, Table S3); while PCA analysis considered all proteins quantified in each individual sample. The CSF proteome of the two disease states were markedly different from each other (Fig. 2A and B). Individual patients also showed consistent patterns of protein abundances discriminating CFS from nPTLS (Fig. 2A). These results demonstrated that it is unlikely that any single subject's CSF sample in the pooled analysis contributed disproportionately to the differential proteome distributions observed between the disease groups. Moreover, the individual analyses also highlighted the potential for diagnostic marker confirmation upon extension to larger sample sets in validation studies.


Figure 2. Comparative analysis of individual CFS and nPTLS CSF proteomes.

A) Unsupervised hierarchical clustering of 59 proteins (see Table S3) that are differentially abundant as determined by ANOVA (p<0.01) clearly separates these two disease states with the exception of onenPTLS sample clustering with CFS patient samples. B) Principal Component Analysis (PCA) of CFS and nPTLS samples demonstrates that the CSF proteomes, and by extension of the CNS status, differ between CFS and nPTLS.


Illustrative pathway analyses of protein results from CSF samples

We utilized pathway analysis as an exploratory tool to assess the value of our data, beyond distinguishing the two syndromes from each other, to see if the data was amenable to analysis that would help generate hypotheses of pathogenesis. We chose representative pathways to analyze for illustration based in part on their quantitative ranking (Table S4) and in part by the potential relevance of the pathway involved. Even this limited investigation demonstrated that there is a wealth of proteome information that can be leveraged for hypotheses generation.

Example of proteins in common and elevated in abundance in the two disease conditions, compared to normal, but at different levels.

An illustration, where the same proteins are elevated in abundance in both conditions, but at different magnitudes, is provided by inspection of proteins in the complement system. This is of interest because both syndromes may be triggered by infections (nPTLS in all cases by B. burgdorferi; many CFS cases by one or more microbes yet to be identified). We found that the complement cascade related proteins were identified and significantly enriched in both CFS and nPTLS pooled CSF proteomes by the Fisher Exact test (p = 0.005) implemented in Ingenuity Pathways Analysis (Figure S1A). In individual patient samples analyzed, we identified and quantified 4 components (C1S, C4B, C1QB, C1QC) which are seen with activation of the complement cascade and which were differentially increased in abundance consistently across the nPTLSpatients compared to CFS (Figure S1B and C). This represents the type of data that can be useful in the formulation of pathogenetic hypotheses because the role of complement in these disorders is under-explored.

Example of proteins solely identified in one condition.

Analysis of the highly fractionated pooled patient samples led to the identification of proteins solely identified in each of the disease states. To investigate if these disease specific proteins have common annotated functional properties, we performed pathway analysis (Tables S5 and S6). As an example, the CDK5 signaling pathway, was found to be significantly enriched (p = 0.00009) for proteins identified only in the pooled CFS proteome. This signaling pathway has been linked to Parkinson's [30] and Alzheimer's diseases [31].

Example of proteins in common and decreased in abundance in the two disease conditions, compared to normal, but at different levels.

In certain cases, proteins were found to be decreased in both CFS and nPTLS compared to healthy normal controls. However, quantitative distinguishing differences could still be found between the two conditions. A specific example relates to networks relevant to neurological function such as axonal guidance (Figures S2A and B), where the proteins in CFS were further decreased relative to nPTLS. These findings highlight quantifiable differences between CFS and nPTLS that may be found, with respect to certain proteins such as those that are known to effect the dynamic changes in CNS cellular architecture, such as axon, neurite, and dendritic spine growth and organization.

Discussion Top

Our results support the concept that CFS and nPTLS are distinguishable disorders with distinct CSF proteomes, where one can be separated from the other. The results also demonstrate that each condition has a multitude of candidate diagnostic biomarkers for future validation and optimization studies. The discovery of many of the same proteins in each proteome is important because it allows comparative pathway analysis, so that useful hypotheses of pathogenesis can be formulated and tested.

Our results represent the most comprehensive analysis of the whole CSF proteome to date for both CFS andnPTLS. These two disorders have similar symptoms that have created diagnostic dilemmas. It has been speculated that one (nPTLS) is a subset of the other, but our results do not support that notion. Our findings alone do not describe why CFS or nPTLS occur, but are provided to illustrate that CSF proteome analysis may provide important and meaningful insights into the biological processes modulated as a function of disease and facilitate the identification of protein candidates for further investigation. Analytical strategies need to be developed for application to those proteins and their pathways that may not have been described yet. Nevertheless, in toto, these results are encouraging because there is an abundance of data now that can be analyzed with existing tools and future methods to develop hypotheses on pathogenesis [9][32].

We regard the proteins that were identified only in one group or differentially abundant between groups, as possible or candidate biomarkers that can be subjected to further analysis in validation and verification studies. The clinical significance of the proteins identified in each pooled sample is difficult to determine in the current discovery phase. As with most technologic methods, we expect multiple replicate analyses of the highly fractionated samples would result in a reduction of the number of seemingly unique proteins identified for each disease group [33].

An important strategy that can be used post-discovery towards validation, is the use of targeted approaches that are either MS-based, immuno-based, or a combination of these approaches [12][34]. One approach, selected reaction monitoring (SRM) MS, allows for much higher sensitivity and specificity, more accurate quantification, and much higher throughput to be achieved for simultaneously measuring many biomarker candidates in large clinical cohorts [35][37]. This approach also compensates for any theoretical over-representation of proteins in pooled samples by a single or small number of individuals. This is a strategy that we plan to use not only for these diseases, but in the investigation of other diseases with neuropsychiatric features. SRM-MS analysis will permit us to directly use small-sized samples, such as the individual CSF samples, enable verification of marker candidates that currently do not have available antibodies (hence not amenable to conventional analyses such as ELISA or Western blots), and provide robust statistical analyses on individual candidate markers or combinations of them to determine which would make the best biomarker(s) for a particular disease condition. Immunobased assays such as ELISA or Western blots may also be used for targeted approaches, but will likely have more utility during a clinical validation phase where much larger sample cohorts are used. Some may choose to apply these methods for additional orthogonal confirmation of a result. However, its greater value may lie in its widespread use as a common diagnostic platform. Regardless of the method chosen, identification of diagnostic CSF biomarkers may be the necessary prelude to a search for the same markers in the highly complex blood, because it permits targeted searches for markers that might otherwise be obscured or have uncertain relevance.

With respect to biomarkers, we believe our proteomic strategy [10], that did not require prior knowledge of which proteins might be present in the CSF, will accelerate the transition from a discovery phase of candidate biomarkers, as described in this study, to full validation for clinical application. We and others have cited important elements that should be considered when an assay or biomarker is being developed for preliminary or full validation [38][40].

Distinguishing CFS and nPTLS will have etiologic implications which could lead to novel diagnostics and therapeutic interventions. On a broader level the strategy we employed may prove useful in providing investigative foundations in other poorly understood neurological conditions.

Supporting Information Top

Figure S1.

Illustrative example of pathway analysis with respect to complement pathways. Protein network and pathway analysis was performed employing Ingenuity Pathways Analysis tools (v8.6-www.ingenuity.com). A) Proteins that participate in complement signaling were significantly enriched (p = 6×10−20) in the CSF proteomes for pooled disease-specific samples. A comparison of protein abundance determined by spectral counts reveals difference between disease states and normal healthy control CSF. Proteins with an increased abundance are colored red and those that decrease in abundance relative to normal healthy control are colored green. B) Proteins annotated as participating in complement that were detected in individual patient analysis are shown the heatmap. Protein abundances measured by ion intensity transformed to Z scores clearly show differences between CFS and nPTLS patients. C) Receiver operator characteristic (ROC) curves demonstrate the discriminating power of the select set of proteins that were detected as having statistical differences by ANOVA (p<0.05) in abundance in the analysis of individual patient samples.


Figure S2.

Illustrative example of pathway analysis with respect to axonal guidance pathways. Protein network and pathway analysis were performed employing Ingenuity Pathways Analysis tools (v8.6-www.ingenuity.com). A) Proteins that associated with axonal guidance and signaling were significantly enriched (p = 6×10−20) in the CSF proteomes for all pooled samples. A comparison of protein abundances determined by spectral counts revealed differences between disease states and normal healthy control CSF. Proteins with an increased abundance are colored red and proteins with decreased abundance relative to normal/controls are colored green. B) Normalized protein abundance clearly differs between CFS and nPTLSpatients. C) Receiver operator characteristic (ROC) curves demonstrate the discriminating power of the select set of proteins that were detected in individual CSF samples as well as in the pooled proteome.


Table S1.

Proteins identified in normal, CFS, and nPTLS pooled samples.


Table S2.

Proteins (n = 474) identified in the analysis of non-fractionated and immunodepleted individual patient samples.


Table S3.

Proteins (n = 59) that were quantified and determined to be significantly different in abundance by ANOVA (p ≤ 0.01) when comparing CFS from nPTLS subject samples and allow for separation of these two syndromes when performing unsupervised hierarchical cluster analysis.


Table S4.

Pathway enrichment determination using Ingenuity pathways analysis tools for proteins present in nPTLSand CFS proteomes. Analysis of proteins detected in the highly fractionated, immunodepleted, pooled CSF samples led to the identification of pathways that are significantly enriched (p ≤ 0.05) by the proteins from the CSF proteomes.


Table S5.

Pathways significantly enriched by proteins identified only in the pooled sample proteome for nPTLS patients.


Table S6.

Pathways significantly enriched by proteins identified only in the pooled sample proteome for CFS patients.


Author Contributions Top

Conceived and designed the experiments: SES TEA TL RDS. Performed the experiments: SES TEA TL AAS TRC. Analyzed the data: SES TEA TL JNA RDS PKC BAF BHN DGC BKH JB. Contributed reagents/materials/analysis tools: SES TEA TL JNA RDS PKC BAF BHN DGC. Wrote the paper: SES TEA TL JNA RDS PKC BAF BHN DGC.

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