Despite many successful applications of Chinese herbal medicine (CHM) in thetreatment and prevention of neurological diseases (ND), the fully scientific understanding of CHM's action mechanisms had been hampered for lack of appropriate methods to explore the combinatorial rules, the synergistic mechanisms, and the molecular basis of CHM. As an improved pharmacology approach, cerebrospinal fluid pharmacology (CSFP), based on the fact that cerebrospinal fluidplays an important role in the health maintenance of specific survival environment for neurons and glial cells, has been constructed and applied to CHM research for treating ND. In the present review, the concept and advantages of CSFP are briefly introduced. The approaches and key technologies of CSFP in CHM research are also collated and analyzed. Furthermore, the developing tendency of CSFP is summarized, and its framework in CHM research is also proposed. In summary, CSFP provides a new strategy not only to eliminate some barriers of CHM research for treating ND, but also to broaden the pharmacology research for bridging the gap between CHM andmodern medicine. Moreover, the advancements in CSFP will bring about a conceptual move in active ingredients discovery of CHM and make a significant contribution to CHM modernization and globalization.  Many neurological diseases (ND) are caused or exacerbated by disparate physiological, pathological, environmental, and lifestyle factors. It is obvious that the hypothesis to search for highly selective single-target drugs interacting with a well-defined molecular target is meeting the challenges owing to the complex etiology and pathogenesis of ND. As the leading cause of disability, ND has attracted unprecedented attention from medical researchers around the world, and different types of drugs and medications are used for treating ND, such as intensive medication, rehabilitation therapy, and traditional Chinese medicine (TCM). TCM has been reevaluated and considered as one of the most important complementary or alternative medicine in most western countries and has been increasingly accepted worldwide . PharmacSerum pharmacology is an external testing method to explore the mechanism of CHM at the cellular and molecular levels. However, owing to the presence of blood-brain barrier and the specific survival environment of neurons and glial cells in central nervous system (CNS), serum pharmacology has some limitations in CHM researches for treating ND. As a methodology and technology, cerebrospinal fluid pharmacology (CSFP) is an emerging pharmacological method mainly to the research of drugs for treating ND . Since CIn this present review, the concept and significance of CSFP are briefly introduced. Its application and potential role in CHM research are also collated and analyzed. Furthermore, the limitations and problems of CSFP are also discussed, and its framework for bridging the gap between CHM and modern medicine is proposed.Pharmacology has provided fresh insight into the drug discovery. However, addressing the scientific suspense on the material basis and the efficacy mechanism of CHM is urgent in order to develop a pharmacological methodology based on the characteristics of CHM. Yan et al.  proposedIn order to investigate the action mechanism of CHM at the cellular level in vitro, serum pharmacology was adopted in the pharmacological investigation and the drug screening of the CHM . serum pThe CSFP was informally established based on the study of Qingnao Yizhi prescription's effect on neurons damaged by glutamate. CSF and serum were, respectively, taken in 1–1.5 h after administration of CHM and added into the culture medium of astrocytes. After 48 h, a certain amount of the incubated medium was transferred into the neuron culture medium. The serum, but not the CSF, showed a certain degree of cytotoxicity on the astrocytes. The astrocytes medium stimulated by the CSF containing Qingnao Yizhi prescription could promote the neuron axon growth. The experiment verified the superiority of CSF to serum and the validity of the CSFP to evaluate the action mechanism of CHM in vitro, thus indicating that the CSFP is suitable CHM study . BrieflyCSFP provided a new method to broaden the pharmacology research of CHM. This method has the following advantages. Firstly and most obviously, the physical and chemical properties of CSF are very similar to the survival environment of neurons and glial cells in CNS. CSFP could eliminate the interference caused by the physicochemical properties of the other solvent itself containing CHM, thus improving the credibility of CHM pharmacology. Secondly, metabolic inactivation, without being absorbed into the body, indirectly exert their therapeutic effects through signaling cascades such as 2, 3 messenger, which are the major pathways transformed into active ingredients from chemical ingredients of CHM. Thus makes the research for material basis and action mechanisms of CHM very difficult . Both thAs an emerging pharmacology used mainly to evaluate the material basis and action mechanisms of CHM in treatment of ND, the experimental conditions for CSFP are very immature and needed further improvement.β1−40. Surprisingly, there is no evident difference of the cell viability among the CSF containing LDP extracted from human group, the CSF containing LDP extracted from rabbit group, and the CSF containing LDP extracted from rat group. Furthermore, further studies on selection of animals in CSFP are needed to be validated.The first key of CSFP is how to obtain effective CSF containing CHM. Theoretically, the animal used to extract CSF and the animal used to isolated cells should belong to the same genus, thus narrowing the differences in the physical, chemical, and biological characteristics of the two kinds of CSF and reducing the immune response owing to the species differences. Ma and Tian  exploredIn order to obtain the high-quality CSF containing drug, determining the timing of administration to animals should depend on the half-life of drug. CSF was taken from rabbits mainly in the period of 1–1.5 h , 1 d 3333, 3 d –37, 3.5 In order to obtain CSF containing higher concentration drugs, most of the experimental researches intend to increase the dose to administration. It is noted that simply increasing the dose of administration to the animals is not necessarily a benefit to absorption, distribution, and metabolism of CHM, and thus increasing the CHM components in CSF. Conversely, CSF containing very high concentrations of drugs inhibited the vitality of neurons . TherefoSince the different process of absorption, distribution, and metabolism are caused by various features of CHM and its multiple pharmaceutical formulations, thus, the active ingredients and thier amounts in CSF containing drugs are different to collect at different times. To verify the optimal acquisition time of CSF containing Tongsaimai tiny pill extracted from rats, Zhang et al.  exploredP < 0.01) [Precise location for puncture is the key point of drawing CSF from experimental animals. Fu et al.  observed < 0.01) .The impact probably caused by preservation, processing, and additive amount of CSF containing drugs cannot be ignored at some main approaches in the drug development process. To establish the standard conditions in preservation, processing, and additive amount of CSF is very important to the CSFP method. How to process CSF is the first issue to consider. Surprisingly, The study of Zhang et al.  illustraPreservation of CSF containing drugs is the key point of ensuring the authenticity with the experiment results. The study of Zhang et al.  demonstrIn summary, in order to make the test conditions closer to the environment of the drug effect in the body, it is suggested that the CSF containing drug may be prepared with 10% or 20% CSF without treating after being extracted from animals and preserved at −20°C no more than 30 days.CHM, the ancient traditional treatment methodology popular in China and surrounding areas, has been recognized as a potential important pharmaceutical area of TCM and holds promise for preventing diseases in a holistic way . In a veTo explore the efficacy and action mechanism of Qingnao Yizhi formula (QYF) for treating vascular dementia and multiinfarct dementia, Mei et al. , 56 utilTo investigate the protective effects of Jiawei Wuzi Yanzong formula (JWYF) on hippocampal neurons injured by beta-amyloid, an experimental model of Alzheimer's disease in vitro, Zeng et al.  used CSFβ-amyloid protein (Aβ25–35), an experimental model of Alzheimer's disease in vitro, several studies manifested that CSF containing Dihuang decoction (CSF-DHD) can effectively reduce calcium ion internal flowing, reduce the protein expression of c-jun, c-fos, bax, caspase-3, and APP, enhance the expression of bcl-2, and improve the survival condition and the vitality of PC12 cells, following injury by Aβ25–35, thus indicating that CSF-DHD has a protective effect on injured PC12 cell induced by Aβ25–35 [β25–35 [To investigate the protective effects of the CSF with ancient prescriptions on PC12 cells injured by Aβ25–35 –62. In a[β25–35 .PC12 cell induced by MPP+, an experimental model of Parkinson disease in vitro, was utilized to investigate the mechanism of ancient prescriptions on Parkinson disease. Studies showed that CSF containing Zhengan Xifeng decoction (CSF-ZGXFD) could remarkably restrain the expression of caspase-3 mRNA, Cyt c protein, phospho c-jun protein, mRNA and protein of Bax, and mRNA and protein of Bcl-2 and significantly upregulate mRNA and protein expression of Bcl-2 in PC12 cell induced by MPP+, thus indicating that CSF-ZGXFD has protective effects on injured PC12 cell induced by MPP+ –66.The injured PC12 cells, cell models of ischemic stroke induced by hydrogen peroxide, sodium dithionite and glutamic acid respectively, was used to clarify the mechanisms of CHM on ischemic stroke. Yan et al.'s  experimeAstrogia CRL-2541 cell injured by the high ammonia, a cell model of hepatic encephalopathy (HE), was utilized to explore mechanism of Ruhuang pill on hepatic encephalopathy. Studies , 68 showEffect of CHM on neurological disease is based on comprehension of effective constituents of CSF containing CHM, which utilizes the CSFP analysis to speculate and estimate effects of drugs. It focuses on the specific component comparison between CSF containing CHM and serum containing CHM and provides assistance for drug effective evaluation and research. To confirm the main effective constituents in Sinisan freeze-dry powder with the improving sedative-hypnotic function, Li et al. , 70 usedThe chronomedicine theory of TCM is an integral part of the treatment in CHM –73. FromThe injured neurons induced by thrombin and hypoxic-ischemic, a cell model of acute intracerebral hemorrhage (AICH) in vitro, were used to observe the therapeutic time window of AICH intervened by Beagle dog CSF containing DSQ-03 herbal medicine. Guo et al.'s experiment revealed that beagle dog's CSF containing DSQ-03 was able to inhibit the neural apoptosis; the therapeutic time window of AICH treated with DSQ-03 was at a time from 0 h to 24 h following intracerebral hemorrhage. Meanwhile, the study laid the foundation to further investigate the efficacy mechanism and to determine the clinical dose .Monarch, minister, assistant, and guide in CHM prescription contain many principles of system theory, and the aim of coordination and cooperation in several kinds of CHM is to regulate body functional imbalances and disorders . CSFP coDue to limited space, only 4 kinds of literature reports related to CSFP containing CHM were provided, each of which was published by mainly Chinese scholars in recent years. Although the studies introduced in this paper are not thorough and systematic enough, there is still no doubt that the CSFP plays an effective role in studying the efficacy mechanism of CHM and solving the problems of administration in vitro experiment. Brief information of CSFP technologies and tools is shown in CHM has a unique theoretical system which belongs to the dialectical and philosophical medical system and mainly reflects the features of the experience-based medicine. Its clinical practice is based on the theory of the holistic concept and syndrome differentiation . In termOwing to the presence of blood-brain barrier and the specific survival environment of neurons and glial cells, it is no doubt that serum pharmacology is not suitable to evaluate the effects of CHM for treating ND. Fortunately, as a novel approach, CSFP imitated the survival microenvironment of neurons and glial cells to optimize the discovery of drug for treating ND. Since the CSFP was proposed, it is gradually becoming an important approach to explore the details of drug-target, material basis, and efficacy mechanism of multiple active components in CHM for treating ND. As seen in the above studies, CSFP was suitable to such studies, providing a robust basis and direction for basic research. In addition, CSFP can truly reflect pharmacological effect and increase the relevance of active ingredients and should be adopted in the pharmacological investigation and the drug screening of CHM. So it is thought that CSFP has an important scientific significance at the cellular level.The application of CHM is inseparable from the guiding of CHM theory. It does not matter whether CHM could pass through BBB, it is likely to play a role in treating ND , thus alWe can draw a conclusion that all these roles may be the result of maintaining balance and harmony of the internal environment through the regulation of neuro-endocrine-immune network. However, the precise mechanism is still unclear. Here we want to emphasize that CSFP belongs to a new pharmacological experimental method, which is preliminary to solve the problems in vitro experiments of CHM for treating ND and needed further to clarify its action mechanisms.CSFP has become a helpful tool in understanding the fine details of drug-target interactions, efficacy, and mechanism of drugs. However, the CSFP still has some limitations. Firstly, whether the donor animals to extract CSF containing drugs are necessary for modeling, the difference of herbal ingredients in CSF between normal animal and model animal are still an unresolved issue. Secondly, in view of the complexity of CSF ingredients, more studies are required for better understanding the mechanism whereby the CSFP works. Finally, how to make an effective of these active ingredients in CHM, which could not passed through the blood-brain barrier, is still a puzzling question. Using CSFP to investigate CHM pharmacological effects and drug targets, attention should be paid to verify the results of CSFP analysis and mutual authentication. A diagram is proposed to exhibit the research approach of CSFP for CHM research . This apIn summary, CSFP, as an improved pharmacology method to explore the material basis and action mechanism in vitro, not only breaks the theory that pharmacokinetics studies are limited to the overall function, but also offers new ideas for the selection of the appropriate cells in vitro culture system to study material basis and efficacy mechanism of CHM for treating ND. Moreover, the advancements in CSFP undoubtedly bring about a conceptual move in active ingredients discovery of CHM, make an operational shift in CHM research, and make a significant contribution to CHM modernization and globalization.


We and other investigators have postulated deterioration of essential choroid plexus (CP) functions in some elderly and especially Alzheimer’s disease patients based on apparent anatomical, histological and pathological changes in CP. We have termed this putative phenomenon CP failure. By focusing on four essential energy-requiring CP functions, specifically ascorbic acid (AA) and folate transport from blood into CSF, transthyretin synthesis and secretion into CSF, and electrolyte/acid–base balance in CSF, we were able to evaluate the hypothesis of CP failure by reviewing definitive human data. In both healthy elderly and Alzheimer’s disease patients, the CP functions normally to transport AA and folates actively from blood into CSF, synthesize and secrete transthyretin into CSF, and maintain CSF acid–base balance and ion concentrations. These human CSF compositional data provide no support for the notion of CP failure in elderly humans and Alzheimer’s disease patients. The human choroid plexus (CP), a locus of the blood-CSF barrier (BCSFB), is an organ in the cerebral ventricles weighing approximately 2 grams. The CP In order to perform these functions, the human CP has an estimated blood flow of 4 to 5 ml/min/g and normally produces CSF at a rate of ~ 0.4 ml/minute, although there is substantial diurnal variation (see below). The anaSoon after our work on the vitamin transport systems in animal CP almost forty years ago, one of us (RS) and colleagues proposed that the non-stroke related dementias were due to a central vitamin deficiency state secondary to CP failure–analogous to renal failure. This hyIn order to evaluate CP function in the elderly and Alzheimer’s disease patients, we focus on four essential functions of the CP–AA and folate transport from blood into CSF via the CP, transthyretin synthesis/secretion into CSF, and CSF14C-ascorbate distribution in mice, first proposed that AA was transported into CSF, and then brain, via the CP close to 2.9 mM (or mEq/L) and [Na+] about 150 mM. Acid–base transporters in the BCSFB stabilize CSF pH near 7.3[3-] at about 24 mM. Perturbations in plasma electrolytes are not mirrored in CSF due to: i) the diffusion restriction imposed by the BCSFB tight junctions and, ii) the transporter activity by CP epithelium that tightly controls ion concentrations in secreted CSF and [K+] compared to young subjects and age-matched controls[2 levels[3- and H+ transporters at the BCSFB. Accordingly, although the information is somewhat limited, the available evidence  strongly suggests that regulatory Na+, K+ and H+/HCO3- transport at the BCSFB is maintained in Alzheimer’s disease[In Alzheimer’s subjects, there is compelling evidence for unaltered concentrations of CSF [Nacontrols  strongly support the notion that the CP in elderly and Alzheimer’s patients generally functions well. This is in the face of clear histological, pathological and anatomical modifications in CP tissue of some elderly and Alzheimer’s disease patients,12,13,31AA: Ascorbic acid; Alz’s: Alzheimer’s disease; BBB: Blood–brain barrier; BCSFB: Blood-CSF barrier; CP: Choroid plexus; CSF: Cerebrospinal fluid; KO: Knock out; Na-K-Cl cotransporter: Sodium-potassium-chloride cotransporter in CP; PCFT: Proton-coupled folate transporter; RFC: Reduced folate carrier; SVCT2: Sodium-dependent vitamin C transporter 2; TTR: Transthyretin.The authors declare they have no competing interests.Both RS and CEJ analyzed the literature and wrote the manuscript. Both authors have read and approved the final manuscript.


Neuroendoscopic system is the best choice for detection and assessment of intracranial cysts and intraventricular tumors and cysts. There are some reasons and features for neuroendoscopy to be the number-one option for these dysfunctions such as entrapment of cerebrospinal fluid (CSF) inside the arachnoid cysts, location of intraventricular tumors within the CSF, and generally any ventricular enlargement because of CSF pathway obstruction. These features provide optimal space for visualization and working by neuroendoscopy. However, in the context of recent technologies such as robotic assisted neuroendoscopy and frameless stereotactic based neuroendoscopy, the ventricular enlargement is no longer an essential prerequisite for neuroendoscopy practice.This study presents an overview of history and current advancement of neuroendoscopy as a safe and effective management modality for the treatment of a variety of intracranial disorders, its indications and limitations which underpin fundamental basis of modern neurosurgery. Furthermore, we present our new technique for the treatment of intracranial arachnoid cyst using endovascular stent for patency maintenance of opening pathway.Neuroendoscopy, Arachnoid cyst, Endoscope, Intraventricular lesions, Endovascular stent


The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD).Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99).We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control. The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers. Synthesis by adenylate and guanylate cyclases and degradation by phosphodiesterases (PDEs) regulate the concentrations of cAMP and cGMP In the brain, cAMP and cGMP signaling is involved in a multitude of mechanisms in neurons, astrocytes, oligodendrocytes and microglia. Examples are signal transduction in synapses, communication between neurons and glia cells or inflammatory processes To date, clinical or even preclinical biomarkers for neurodegenerative diseases such as PD, ALS, CJD are desirable and in the focus of biomedical research The aim of the present study is to investigate cAMP and cGMP in CSF of PD, PDD, ALS, CJD and control patients. The analysis of cAMP and cGMP in CSF is carried out with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). To evaluate the suitability of cAMP and cGMP as biomarkers we use receiver operating characteristic (ROC) curves and the Youden index All patients included in this study attended to the general outpatient clinic, the outpatient memory clinic, the outpatient clinic for motoneuron diseases or the surveillance unit for transmissible spongiform encephalopathies . The Ethics Committees of the Universities Göttingen and Ulm approved the collection and analysis of CSF samples and specifically approved this study (Ulm). Samples of CJD patients were obtained from the surveillance and therapy trail The group consisted of 14 patients diagnosed with ALS. M/M) at the PRNP codon 129, one was heterozygous (M/V) and two patients were homozygous for the valine (V/V) allele. Five of the definite cases had a positive 14-3-3 immunoblot and one of them was diagnosed as familial CJD (fCJD). Tau protein exceeded the proposed cut-off of 1300 pg/mL in all but the fCJD case PRNP codon 129 of the five neuropathologically-defined probable CJD cases is unknown except for one (M/M). All of them had a positive 14-3-3 immunoblot and tau protein in the CSF above the cut-off. A last case underwent no histopathological examination but was diagnosed as fCJD.p = 0.70 for cAMP and p = 0.38 for cGMP, Kruskal-Wallis test). The cAMP and cGMP concentrations did not correlate with age as well.We investigated different control groups for the three neurodegenerative diseases to account for age differences. The control patients had the following diagnosis: polymyositis (two patients), diabetes mellitus (two patients), amnesia, sinusitis. In three patients the lumbar puncture was carried out to exclude an acute or chronic inflammation.The 14 patients in the control group had the following diagnoses: migraine with aura, episodic headache and symptomatic epilepsy, transient ischemic attack, borreliosis, anterior ischemic optic neuropathy (AION), PD, vestibular neuritis with depression and iron deficiency anemia, pseudotumor cerebri (PTC), migraine and polymyalgia rheumatica. For one of the control patients a lumbar puncture was carried out to exclude an acute or chronic inflammation.The patients in the control group had the following diagnoses: migraine, vestibular neuritis, ALS, vertigo, epilepsy, polyneuropathy, subcortical vascular disease and ischemia, recessive transient attacks, lower body parkinsonism, neuritis retrobulbaris and for one case the lumbar puncture was carried out to exclude an acute or chronic inflammation.Total tau protein concentrations in CSF samples of the CJD cohort were measured by a commercially available ELISA  The CSF samples (35 µL) were mixed with 0.4 M perchloric acid (1∶1) and centrifuged at 20000× g for 10 min at 4°C to precipitate and remove proteins. The supernatant was analysed in duplicate by LC-MS/MS as described previously Eluates were detected with an API 4000 triple quadrupole mass spectrometer  in the positive electrospray ionisation (ESI) mode by multiple reaction monitoring (MRM). The following transitions were used: 330.08 → 136.10 (cAMP) and 346.15 → 152.10 (cGMP). The injection volume was 25 µL.2, 0.85 mM MgCl2, and 1 mM Na2HPO4, pH 7.0–7.4) and 0.4 M perchloric acid. The stable isotope-labelled internal standards 13C5-cAMP (transition: 335.15 → 136.20) and 15N5-cGMP (transition: 351.03 → 157.00) were added to the standard solutions and CSF samples in a concentration of 100 nM to account for matrix effects and variations in ionisation.Standard solutions were prepared in a mixture (1∶1) of artificial CSF  for the indicated time see . Three ap-value<0.05 was regarded as statistically significant. Cut-off levels of cAMP, cGMP, tau protein and tau/cAMP for diagnosis of CJD were selected by calculating the Youden Index of the ROC curves.All statistical analyses were carried out with GraphPad Prism 5.03 . The data of the stability measurement were analysed by a one-way ANOVA and compared with the control sample by Dunnett's multiple comparison test. The cAMP and cGMP concentrations of the different groups of patients were compared by a Mann-Whitney or Kruskal-Wallis test. Spearman's rank correlation coefficient (r) was calculated for cAMP or cGMP with the Hoehn & Yahr score, MMSE, age (PD and PDD), tau protein as well as survival (CJD). A Different storage and handling conditions during and after CSF collection could affect the results. We checked the stability of cAMP and cGMP in CSF under diverse conditions to reduce the risk of false positive or negative results.The concentrations measured in the differently treated CSF samples were all within the range of ± 1 SD of the control sample . This isWe measured the concentrations of cAMP and cGMP in CSF samples of patients suffering from PD or PDD and control patients (CON) . Mean CSp = 0.80 for cAMP, p = 0.70 for cGMP), MMSE  or age .A correlation analysis of the measured CSF concentrations in PD and PDD patients revealed no correlation between cAMP or cGMP and the Hoehn & Yahr score  and 10.81 nM (ALS) for cAMP and 2.00 nM (CON) and 1.64 nM (ALS) for cGMP . NeitherThe LC-MS/MS analysis of cAMP and cGMP in CSF of patients with CJD showed a marked reduction of cAMP of about 70% compared with controls . Mean cop = 0.04) between cAMP and tau protein concentrations in the CSF of CJD patients (Since tau protein is suggested for the diagnosis of CJD we looked for a correlation with the cyclic nucleotides patients . Concentpatients .p = 0.35) or cGMP (p = 0.48).We further investigated, whether cAMP and cGMP concentrations correlated with the survival of the CJD patients. The patients had a survival time of 50.9±25.1 d (mean ± SEM) after lumbar puncture. There was no correlation with cAMP .In this study, we investigated the concentrations of the cyclic nucleotides cAMP and cGMP in CSF of patients suffering from PD, PDD, ALS or CJD in comparison with controls. The measured values for cAMP and cGMP in the control groups agree with previously reported data showing concentrations in the range of 8–14 nM (cAMP) and 2–3 nM (cGMP) Both nucleotides cAMP and cGMP are stable in human CSF during different handling and storage conditions as shown in The impact of the cyclic nucleotides cAMP and cGMP in CSF of PD cases has been questioned for several years In contrast to PD, there are no studies of the cyclic nucleotide concentrations in CSF of PDD patients. Similar to the observation in PD patients without dementia we did not find differences in PDD cases. There are reports of alterations in other types of dementia but the findings are not consistent. The discrepancies could be ascribed to distinct primary disorders causing the dementia Two studies investigated CSF cGMP concentrations in ALS patients so far with conflicting results. Ilzecka (2004) showed a 50% decrease of cGMP in CSF of ALS cases but an earlier study by Ikeda et al. (1995) observed no difference For the first time we investigated concentrations of cAMP and cGMP in CSF of Creutzfeldt-Jakob disease patients. Since there is a substantial degeneration of the whole brain in prion diseases in a relatively short time-span In our study, the CJD patients distinguish from PD and ALS cases by the reduction of cAMP and cGMP concentrations in CSF. Therefore, we evaluated the suitability of cAMP and cGMP as biomarkers for CJD. The ROC analysis showed that both cyclic nucleotides have the potential for a diagnostic marker. With a sensitivity of 100% and specificity of 63.6% for cAMP and a sensitivity of 66.7% and specificity of 100% for cGMP they do not reach the potency of other CJD biomarkers, e.g. tau protein, 14-3-3 protein or S-100 protein In contrast to cGMP, there is a significant correlation between tau protein and cAMP concentrations. The combination of tau with cAMP in a single ratio tau/cAMP again resulted in an improvement of the diagnostic potency although sensitivity and specificity where similar. We assume that the potential of the combination of tau protein and cAMP is underestimated in our study since the potency of tau protein itself is very high in our cohort. Using cAMP in this ratio seems to be more useful than cGMP because of the more pronounced decrease of cAMP in CJD patients.Although the diagnostic potency of cAMP and cGMP alone or in combination is lower than for other recently established potential biomarkers of CJD such as ERK2 or ubiquitin, there are still some advantages of the cyclic nucleotides Immunological assays, which are often preferred in the clinic, are commercially available for the determination of cAMP and cGMP if LC-MS/MS analysis is not possible. They enable a simple and fast analysis without involving expensive equipment. However, Zhang and colleagues (2009) showed the advantage of LC-MS/MS over enzyme based immuno assays by comparing a competitive enzyme immunoassay (EIA) for the analysis of cGMP in plasma samples and a LC-MS/MS assay for the same analytes. The LC-MS/MS method showed a better precision and accuracy as well as reduced matrix effects The combination of cAMP and cGMP or tau protein and cAMP is a good example for the combination of different markers to obtain a higher diagnostic potency. This is important since biomarkers related to the pathological process are not always available. Hence, “secondary” markers that indicate downstream effects of the pathological events have to be used. However, frequently they lack specificity for the disease as described above for tau protein. Such weaknesses could be solved or reduced by combinations of biomarkers. In this context, LC-MS/MS analysis is a beneficial tool as shown in the present study. It allows the simultaneous measurement of several analytes in a single run.In conclusion, we showed reduced concentrations of cAMP and cGMP in CSF of CJD patients but not in PD, PDD or ALS. This may be owing to the widespread brain atrophy in CJD compared with the localised neurodegeneration in the other diseases. Especially in combination, cAMP and cGMP have a diagnostic potential for CJD. In combination with tau protein, cAMP led to a further improvement of this marker. Since there are already fast and reliable immunological assays for cAMP and cGMP available the cyclic nucleotides can easily be included into routine analysis and may improve the diagnosis of CJD if LC-MS/MS is not applicable. The measurement of cAMP and cGMP CSF concentrations may be useful to monitor disease progression and therapy control. Our study also highlighted the advantages of LC-MS/MS for the combination of different biomarkers.


The microtubule-associated protein ASPM  plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes. In mammals, meiosis is unique to germ cells and is critical for sexual reproduction These asymmetric cell divisions ensure the maximal retention of the maternal cytoplasmic components that are required for early development Meanwhile, meiotic spindle assembly and migration is crucial for meiotic progression and the asymmetry of the meiotic division. The spindle, which is mainly composed of microtubules, is an essential cellular structure that is responsible for the accurate segregation of chromosomes in both mitosis and meiosis Drosophila, ASPM is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules, and it is involved in microtubule organization during spindle formation and cytokinesis C. elegans, ASPM and calmodulin work together to promote meiotic spindle organization and the accumulation of LIN-5 at meiotic and mitotic spindle poles. These proteins also contribute to meiotic spindle positioning in conjunction with the microtubule motor dynein ASPM is a structurally and functionally conserved microtubule associated protein. In In ASPM mutant mice, truncated ASPM proteins similar to those that cause microcephaly in humans fail to localize to the midbody during M phase. This causes not only mild microcephaly but also a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by low fertility To investigate the role of ASPM during mouse oocyte meiotic maturation, we examined its expression and subcellular localization. An analysis of ASPM expression in mouse oocytes during meiosis by western blot revealed four bands, corresponding to 364, 212, 130, and 70 kDa,each with similar expression levels among different stages  . TherefoTo examine the subcellular localization of ASPM, we performed immunofluorescence staining on oocytes at different stages of maturation and also on mouse embryonic fibroblasts (MEFs). At the GV stage, ASPM was distributed evenly in the cytoplasm. At GVBD, when chromatin was condensing and microtubules were beginning to polymerize, ASPM congregated around the chromatin and colocalized completely with the microtubules. When oocytes progressed to pro-MI and MI, ASPM was concentrated at the total spindle apparatus; for MII-arrested oocytes, ASPM was concentrated at the spindle and the polar body . ThroughTo clarify the correlation between ASPM and microtubules, we treated MI oocytes with spindle-perturbing drugs (nocodazole and taxol). First, we treated the MI oocytes for 5, 10 and 15 min with the microtubule-depolymerizing agent nocodazole. We found that the microtubules were gradually disassembled with an increase in treatment time, and no intact spindles were observed in the oocytes after 15 min; ASPM remained colocalized with Ac-tubulin during the entire process .Next, the MI oocytes were treated with the microtubule-stabilizing reagent taxol (10 µM) for 45 min. After treatment, the microtubule fibers in taxol-treated oocytes were excessively polymerized, and numerous asters were observed in the cytoplasm. Again, ASPM remained colocalized with Ac-tubulin .Gene-specific morpholino oligonucleotides have proven to be an effective approach for gene knockdown in mouse oocytes After 18 h of culture, DAPI-labeled DNA configurations were assessed to determine the progression of meiosis in each group of mouse oocytes . Of the To further evaluate the functional effects of the downregulation of ASPM expression, the oocytes were examined by immunofluorescence. As shown in To quantitatively compare the behavior of the spindle in control oocytes and in ASPM morpholino-injected oocytes, we measured the spindle length (S), the distance from the spindle pole to the closer cortex (D1), and the distance from the other spindle pole to the further cortex (D2) at the end of culture, as previously described The detection of ASPM at the meiotic spindle and downregulation of ASPM led to an abnormal meiotic spindle and inhibited meiotic progression prompted us to investigate whether ASPM co-localized with specific spindle-associated proteins to control spindle assembly. Lysates from MEFs and mouse MI-stage oocytes were used for the studies. The immunoprecipitation (IP) of ASPM from lysates followed by mass spectrometry identified a peptide absent from control IPs that corresponded to calmodulin (data not shown). Further western blot analysis confirmed the expression of calmodulin as a 20-kDa band in the IP elution lane; the same bands were detected in control MEF cell lysates but not in the IP-control lane .These results indicated that ASPM co-immunoprecipitated with calmodulin in the MEFs and in the mouse oocytes. Therefore, we next tested the localization of ASPM and calmodulin during mouse oocyte meiosis. We found that ASPM and calmodulin colocalized at the spindles of MI and MII oocytes .In this study, we provide the first evidence that ASPM is a conserved microtubule-associated protein that plays an essential role in the control of spindle organization during mouse oocyte meiotic maturation. The perturbation of ASPM function causes meiotic spindle assembly defects, and first polar body extrusion (PBE) greatly decreased when ASPM was partially inhibited.DrosophilaPrevious reports have shown that ASPM colocalizes with γ-tubulin at the spindle poles during mitosis in The subcellular colocalization of Ac-tubulin and ASPM during meiotic maturation prompted us to further explore the relationship between microtubules and ASPM using the spindle-perturbing drugs nocodazole and taxol. After nocodazole treatment, microtubules were depolymerized; ASPM and Ac-tubulin disappeared from the spindle upon complete spindle collapse. After taxol treatment, microtubule fibers were excessively polymerized and formed a large spindle, together with numerous asters in the cytoplasm of treated oocytes, ASPM and Ac-tubulin colocalized to the spindle and cytoplasmic asters. Therefore, the localization pattern of ASPM is similar to that of other proteins that are involved in spindle formation, such as septin1, which is most likely attached to the spindle components, perhaps participating in meiotic spindle assembly and maintenance C. elegans, ASPM, as a novel LIN-5 binding partner, acts together with calmodulin to promote meiotic spindle organization Xenopus embryo mitosis indicated that unconventional myosin ensured pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle and also reported increased spindle length upon knockdown of myosin X, presumably due to the activation of the spindle assembly checkpoint (SAC) To dissect the role of ASPM in mouse oocyte maturation, we used gene-specific morpholino injection to knock down ASPM protein expression and assess the loss of function phenotype. Western blot confirmed that the ASPM protein level was reduced by 49.14% after ASPM morpholino injection. Previous reports showed that some conserved proteins involved in the regulation of spindle organization and spindle assembly are relatively stable, such as LGN and p38α, but even moderate downregulation can cause functional effects C. elegans, the same phenomenon was observed in ASPM-deleted oocytes Drosophila, abnormal spindle (asp) contains six consensus sites for phosphorylation by p34cdc2 and four consensus sites for phosphorylation by MAP kinase Drosophila asp mutants exhibit mitotic metaphase checkpoint arrest with abnormal spindle poles In addition, the downregulation of ASPM significantly decreased the PBE rate; most of the oocytes remained blocked in MI. In Taken together, our ASPM subcellular localization, interaction and function data strongly suggest that ASPM plays a critical role in meiotic spindle organization and meiotic progression. Understanding the nature and function of ASPM on mouse oocyte meiosis will enrich our knowledge of the regulatory mechanisms involved in spindle organization and lay the foundation for further exploration of the intricacies of the meiotic cell cycle.The experimental procedure was approved by the Animal Care Commission of the College of Animal Sciences and Technology, China Agricultural University. The adult (8-week-old) C57BL/6J female and DBA/2J male mice purchased from laboratory animal research center of the Academy of Military Medical Sciences. They were housed in a standard animal facility under conditions of controlled temperature (20°C) and photoperiod (a 12∶12-hour light/dark schedule), with food and water provided ad libitum.All chemicals were purchased from Sigma-Aldrich  unless otherwise indicated.2 in air. At different time points during the culture, the oocytes were collected for immunofluorescence, drug treatment, microinjection and western blot.Oocytes were isolated from 21- to 23-day old female B6D2F1 (C57BL/6J×DBA/2J F1) mice that had been injected with 5IU of pregnant mare serum gonadotropin (PMSG) to stimulate pre-ovulatory follicle development. The oocytes were collected in M2 medium with or without 2.5 mM milrinone to keep the oocytes at the GV stage. The oocytes were then washed thoroughly and cultured in M2 under liquid paraffin oil at 37°C in an atmosphere containing 5% CO2 in air. After treatment, the oocytes were washed thoroughly and prepared for immunofluorescence. Control oocytes were treated with the same concentration of DMSO in the medium before examination.MI-stage oocytes were treated with nocodazole or taxol. For nocodazole treatment, 10 mg/ml nocodazole in dimethylsulfoxide (DMSO) stock was diluted in M2 medium to a final concentration of 20 µg/ml, and oocytes were incubated in this medium for 5, 10 or 15 min; for taxol treatment, 5 mM taxol in DMSO stock was diluted in M2 medium to a final concentration of 10 µM, and MI-stage oocytes were incubated in this medium for 45 min at 37°C in an atmosphere of 5% COFor the immunofluorescence staining of ASPM, calmodulin and Ac-tubulin, oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) and permeabilized with 0.5% Triton X-100 for 45 min at 37°C. After three washes with 0.1% PVA/PBS at 37°C for 5 min, the oocytes were blocked in PBS supplemented with 1% BSA for 1 h and incubated overnight at 4°C with 1∶500 rabbit anti-ASPM antibody , 1∶200 mouse anti-calmodulin antibody  or 1∶10000 anti-Ac-tubulin antibody. The oocytes were then washed three times in 0.1% PVA/PBS containing 0.02% Triton X-100 at 37°C for 20 min each and labeled with Alexa Fluor 594 Goat Anti-Rabbit or Alexa Fluor 488 Goat Anti-Mouse (Molecular Probes) for 1 h at room temperature in the dark. After another three washes in 0.1% PVA/PBS containing 0.02% Triton X-100 at 37°C for 20 min each, the oocytes were costained with DAPI (Sigma). Finally, the oocytes were mounted on glass slides and examined with a confocal laser scanning microscope .Co-immunoprecipitation studies were undertaken to determine whether endogenous ASPM interacts with specific proteins. Lysates were prepared from MEFs and mouse MI-stage oocytes. Analysis was performed using the ProFound™ Mammalian Co-Immunoprecipitation Kit  in accordance with the manufacturer’s instructions. Briefly, rabbit anti-ASPM antibody was immobilized on the coupling gel. Non-related rabbit IgG (Sigma) was used as an immunoprecipitation control. The co-immunoprecipitation complex was eluted and processed for mass spectrometry and western blot analysis.Denuded GV-, GVBD-, MI- and MII-stage oocytes were collected and frozen in 2X Laemmli buffer (Bio-Rad) with protease inhibitors. Prior to analysis, the samples were thawed and subsequently heated to 100°C for 5 min. The proteins were separated on 7.5% or 12% acrylamide gels containing 0.1% SDS and then transferred onto hydrophobic PVDF membranes . The membranes were blocked with 5% non-fat dried milk in TBS containing 0.05% (v/v) Tween-20 overnight at 4°C and incubated with a diluted rabbit antibody against ASPM (1∶2000) and mouse antibody against calmodulin (1∶1000) for 2 h at room temperature, followed by three (20-minute)washes in TBS containing 0.05% (v/v) Tween-20. A peroxidase-conjugated secondary antibody  was added for 1 h, and protein bands were then detected using an ECL-plus system . The densitometries of bands were analyzed with Image J software.TAGAAGCCGAGCCACCAGAGGTCAT, Gene Tool), 25 nucleotides in length, were used to knockdown ASPM translation levels in oocytes. Control groups included: (i) non-injected oocytes that were subject to the same culture conditions and (ii) oocytes injected with standard control morpholinos . For each group, 10pl of 1 mM morpholino solution (Gene Tool) was microinjected directly into the cytoplasm of denuded, fully grown oocytes arrested at GV in medium supplemented with 2.5 µg/ml milrinone. Sterile Femtotip capillaries and a FemtoJet microinjector  were used to standardize the injection volumes. Following oocyte microinjection, oocytes were maintained in GV arrest for 30 h. The oocytes were subsequently washed thoroughly, transferred to fresh medium and cultured for an additional 18 h. Finally, the oocytes were collected for further western blot and immunostaining analysis.To assess ASPM function in oocyte meiosis, ASPM-specific morpholinos (All data are presented as the mean percentages (± SEM) of a minimum of 3 independent experimental replicates. The different groups were analyzed by one-way ANOVA using the program SAS . Significance was assigned at P<0.05.


Successful hand and face transplantation in the last decade has firmly established the field of vascularized composite allotransplantation (VCA). The experience in VCA has thus far been very similar to solid organ transplantation in terms of the morbidity associated with long-term immunosuppression. The unique immunological features of VCA such as split tolerance and resistance to chronic rejection are being investigated. Simultaneously there has been laboratory work studying tolerogenic protocols in animal VCA models. In order to optimize VCA outcomes, translational studies are needed to develop less toxic immunosuppression and possibly achieve donor-specific tolerance. This article reviews the immunology, animal models, mixed chimerism & tolerance induction in VCA and the direction of future research to enable better understanding and wider application of VCA. Two areas of transplantation that posed significant obstacles to clinical application were vascularized composite allotransplantation (VCA) and donor-specific tolerance. However, over the last decade, it is heartening to note the progress that has been made in both of these fields. VCA has achieved acceptance in the field of transplantation  and promClinical feasibility of VCA has been established with the long-term success of hand and face transplantation. Over 50 hand and 14 face transplants have been performed worldwide with excellent outcomes . The sucVCA is composed of skin, muscle, vessels, nerves, tendon, bone, and so forth—each with differing immunogenic potential. Skin is probably the most immunogenic of all human tissues . Lee et In addition, the other theoretical advantage of VCA is the potential to transplant vascularized bone marrow present in the skeletal component of the allograft. The bone marrow is transplanted with its microenvironment. This has been postulated to confer an immunomodulatory effect that could lead to an improved long-term graft survival . AlthougThe high antigenicity of skin can be traced to the high proportion of potent antigen-presenting Langerhans cells. These and skin keratinocytes express MHC class I constitutively and upon stimulation present MHC class II, intercellular adhesion molecule 1 (ICAM-1), and proinflammatory cytokines. In addition, skin bears similarity with solid organs such as lung and intestine which have the highest rates of acute rejection , 21. SkiClinically, episodes of rejection are manifested by the appearance of characteristic cutaneous lesions—rash, edema, vesiculation, desquamation, necrosis, and ulceration . AtypicaThere are two aspects that are well established in solid organ transplantation that are yet to be clearly delineated in VCA. These are the roles of HLA antibodies and the occurrence of chronic rejection. In renal transplantation, humoral rejection is diagnosed by the presence of (1) histological injury—neutrophils in capillaries, acute tubular injury and fibrinoid necrosis; (2) evidence of antibody interaction with tissue—C4d deposition in peritubular capillaries; (3) serological evidence of antibodies to donor HLA (DSA). The occurrence of this triad is clearly related to organ dysfunction. The incidence and occurrence of humoral rejection in VCA have not been studied. Although C4d deposition has been documented in VCA literature, it has been described in the absence of donor-specific antibodies and histological tissue injury .Similarly, chronic rejection is poorly defined in VCA. Histological and clinical features indicative of chronic injury in VCA include vascular narrowing, loss of adnexa, skin and mucosal atrophy, fibrosis of deep tissue, myointimal proliferation, and nail changes . TransplFunctional outcomes after hand transplantation have been excellent. In the report of the international registry on hand and composite tissue transplantation , protectIn contrast, functional outcome assessment in face transplant recipients is more difficult to standardize due to the uniqueness and complexity of the defect in individual patients. Results from the early recipients are very encouraging: the first four patients were able to eat, drink, and speak within 10 days of transplantation . As new Unlike solid organ transplantation, greater scrutiny has been placed on immunosuppression-induced complications in VCA recipients. This is largely appropriate as VCA has been deemed life enhancing as opposed to a life saving intervention. On this basis, recipient selection has been very stringent thus far in VCA. The majority is physically healthy individuals suffering from severe tissue defects of the face or limbs . DespiteMetabolic complications have been reported in 69% of hand recipients and include diabetes, hypertension, and renal dysfunction including the need for renal replacement therapy in 1, Cushing's syndrome and aseptic vascular necrosis of both hips needing replacement . PosttraGraft loss has been reported in the hand transplant literature: 7 patients from China due to an inadequate immunosuppression; 3 patients from the West from the cessation of immunosuppression, transplant vasculopathy, and bacterial infection . More coClearly, there is a significant price to pay for the immunosuppression currently essential for the successful VCA. While we strive to gain better understanding of the immunology of VCA, the results thus far urge us to find ways to minimize the need for immunosuppression in these recipients. It is time to consider a clinical application of tolerance data accrued from animal experiments and the clinic.αβ T-cell dose has been defined. Subsequent patients have demonstrated durable multilineage chimerism and have been successfully weaned off all immunosuppression. None of the recipients developed GVHD or donor-specific antibodies. Some of the complications that have been reported in the above tolerance induction studies include engraftment syndrome with reversible acute kidney injury [Induction of chimerism has been shown to be a reproducible method to induce tolerance in the laboratory. Studies published in 2008 supported strategies to achieve tolerance by donor bone-marrow infusion in living donor renal transplant recipients. The limitations of the studies were success only in HLA matched pairs in one study . A receny injury  and the y injury , 34. Thus this approach appears very promising as a potential way forward in solid organ transplantation. But, more importantly, it may hold important lesions to enable a wider application of VCA. The reluctance on the part of the plastic and reconstructive surgeons in embracing VCA is the fear of long-term complications that are part and parcel of conventional immunosuppression. Future refinement of the above-mentioned tolerance strategies to enable use with deceased donor transplantation, further elucidation of the mechanistic components of these studies and a longer-term followup of the “tolerant patients” could help persuade reconstructive surgeons to use VCA. It is known that the current challenge in the widespread clinical applicability of VCA is the toxicity of high-dose postoperative immunosuppression. The use of preclinical animal models is essential in developing novel cellular immune-reduction therapies that will ultimately make VCA a safer and viable treatment option. Both small and large animal models have been employed in studying VCA immunology thus far. Small rodent animals are widely preferred for VCA studies for certain reasons. Logistically, such animals are relatively inexpensive and easy to maintain and handle. Biologically, these animals have short lifespans and accelerated reproduction rates that allow for VCA-related immune activities to be observed along varied time points of the life history . VCA is Mice and rats are the predominant small animal models used in VCA studies. The mouse model is a more valuable tool for basic immunologic research due to the numerous genetic variations of inbred and knockout strains and the commercially available genetic probes and antibodies , 36. MouHowever, there are limitations of small animal models as it pertains to VCA. The most significant obstacles have been the technical challenges during microsurgery. Dissection and microanastamosis in small vessels in mouse models are problematic due to the fragility of the vessel walls , 39. SimDespite the technical limitations of small animal models, such animals continue to be a cornerstone of VCA research. Various composite allografts have been performed in mice and rats in the past decade, including hind-limb transplants, bone-marrow transplant (BMT), a hemiface allograft, and even an allograft of the groin region has been performed , 41.Immune-reduction protocols that have been successfully demonstrated in small rodent models are further evaluated in large VCA animal models for efficacy and safety before clinical trials in humans. Large animal models allow for immune-reduction therapies to be further examined in more complex biological systems that are more realistic and representative of the human immune system , 42–45. Tolerance of VCA can be induced in small rodent models, but tolerance protocols established in rodents are difficult to translate to preclinical large animal models and eventually clinical human trials. In contrast to the isolated, pathogen-free lab rodents, large animal models are exposed to uncontrolled environmental factors over longer lifespans, resulting in immune-reduction protocols that are challenging to stabilize. Further, unlike rodent models, the complex immune system in large animals requires significant potent doses of immunosuppression for graft survival in unrelated donor/recipient pairs . ExampleA major factor limiting VCA is the requirement for lifelong immunosuppression and the toxicities associated with the use of these agents . VirtualThe establishment of donor hematopoietic chimerism in organ transplant recipients leads to donor-specific tolerance –52. Chimαβ-TCR mAb, FK-506, and antilymphocyte serum). The BMT conditioning strategies based on costimulatory blockade of CD28 or CD40 ligand in combination T-cell depletion and low doses of irradiation have also reported to induce long-term acceptance of VCA in rat [αβ-TCR mAb, FK-506, and anti-lymphocyte serum and 300 cGy TBI for BMT [Mixed chimerism induces donor-specific tolerance to virtually all the organs or tissues tested including skin, heart and lung, kidney, intestine, pancreas, islets, and composite tissue allografts , 64–66. A in rat . The advA in rat . The chi for BMT . There wreg) they generate, maintain self-tolerance through regulatory feedback loops. They also show promise as a cell-based therapy to promote allograft acceptance. Bone marrow has long been appreciated to possess immunomodulatory properties [One unique feature that distinguishes VCA from other transplants is the presence of its own hematopoietic microenvironment and supportive stromal cells from accompanying donor bone. Bone-marrow-derived cells, especially plasmacytoid precursor dendritic cells (p-preDC) and the regulatory T cells  which is closer to the clinical reality. Although simultaneous HSCT-induced mixed chimerism offers an opportunity for tolerance induction, there are obvious drawbacks that would prevent its clinical application. The major concern is that simultaneous BMT and VCA may increase the risk of complications from combined major operation and conditioning for BMT. Moreover, nonmyeloablative conditioning regimens usually require a period of time from days to condition the recipient, which takes five to six days. Attempts to compress the conditioning to ≤2 days would result in unacceptable toxicities. As such, an approach to establish chimerism electively after recovery following VCA  and to syngeneic cells (9.9% ± 0.8%) occurred and significant cytotoxicity to third-party cells  was detected in mixed chimeras [The pluripotent hematopoietic stem cells engraft and co-exist with recipient stem cells to give rise to all hematopoietic lineages in the recipient. Mixed chimerism is a hybrid immune system. The mutual tolerance in this hybrid immune system must be systemic as suggested by stable and durable coexistence of genetically different donor and recipient hematopoietic components in mixed chimerism. The mutual tolerance in mixed chimeras should be systemic including adaptive immune tolerance (T and B cells) and innate immune tolerance. T-cell tolerance in mixed chimeras has been well studied e thymus , 88. Funchimeras , 90. T-cchimeras –93. As achimeras . These r6 T-cell-depleted ACI donor bone-marrow cells, and treated tacrolimus, antilymphocyte serum, and anti-αβTCR showed between 1.8% and 35% donor chimerism 1 month after BMT. Donor engraftment was multilineage in these chimeric recipients 1 to 2 months after BMT. Chimeric animals were then subjected to heterotopic osteomyocutaneous flap transplantation 4–6 weeks after BMT. Over 57% of animals conditioned with 400 cGy TBI and 33% of animals conditioned with 300 cGy TBI showed long-term VCA acceptance though peripheral blood chimerism was lost 5 months after BMT. Interestingly, when donor chimerism was analyzed in various hematopoietic compartments in long-term VCA acceptor animals, there was significantly higher donor chimerism detected in the transplanted donor bone , recipient bone , spleen , mesenteric lymph node  , and thymus  compared to peripheral blood (0.09% ± 0.06%). The highest level of donor chimerism was detected in the transplanted donor bone. The authors hypothesized that the donor bone may either serve as a tolerizing source of donor lymphoid cells for systemic microchimerism as previously observed [reg) [Mixed chimerism achieved by nonmyeloablative conditioning has been shown to induce donor-specific tolerance in fully MHC-mismatched VCA recipients. In a paper published by Rahhal et al. , WF reciobserved  or have ed [reg) .+ CD25+/FoxP3+  Treg play a principal role in regulating immune responses to allogeneic antigens and are robust suppressors of T-cell activation [reg in peripheral tolerance to VCA. WF recipients were conditioned with 400 cGy TBI, transplanted with 100 × 106 T-cell-depleted ACI donor bone-marrow cells, and treated with tacrolimus, anti-lymphocyte serum, and anti-αβTCR. Recipients were monitored for engraftment and then transplanted with a heterotopic osteomyocutaneous flap. Peripheral blood donor chimerism at 1 month after BMT was approximately 30% and was multilineage for both lymphoid and myeloid cells. Sixty-seven percent of transplanted animals displayed long-term acceptance. The group demonstrated that sorted CD8−CD4+/CD25+  Treg from spleens of VCA transplanted animals could significantly suppress cell proliferation when plated in a 1 : 1 ratio with either WF responders/ACI stimulator or WF responder/F344 stimulator. Interestingly, when these sorted cells were restained for FoxP3+  Treg, VCA rejector animals demonstrated higher absolute numbers of FoxP3+  Treg. Similarly, Bunnag et al. showed higher levels of FoxP3 mRNA levels in rejected human renal tissue compared to nonrejected tissue [+  Treg 6 months after-CTA which suggested a potential role for Treg in peripheral tolerance whereby newly induced Treg could potentially migrate to target tissue in high numbers as needed. Because FoxP3+ cells were detected in the peripheral blood and appeared to increase over time, immunofluorescent assays were carried out to investigate the presence of FoxP3+  Treg at the VCA graft site. Though FoxP3+  Treg were not detected in skin samples from VCA-rejected animals, skin samples from long-term VCA acceptor animals stained positive for CD4+FoxP3+  Treg (+  Treg were detected in biopsies from human hand allografts [+ cells in the VCA acceptor animals was not due to the VCA transplant itself, syngeneic controls were performed where WF rats were conditioned in a similar manner but received WF bone-marrow cells and a WF CTA. No CD4+FoxP3+ cells were detected in the transplanted graft samples from these animals.CD4tivation . Using ativation  evaluated tissue . In addi3+  Treg . Recentllografts  and in h+  Treg expression in the skin of CTA-rejector animals correlated with the increase in FoxP3+  Treg expression in the spleen of rejected animals. Lu et al. showed elevated numbers of mast cells and Treg in tolerant skin grafts in a donor-specific transfusion/anti-CD154 model [+FoxP3+  Treg in the skin and muscle of tolerant composite allografts [+  Treg have also been detected in rejected allografts. Biopsies from human hand transplant recipients undergoing severe rejection demonstrated elevated FoxP3+  Treg [reg were detected in skin biopsies from patients with acute GVHD compared to patients without GVHD [reg are recruited to the site of antigenic challenge early after transplantation to effectively prevent the infiltration of effector T cells [reg suppress the induction phase of immune responses in draining lymph nodes rather than the effector phase in the periphery [+  Treg that home to various tissue sites as needed to induce tolerance early on after antigenic challenge and then maintain peripheral tolerance. Recently, Hoerning et al. demonstrated that circulating CD4+FoxP3+CXCR3+  Treg correlate with renal allograft function and that peripheral immunoregulation depends on Treg allograft homing [+  Treg are detected in tolerant or rejected tissues. Bunnag et al. showed that in human renal transplants, FoxP3 expression increased with time after-transplant. As such, late biopsies had greater FoxP3 expression than early biopsies [+  Treg may be important in tolerance induction and long-term VCA graft survival. The lack of FoxP354 model . Similarlografts . However3+  Treg . In addiout GVHD . Time-co T cells . Similareriphery . As sucht homing . In addibiopsies . Taken tSignificant progress has occurred in VCA in the past decade. Alexis Carrel, Peter Medawar, and Joseph Murray would be pleased to find that their pioneering work would one day make hand and face allotransplantation a reality. However, the next major advance to make VCA widely available is to minimize or avoid the toxicities of the immunosuppressive agents altogether. Based on the recent clinical success in renal transplantation, tolerance induction may be a path ahead. Future research should focus on establishing safe, simple, and durable donor-specific tolerance in HLA-mismatched recipients of VCA. Drug-free graft approaches to achieve acceptance have been termed the “holy grail” in transplantation and would represent a transformational achievement for VCA to reconstruct traumatic combat-related and civilian injuries, allowing unlimited tissue for repair.


We present a theoretical study on the impact of an interface on the FRET efficiency of a surface-bound acceptor-donor system. The FRET efficiency can be modified by two effects. Firstly, the donor’s electromagnetic field at the acceptor’s position is changed due to the partial reflection of the donor’s field. Secondly, both the donor’s and the acceptor’s quantum yield of fluorescence can be changed due to the interface-induced enhancement of the radiative emission rate (Purcell effect). Numerical results for a FRET-pair at a glass-water interface are given. Förster Resonance Energy Transfer or FRET  has becoFrequently, smFRET experiments are performed with the molecules immobilized on a surface ,10. ThisIn the past, several studies have been published that focus on the strong change of the FRET efficiency due to the coupling of the donor and acceptor fluorescence to the local density of states of the electromagnetic field as modified by the presence of a cavity  or a micBefore considering the problem of the FRET efficiency in the vicinity of an interface, we have to first briefly recall the classical theory of FRET, because the details of this derivation will be needed for a close understanding of the modifications induced by the presence of an interface that divides to half-spaces with different refractive index. In the second subsection, we will then study the change in acceptor excitation rate by the presence of an interface. In the third and last subsection, we will consider the interface-induced changes in fluorescence quantum yield of both the acceptor and the donor, which are important when calculating FRET efficiencies from the ratio of donor and acceptor intensities.q that oscillate, the oscillation amplitude and orientation described by the dipole vector a, and the oscillation circular frequency ω, which is also the frequency of the emitted light. The product of the charge q times the amplitude vector a is called the dipole moment pd , i.e., pd = qa. When plugging such an oscillating electric dipole as a source into Maxwell’s equations, one obtains the electromagnetic field of the well-known Hertzian dipole emitter. In particular, the electric field amplitude readsA light emitting molecule can be fairly well described by the classical picture of an oscillating electric dipole. Such a dipole is characterized by the charges r is the distance vector from the dipole to the observation point where the field is calculated, r is its modulus, k is the modulus of the wave vector, k = nω/c, with c being the vacuum speed of light, er is a unit vector pointing from the dipole towards the position r, and n is the refractive index of the medium. Here, and in all that follows, we omit the factor e−iωt describing the temporal evolution of the electric field. The far-field emission rate of such a dipole is given bywhere k0 is the length of the wave vector in vacuum (k0 = 2π/λ), with λ being the vacuum wavelength of light. When a second molecule  is brought very close to the emitting molecule (the donor), the field contributions falling off proportional to r−1 and r−2 can usually be neglected if kr ≪ 1, and one has to take into account only the near-field componentwhere σa and the number of  photons per area per second coming from the donor. This photon flux is given by the electromagnetic energy flux density associated with the donor’s electromagnetic field divided by the energy per photon. This energy flux density, averaged over one oscillation period, is equal to the modulus of the Poynting vector or (nc/8π) · E2d, and the energy per photon is equal to hc/λ, where h is Planck’s constant. Furthermore, one has to take into account that the acceptor’s excitation rate is also orientation-dependent and proportional to the scalar product of its dipole vector pa and the vector of the exciting electric field Ed. One minor complication comes in by the fact that when one measures absorption cross-sections σa on ensembles of molecules, one averages over all possible dipole orientations, so that the absorption cross-section of a molecule with its dipole aligned with the electric field vector of the exciting field is three times larger than the measured value of σa. Taking all that together, one arrives at the following expression for the acceptor’s excitation rate ke:The excitation rate of the second molecule (the acceptor) is given by the product of its absorption cross section p̂a is the unit vector along the acceptor’s dipole moment. Inserting the explicit expression for Ed yieldswhere k. If both donor and acceptor can freely rotate, one can average the orientations p̂a and p̂d of donor and acceptor dipole, resulting in k2 = 2/3.which also defines the so-called orientation factor ke still contains the a priori unknown donor dipole amplitude pd. It can be found by scrutinizing the total energy emission of the free donor. From a quantum mechanical point of view, the totally emitted energy is that of one photon, hc/λ. From a classical electromagnetic point of view, it is given by the energy emission rate of the donor times the mean radiation-related lifetime of the donor’s excited state , which is given by the inverse of the radiative transition rate kf;d from the excited to the ground state of the donor. The inverse of the sum of the radiative and the non-radiative transition rate, knr;d, determines the observable excited state lifetime τd, whereas the ratio of kf;d and kf;d + knr;d define the donor’s fluorescence quantum yield φd . Thus, k−1f;d is given by the ratio of the observable excited state lifetime τd divided by the fluorescence quantum yield φd. Setting both the quantum-mechanical and the classical-electromagnetic energy emission equal to each other, one obtainsThe equation for Sd. Thus, one findswhere we have used so that the acceptor’s excitation rate now readsFd(λ), and of the acceptor’s excitation strength as given by its absorption spectrum σa(λ). This leads toUp to this point, donor emission and acceptor excitation was assumed to happen at exactly one and the same wavelength. To obtain the actual acceptor excitation rate, one has to integrate the found expression over all wavelengths, taking into account the wavelength-dependence of the donor’s emission strength as given by its emission spectrum Fd(λ)/∫ dλFd(λ) is the normalized emission spectrum of the donor, and where we have replaced k0 by 2π/λ. The so-called Förster radius is defined bywhere so that one finally findsσa (in cm2) and molar extinction coefficient ε (in cm−1 M−1),Taking into account the relation between absorption cross section NA is the Avogadro–Loschmidt number, one finds the usual textbook expression for R0where Finally, we have to discuss the two most common methods of experimentally determining FRET efficiencies. The first one measures the fluorescence intensity of the donor and the acceptor upon donor excitation. For the sake of simplicity we assume that one can neglect direct excitation of the acceptor by the excitation light, and any bleed-through of donor emission into the acceptor detection channel. Also, the detection efficiencies for both donor and acceptor fluorescence are assumed to be the same. Then the observable fluorescence intensity from the donor in the presence and in the absence of the acceptor will be proportional toandrespectively, and that of the acceptor is proportional tokf and knr are the radiative and non-radiative transition rates, respectively, and the additional subscript d refers to the donor and the subscript a to the acceptor. Accordingly, φa is the fluorescence quantum yield of the acceptor. Thus, we find the textbook relation for the FRET efficiency E:Here, the kf;d + knr;d = τ−1d. The second method is based on the measurement of the donor’s excited state lifetime. In the presence of the acceptor, this lifetime is given bywhere we have used E asThus, one finds a second relation for the FRET efficiency L apart. One of the molecules (let us assume the donor) is kept at a distance zd from a surface; the half-space below that surface has the refractive index ng, and the half-space above the surface, containing the smFRET system, has refractive index nm. As before, both the donor and the acceptor are considered to be ideal electric dipole emitters/absorbers. Furthermore, we will assume that both donor and acceptor are free to rapidly rotate around their center positions so that one can average over donor and acceptor orientation when calculating FRET efficiencies. As will be seen, an extension of the theory beyond this assumption is straightforward but would introduce another four degrees of freedom. But we will assume that the connecting line between acceptor and donor keeps a fixed angle towards the vertical. For example, in case of a charged surface and a charged smFRET system, one of two limiting cases may be expected: either both molecules are adsorbed to the interface and θ = π/2 due to attractive electrostatic interaction, or one finds θ ~ 0 due to electrostatic repulsion between surface and smFRET system. Our results will also be applicable to the more general case of orientational flexibility of the donor-acceptor axis. One has then only to average over different orientations of this axis with an appropriate weight function that describes the chance that a particular orientation occurs.Consider the system depicted in ke is proportional to the donor-generated local electric field strength at the position of the acceptor. Due to partial reflection at the interface, this field strength will be different from the situation without interface. For calculating this field strength, we start by expanding the electric field of the free donor into a Weyl representation [As we have seen in the preceding subsection, the energy transfer rate entation  of planeq is the part of the wave vector parallel to the interface, and e±p and es are pointing along the electric field polarization of p- and s-waves, respectively, as shown in z > zd, and the minus sign for z < zd. The integration over q extends over the whole two-dimensional q-plane. The electric field as given in nm and yields, after explicit integration, the result of where q-dependent Fresnel coefficients rp;s for the reflection of plane p- and s-waves at a planar interface, the reflected field can be written asIn the presence of an interface, each plane wave component in the Weyl representation is partially reflected, adding to the total field strength in the upper half space. Using the standard, E′d = Ed + Er. Whereas the integrals in i.e., proportional to |E′d · pa|2, where ρa and za are the lateral and vertical position of the acceptor, respectively, and pa its excitation dipole moment. What we are interested in is a comparison of the FRET efficiency of our acceptor-donor system in free space with the same system attached to the surface. For the sake of simplicity, we will here compare both situations in the limit of rapidly rotating acceptor and donor orientation. As already mentioned, we could equally well consider particular fixed orientations of donor and acceptor, which would, however, add another four degrees of freedom without making the general effect of an interface on the FRET efficiency more clear. As will be seen from the calculations of the emission rate enhancement below, all the considered electrodynamic effects are strongly orientation-dependent, so that a fixed donor and/or acceptor orientation will lead, in general, to an even stronger modification of the FRET efficiency than in case of rapid rotational diffusion of donor and acceptor. Thus, we define the ratioso that the total electric field strength in the upper half space is given by Q > 1) or reduced (Q < 1) when compared with a measurement in free solution. Here, the angular brackets with subscript Ω indicate averaging over all orientations of donor and acceptor. Due to symmetry, the ratio Q depends only on the modulus ρa = |ρa| of the lateral position vector, but not its two-dimensional orientation. Moreover, the problem is reciprocal: the ratio Q in which tells us how much the acceptor’s excitation rate of the surface-bound system is enhanced which adds an additional term to the energy loss per time exhibited by the dipole. Thus, the emission rate is changed toγ in emission rate alias radiative transition rate is given byso that the relative change p̂d. The factor γd is the interface-induced change of the radiative transition rate of the donor. An identical relation holds also for the emission rate of the acceptor, where one has only to exchange all subscripts d by a. If one can neglect the optical dispersion of the materials in the upper and lower half spaces, then the resulting factor γ is identical for both donor and acceptor if they are at the same distance from the surface. Knowing both the factor Q and γ, the interface-modified acceptor excitation rate is given bywhere we have again averaged over all possible orientations of the emission dipole, The modified radiative emission rate changes also the quantum yield of both the donor and the acceptor, according to the formulad or a.where we have omitted the subscript E by the intensity ratio according to When calculating the FRET efficiency However, if one uses the quantum yield values for the dyes in free solution, the calculated FRET efficiency isφa = φd. Similarly, when using the lifetime-based τd of the donor in free solution, or the interface-modified value τ′d = τd/(γφd + 1 −φ′d). When using τ′d, one recovers τd, one findswhich becomes identical to γ, Q, and E′ for a glass-water interface, where the refractive index of glass was set equal to 1:52 and that of water equal to 1:33. All calculations where done for an assumed emission wavelength of 550 nm. γ. As can be seen, close to the interface (a few dozen nanometers), γdoes not change significantly, reaching a maximum value of 1.18 on the surface . For the sake of completeness, the figure shows also the extreme values of γ for horizontal (‖) and vertical (⊥) emission dipole orientation. An identical figure is obtained for the radiation enhancement of an acceptor, where the x-axis has only to be rescaled by the ratio of the acceptor’s to the donor’s emission wavelength. Taking into account the dependence of the fluorescence quantum yield on the enhancement factor γ, φ/φ = (1 − φ), Δγ/γ so that a 18% change in the radiative rate γ for a fluorophore with 50% fluorescence quantum yield translates into an approximately 9% change in quantum yield. Of course, the effect becomes smaller with increasing value of quantum yield.We first performed calculations of Q and the FRET efficiency E′ as a function of lateral (ρa) and vertical (za) distance between donor and acceptor, respectively, where it is assumed that the donor sits directly on the surface at ρd = 0. As was already mentioned, an identical figure is obtained when exchanging the positions of donor and acceptor.Next, Q-factor varies between 0.87 and 1.06, depending on the orientation of the donor-to-acceptor axis. For example, for a donor-acceptor distance of 10 nm, if the donor-acceptor connecting line is close to the surface, Q is close to its minimum of 0.87, whereas if this axis is perpendicular to the surface, it reaches its maximum value of 1.06. When using E is given byThe φ = φd for φ = φa for E/E is half that of ΔQ (Q = 1) for dyes with quantum yield of 1.0, and the FRET efficiency is only changed between −7% and +3%. For quantum yield values of 0.5, the relative change in FRET efficiency lies between −2.5% and 4.5%. Thus, although the energy transfer rate ke can vary by up to 13%, and the radiative emission rate by up to 18%, the resulting variation in FRET efficiency is much smaller due to the intricate dependence of E on both these factors.where Q and the FRET efficiency E′ for the same geometry and distances as used in the calculations for the glass/water interface. The enhancement factor Q for the transition rate varies now between 0.71 and 1.2, and the resulting FRET efficiency varies between −17% and +9% for dyes with quantum yield 1.0, and between −9% and +17% for dyes with quantum yield 0.5. Thus, although the impact of a glass/water interface on the FRET efficiency is rather moderate, it is no longer negligible for a sapphire/water interface.In recent years, objectives with N.A.-values of 1.65–1.7 have become available and are used for single-molecule detection and spectroscopy experiments, because they provide exceptional high light collection efficiency and enable efficient TIR illumination with very small penetration lengths of the evanescent field. When using these objectives, one needs special sapphire cover slides and high-index immersion oils with a refractive index of 1.78. We repeated all the above calculations for checking how such a high refractive-index material will influence the smFRET measurement. For a fluorophore close to the sapphire/water interface, the radiative emission rate is now enhanced by 45%  in contrast to the 18% for the glass/water interface. There are two possible and straightforward ways to experimentally test the predictions made in this paper. The simplest way would be to compare FRET measurements of the same system in free solution, and when it is bound to a surface. However, a problem might be that other factors like the different nano-environment close to a surface or the binding chemistry used for surface attachment may also influence the measured FRET efficiency. A more elegant way would be to use a short and rigid DNA double strand with donor and acceptor at both ends as the smFRET system, and to bind it with one end to the surface. If the surface is prepared in such a way that it carries a negative net charge, one can assume that the negatively charged DNA double strand will adopt an orientation orthogonal and away from the surface, whereas for a positively charged surface, the double strand will adsorb to the surface and thus lie flat on it. As can be seen from In the present paper, we have given a complete theoretical analysis of the impact of interface-induced changes in the donor’s electric field as well as the radiative transition rates of both donor and acceptor on the FRET efficiency. Although the donor’s electric field as well as the radiative transition rates can change by more than ten percent, the resulting impact on FRET efficiency for a glass/water interface is rather moderate. This is, however, no longer true when measurements are performed on high refractive-index substrates such as sapphire, which is used in some single-molecule experiments. Finally, we have given some indications of how to experimentally measure the predicted effects.


The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA–ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA–ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA–ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA–ramA genes, strongly suggesting that a secondary regulator may control ramA expression.Tigecycline resistance has been attributed to  Strains with chromosomal and/or plasmid-mediated resistance mechanisms coupled with efflux/influx-related mutations are being increasingly identified K. pneumoniae has precipitated the emergence of clones harbouring a plethora of resistance mechanisms to clinically relevant antibiotics  and emerging pandrug-resistant clones have left few therapeutic strategies available to combat this pathogen K. pneumoniae and Escherichia coliK. pneumoniae and Enterobacter spp. Tigecycline is able to evade the classical mechanisms that confer resistance to tetracycline and minocycline, such as Tet(A–E)-mediated efflux and ribosomal protection conferred by Tet(M) Tigecycline is a new glycylcycline with substantial anti-Gram-negative activity that has been introduced for the treatment of community-acquired Gram-negative infections caused by extended-spectrum β-lactamase-producing acrR mutations are as significant as those seen when acrR is mutated is not clear.Studies into tigecycline resistance in members of the Enterobacteriaceae have shown that it is mediated via upregulation of efflux pumps that are controlled by certain regulatory loci ramA locus is only found in Klebsiella, Salmonella, Enterobacter and Citrobacter spp.; the genetic organisation in Salmonella differs from the others owing to lack of the romA gene. Like MarA, increased ramA expression is linked to upregulation of the AcrAB efflux pump, which confers a multidrug-resistant phenotype to a variety of different antibiotic classes acrAB gene, as ramA overexpression in an acrAB-deleted strain does not produce a similar phenotype RamA is a member of the AraC/XylS family and is closely related to the MarA and SoxS proteins ramA gene is controlled at a transcriptional level. Studies both in Salmonella enterica serovar Typhimurium K. pneumoniaetetR-like gene, called ramR, that lies upstream of the ramA gene and acts as its repressor  and mutations within acrR, the repressor of the acrAB efflux pump ramA locus and whether its overexpression is always linked to changes within the ramR gene.In studies involving clinical strains, 22.1K. pneumoniae and were isolated from intensive care wards in hospitals within Chile, Turkey and Pakistan from 2006–2008. Three strains from Singapore characterised in a previous study acrA) and AG100B ; and for validation of the ramA reverse transcription polymerase chain reaction (RT-PCR), TS67 (deleted for ramA) and TS68 (deleted for ramR) K. pneumoniae Ecl8 using a modified protocol as described by Merlin et al. All isolates were identified as 2.2MICs of tigecycline  for the clinical isolates were determined and interpreted according to British Society for Antimicrobial Chemotherapy (BSAC) protocols 2.3ramA locus was mapped according to the manufacturer's instructions using the 5′ RACE (rapid amplification of cDNA ends) system . Briefly, 0.5 μg of RNA extracted at mid log phase was reverse transcribed into first-strand cDNA, which was then terminal deoxynucleotidyl transferase (TdT)-tailed. Subsequently, the TdT-tailed cDNA was amplified by PCR and was assessed for positive PCR products. A second round of amplification using nested gene-specific primers  for sequencing.The transcriptional start site of the  primers  and Abri2.4ramA and romA genes. RNA samples were extracted from the different clinical isolates at mid log phase [optical density at 600 nm (OD600) 0.5–0.8] using TRIzol®. Total RNA was digested with DNase I to ensure the removal of contaminating genomic DNA prior to cDNA synthesis. For cDNA synthesis, a SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen) was used. Briefly, 300 ng of DNase I-treated total RNA was converted to cDNA and was used in PCRs with gene-specific primers shown in ramA expression relative to the sensitive strain Ecl8 (ramA non-expresser) is shown in RT-PCR was performed to assess the expression levels of the 2.5romA–ramA promoter regions were amplified and subjected to EMSA with both purified RamA and RamR proteins. Purified RamA and RamR proteins were extracted from recombinant pET constructs containing the ramA and ramR genes using metal chelation chromatography on nickel/nitrilotriacetate superflow agarose . Briefly, end-labelled  PCR products were incubated with increasing concentrations (200 nM and 400 nM) of RamA or RamR in binding buffer . The complexes were run on 5% native polyacrylamide gel electrophoresis (PAGE) gels for 2.5 h. The gel was then dried and exposed to the phosphor screen for image analysis. To confirm that the interactions between RamA or RamR and the promoter regions were specific, competition experiments with bovine serum albumin (BSA) as a negative control and with cold promoter were also performed.The 2.6ramA expression was elevated, the ramR and acrR genes and the pI/pII promoter regions were amplified using the primers shown in For those strains where 2.7600 ca. 0.6–0.7) prior to protein extraction using sonication. Then, 15 μg of total protein was loaded onto a 10% NuPAGE® gel (Invitrogen) prior to transfer to a nitrocellulose membrane as per the manufacturer's instructions. The blots were hybridised with the primary antibody anti-AcrA  (1:40 000) overnight at 4 °C. The secondary antibody IRDye-800 goat anti-rabbit IgG  was incubated with the blots for 1 h at room temperature and the membranes were scanned using the Odyssey Infrared Imaging system, which is based on near infrared fluorescence detection. The membrane was scanned in two channels at 700 nM and 800 nM, which detects the green and red channels, respectively. All images were quantified using the reading from the 800 nM channel. Analysis of band intensities to assess AcrA levels was performed using the Odyssey software v3 (Li-Cor Biosciences). Levels of AcrA protein of all test strains were compared against the sensitive K. pneumoniae strain Ecl8 to report a relative fold increase in AcrA levels. Both AG100A and AG100B were also used as controls in the AcrA Western blots.Levels of AcrA were determined by Western blot analyses as described previously by Schneiders et al. 33.150 and MIC90 values  of tigecycline, minocycline, tetracycline, ciprofloxacin and ceftazidime against the strains are shown in 90 values were 128 μg/mL and >128 μg/mL, respectively; however, cross-resistance to tigecycline was not observed, consistent with previous survey data The MIC3.2ramR has been shown to function as its cognate negative regulator ramR and romA–ramA loci between Klebsiella, Enterobacter, Citrobacter and Salmonella, we sought to confirm whether ramR controlled the expression of the romA–ramA locus directly in Klebsiella and whether other accessory binding sites existed for RamR within the romA–ramA locus. Bioinformatic analyses using ClustalW  showed that the palindromic binding sites identified for RamR in Salmonella spp. are conserved within the intergenic region between the ramR and romA coding sequences of K. pneumoniae, Enterobacter and Citrobacter spp.  of the pII promoter region implies that there is a putative but degenerate palindromic site upstream of the ramA gene , autoregulates itself by binding to its own promoter region. The intergenic regions, designated pI and pII, bound both purified RamR and RamA proteins A. The inamA gene A.3.53.5.1ramR gene. Changes within the ramR gene were found in both the DNA- and ligand-binding domains of the protein   . Since tamR gene  and/or tne , which sustained a change within the palindromic sequence  but harboured no changes within the ramR gene, was found. Correspondingly, TS170 exhibited a small increase in ramA expression  have been previously documented ramA, appear not to produce greater levels of the AcrA protein (Of the 27 strains tested, 9 sustained changes within the  protein B.4ramA gene. In this study, we identified in silico that key regulatory features of the ramA locus are conserved amongst Klebsiella, Enterobacter, Citrobacter and Salmonella spp.Recent studies have linked antibiotic resistance, particularly tigecycline resistance, to increased expression of the ramA autoregulates its own expression. In addition, RamR appears to be able to bind regions that either contain a perfect palindrome or more degenerate palindromic sites (Figs. This work also shows that both the RamR and RamA proteins bind the pI and pII promoters specifically, implying the presence of recognition sites for both proteins within these promoter regions. Like other orthologous systems, such as MarRAB and SoxRS es Figs. A and 3A.romA–ramA locus is not entirely identical to that observed in Salmonella as there are two promoters within the romA–ramA locus. The presence of these two promoters supports the possibility that the romA–ramA genes can be regulated independently of each other or as part of an operon (ramA-overexpressing strains (romA and ramA expression are not linked, supporting the finding that two different promoters control regulation of the romA–ramA locus (The present results show that regulation of the n operon A. Consis strains . InteresmA locus A.ramA expression in clinical isolates that were resistant or intermediately susceptible to tigecycline. The results demonstrate that in geographically diverse isolates of K. pneumoniae, 18 isolates exhibited tigecycline MICs ≥ 4 μg/mL (ramR–romAramA locus but it still overexpressed the ramA gene. Two key observations arise from these data: first, ramA is not always associated with ramR-mediated derepression; and second, decreased tigecycline susceptibility is not always associated with ramA expression. We also did not observe any significant mutations within the promoter regions, as has been observed previously ramA overexpression, a similar situation that is noted for the marRAB and soxRS systems ramA and acrA expression has been noted previously We chose to extend the molecular findings by determining the regulation of  4 μg/mL . A notabramA overexpression, we should consider the broader implications of these intrinsic resistance mechanisms in the development and persistence of antimicrobial resistance.Overexpression of transcriptional regulators such as RamA triggers the expression of a multitude of genes that confer pleiotropic phenotypes