In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease. Huntington disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat extension in the coding region of the huntingtin gene, leading to striatal atrophy which later expands to the cerebral cortex and other subcortical brain regions ,2. CliniHdh Q111 striatal cell lines derived from knock-in mice with 111 CAG repeats introduced into the mouse HTT homologue Hdh [Hdh150 knock-in mice and did not find any deficit under resting condition when compared to wild type controls [Hdh150 neurons were more vulnerable to calcium overload than neurons from their wild-type littermates [in situ. To further address the precise nature and the role of metabolic and mitochondrial dysfunction in HD, studies were performed in genetic models of HD, particularly in mice expressing full-length mutant huntingtin (fl-mHtt). As observed in pre-symptomatic and early HD patients, no major impairment in the enzymatic activity of the mitochondrial respiratory chain complexes I–IV was evidenced in either the striatum or the sensorimotor cortex of these mice . By contogue Hdh -23. Howeogue Hdh , suggestcontrols . Howevertermates . These rin situ, directly in the culture well, preserving the neuronal network integrity.To pursue these questions, we investigated mitochondrial respiration in specific primary neuronal subpopulations cultured from a new HD transgenic rat model expressing fl-mHtt and all regulatory elements integrated from a bacterial artificial chromosome (BACHD rats). These rats display a robust, earlier onset and faster progressive HD-like phenotype even in heterozygous transgenic rats compared to previously described HD rats expressing a mHtt fragment . Oxygen All experiments were approved and performed in accordance with internal institutional guidelines; Trophos is an accredited institution for animal experimentation in France  and in strict accordance with national and European regulations . All efforts were made to avoid or minimize animal suffering and to reduce and refine the experimentation.CAG/CAA repeats (line LY.005) [2 overdose. Rats were placed in a hermetic box then exposed to a mixture of O2/CO2 (40-60% respectively) until sleep was induced. The % CO2 was then progressively increased up to 100% while O2 was decreased (0.5L/min every 30 sec. for 3 min up to 5L/min) and then maintained for 4-5 min. Male heterozygous transgenic BACHD rats (HD) expressing high levels of fl-mHtt protein containing 97  LY.005)  were bre5’-ATGGCGACCCTGGAAAAGCTG-3’ and 5‘-AGGTCGGTGCAGAGGCTCCTC-3’ (Eurofins). PCR conditions were: 30 cycles at 94°C (30 s), 60°C (30 s), 72°C (1 min).Rat embryos from a single pregnant female rat were used for each independent experiment. All embryos were genotyped to identify WT and HD animals. During genotyping, brains were maintained in Hibernate® conservation medium (BrainBits) at 4°C. The embryos were genotyped with a PCR-based assay using DNA from tail tissue. The mHTT gene product was detected using the primers 2, 5% CO2. E17 primary cortical neurons were prepared using a similar protocol and seeded at a density of 110,000 cells/well. Striatal and cortical neuronal culture purity was controlled by immunofluorescence using NeuN antibody (Millipore) to identify neurons, GFAP (Millipore) for astrocytes and F4/80 (Santa Cruz) for microglial cells. Both striatal and cortical neuronal cultures were 98% pure. The presence of mHtt, already in the form of aggregates, was confirmed in primary striatal and cortical neuronal cultures derived from HD rats  reflecting lactate release were concomitantly measured using a XF24 extracellular flux analyzer (Seahorse Bioscience) as previously described . p-triflouromethoxyphenylhydrazone  (Sigma) was injected to determine maximal OCR (state 3u). Finally, rotenone  (Sigma) and antimycin A  (Sigma) were injected to inhibit complex 1 and complex 3 respectively and to completely abolish mitochondrial respiration. After each injection step, two measurement cycles (measurement + mix + pause) were performed (Experiments in substrate-rich media were performed using Dulbecco’s Modified Eagle’s Medium (DMEM) containing 25 mM glucose without bicarbonate (Invitrogen) and complemented with 1 mM fresh pyruvate (assay medium). One hour before the start of the respiration measurements, the original culture medium was replaced by assay medium and plates were placed into the XF analyzer calibrated at 37°C. After 10 min of equilibration, OCR was measured for 1.5 min to establish a baseline rate. The medium was then gently mixed for 2 min followed by a 3 min pause to restore normal oxygen tension in the microenvironment surrounding the cells. After baseline measurements, oligomycin  (Sigma) was injected into each well in order to inhibit mitochondrial ATP synthase to determine proton leak-dependent OCR (State 4o). After this, the uncoupling agent carbonilcyanide erformed  and afte4 2 mM, Na2HPO4 1.2 mM, pH 7.4), complemented with 2 mM GlutaMAX™ (Invitrogen). One hour before the start of the assay, the original culture medium was replaced by KHB and the plates were placed in an incubator at 37°C without CO2 and then in the XF analyzer. After 10 min of equilibration in the instrument, OCR was measured for 1.5 min to establish a substrate free basal rate. Medium was then gently mixed for 2 min followed by a 3 min pause to restore normal oxygen tension in the microenvironment surrounding the cells. After baseline measurements, the substrates D-glucose (Sigma), pyruvate (Sigma) or lactate (Sigma) were injected and OCR was measured for 1.5 min followed by injections of oligomycin, FCCP and rotenone plus antimycin A as described above. After each injection, two or three measurement cycles (measurement + mix + pause) were performed. To assay the ability of different substrates to support respiration, experiments were designed using a simple substrate-free modified Krebs Henseleit Buffer (KHB)  were validated and analyzed. Respiration parameters were calculated from data obtained during the steps of OCR measurement. For basal respiration and oligomycin respiration, the last OCR measurement of the cycle was selected for calculation. For FCCP the first OCR measurement of the cycle was selected to avoid the effect of substrates depletion on maximal OCR. For further details please refer to the legend of the respective figures. Because non-mitochondrial oxygen consumption rate (measured after antimycin A injection) was overestimated due to the fact that FCCP injection activated non-mitochondrial oxidase and because the rate was negligible (according to preliminary assays), we decided to not remove non-mitochondrial OCR from all measurements. Cell respiratory control ratio (RCR) was calculated by dividing the maximal rate (uncoupled) by the oligomycin rate (proton leak). Spare respiratory capacity was calculated by subtracting the basal rate from the maximal (uncoupled) rate. Coupling efficiency was calculated by dividing the fraction of basal mitochondrial oxygen consumption used for ATP synthesis  by the basal rate. The substrate response was determined by dividing the OCR obtained after substrate addition  by the substrate-free basal rate in KHB. Data are expressed as means ± SEM. Comparisons between two groups were performed using an unpaired Student’s t test. Comparisons of several groups or differences between treatments and experimental groups were conducted using a one way or two way analysis of variance (ANOVA) followed by Dunnett’s post test. A p-value of less than 0.05 was considered statistically significant. E17 primary striatal neurons from HD and WT rat embryos were cultured for 7 days in Neurobasal™ medium and then placed in DMEM with similar concentrations of amino acids, vitamins and glucose (25 mM) to measure OCR. Their response to the respiration analysis paradigm in substrate rich medium is shown in In first experiments, the medium used to measure respiration contained 25 mM glucose to mimic their original culture conditions. This is a high, supra-physiological concentration compared to the glucose concentrations found in brain that are estimated to be around 2.5 mM in rats . To checIncreasing evidence supports the idea that glucose is not the sole energy substrate for neurons in the brain. Oxidative substrates such as pyruvate or even lactate provided by glial cells could be supplementary fuel for neurons . ResultsTo see whether there is any difference in the capacity of WT and HD striatal neurons to use physiological glucose concentrations, OCR was measured in KHB as shown in We also evaluated HD and WT striatal neurons’ capacity to use alternative substrates, pyruvate and lactate according the protocol set up in To see how neurons respond to a mixture of metabolic substrates, OCR was measured in WT and HD striatal neurons in the presence of 2.5 mM glucose followed by addition of 10 mM lactate or pyruvate . Neither+ extrusion, ECAR could not be used as a measure of glycolysis under uncoupling conditions; therefore, data obtained after FCCP injection was not analyzed. Then, ECAR was measured in WT and HD primary striatal neurons incubated either in rich substrate DMEM or in KHB containing 2.5 mM glucose  attached to the culture plates after 7 days in culture using extracellular flux measurement technology . This alycolysis -44. When HD striatal neurons were incubated in substrate-rich media containing a supra-physiological glucose concentration, no respiratory deficits were detected. These results are consistent with previous results obtained in HD knock-in mouse primary striatal neurons  where miInterestingly, in our study, even though mHtt aggregates are present in both cell types, metabolic defects were only detected in embryonic striatal neurons and not in cortical neurons, a brain region affected later in Huntington disease progression. Indeed, cortical neurons showed neither OCR deficits nor ECAR alterations in the presence of a physiological concentration of glucose. These results provide additional insight into the mechanisms of selective striatal neurodegeneration and into the relative metabolic vulnerability of different cellular populations to mHtt toxicity. In the light of the present study, it might be argued that striatal neurons seem to have different metabolic requirements compared to cortex and may have a reduced capacity to manage substrate deprivation. Indeed, we can observe in In summary, early glycolysis defects are found specifically in HD striatal neurons. These subtle defects, observed only with levels of glucose found in brain, may only have adverse consequences after prolonged stress or in combination with other age-related declines in metabolism, explaining why neurodegeneration only becomes evident in HD gene carriers in middle age. These results can be related to new insights in pre-symptomatic carriers of apolipoprotein E4, who show reduced cerebral glucose metabolism even before Aβ aggregation and decades before the onset of AD pathology ,51. ThesProtocol S1Methods for assessing mhtt expression in primary neuronal cultures.(DOC)Click here for additional data file.Figure S1Confirmation of mhtt expression in primary neuronal cultures. Primary striatal and cortical neurons from HD rats express mhtt. SDS-insoluble and thus aggregated proteins were trapped on a nitrocellulose membrane and probed with a polyQ-specific antibody. The presence of aggregated polyQ-containing protein in primary striatal and cortical cultures from HD rat embryos but not their WT littermates indicates the expression of aggregated forms of mhtt in these neurons. Each dot is the extract from a separate neuronal culture, prepared from an individual WT or HD embryo. (TIF)Click here for additional data file.


In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state.  Voltage-gated potassium (Kv) channels are a family of transmembrane proteins which open and close potassium-selective pores in response to changes in cell membrane potential Functional, biochemical and theoretical studies suggest that the surrounding membrane can influence Kv channel function and localization. For example, Kv2.1 channels were activated in membranes in which sphingomyelin was converted to ceramide-1-phosphate in vivo is challenging as the composition and physical state of the membrane is highly regulated by the cell itself. Consequently, much work has been done to isolate membrane proteins and reconstitute them in membranes of controlled composition. For electrophysiological studies of ion channel activity, several in vitro systems have been developed including black lipid membranes (BLMs) and patch-clamp on multilamellar vesicles Studying the effects of membrane properties on channel function and localization in vitro system. These cell-sized unilamellar liposomes (−7 N/m to 10−3 N/m) Giant Unilamellar Vesicles (GUVs) are a promising alternative iposomes  can be pRealizing the potential of this system requires efficient methods to incorporate functional Kv channels into GUVs. Classically, GUVs have been prepared using a lipid film deposited from organic solvent, a procedure likely to damage all but the toughest proteins This paper presents a method to functionally reconstitute the voltage-gated potassium channel, KvAP, into GUVs. KvAP is a bacterial channel with high homology and strong functional and structural similarities to eukaryotic Kv channels 2 to block K+ permeation). Bacteria were solubilized in 40 mM DM , Affymetrix) and then purified using a polyhistidine-tag affinity column . The hexa-histidine tag was cleaved by incubation overnight with thrombin , and 1 mM TCEP (Invitrogen) added to reduce cysteines prior to final purification on a Superdex 200 10/300 GL size exclusion column . An example of an elution profile is shown in Protein purification and reconstitution in Small Unilamellar Vesicles (SUVs) were performed following published procedures 2) were deposited onto two parallel platinum wires  in a custom-made Teflon chamber  and cholesterol to achieve a final molar ratio of DPhPC∶DPPC∶Cholesterol of 6∶2∶2 (protein∶lipid mass ratio of 1∶20), a composition previously shown to produce phase-separated GUVs at room temperature The growth of GUVs on the wires was observed using phase contrast microscopy on a Zeiss Axiovert 135 microscope . For more detailed observations of vesicle morphology and fluorescence quantification, GUVs were transferred to a chamber containing a solution of glucose, NaCl or KCl and HEPES (pH 7.4) with the same osmolarity as the GUV formation buffer. Confocal images were taken using a Nikon Eclipse TE 2000-E microscope with a D-Eclipse C1 confocal head with two laser lines  and a Nikon Plan Fluor 100× oil objective (1.3 NA).Confocal images of GUVs were used to measure the protein density in the GUV membranes and study vesicle unilamellarity. To calibrate the measured fluorescence intensity as a function of fluorophore density, a classic electroformation protocol +] solution , ∼200 mM glucose); the correction for the liquid-junction potential was calculated using the Henderson equation pipette; I = −Ipipette). For ionic selectivity measurements, sections of current traces consistent with single channel gating events were identified, and a histogram of the current was fitted to two gaussian functions representing the “open” and “closed” current levels. The mean single-channel current and standard error of the mean were calculated for each membrane voltage. Voltage-gating was tested by holding the membrane voltage at −100 mV (30 seconds per cycle) before applying a transient voltage step (5.5 seconds duration) to a more positive voltage. The mean current during the interval from 0.5 s to 1 s after the beginning of the voltage-step was used to estimate the peak patch current and conductance. To characterize the relative concentration of the protein in the GUV and patch pipette, a ProSilica GC1380 camera  was used to record epi-fluorescence images of the protein (marked with Alexa Fluor 488) and the membrane . All the electrophysiological data presented in the main text were obtained at room temperature (∼20°C) using fluorescently labeled KvAP reconstituted into DPhPC GUVs grown in 100 mM KCl.The activity of reconstituted channels was studied using the patch-clamp technique The protocol to produce GUVs containing KvAP is presented before describing the characterization of protein incorporation and activity.GUVs containing reconstituted KvAP were prepared using a protocol based upon a SUV fusion/electro-formation method To confirm the method was suitable for different biophysical applications, GUVs were prepared with several different membrane compositions and different buffers encapsulated inside the GUVs.Clearly, the GUV internal solution is especially important for studies of ion channels. For a relatively “low-salt” buffer , useful GUV yields could be obtained by applying a sinusoidal voltage of 0.7 V (RMS) and f = 10 Hz for approximately two hours. Preparing GUVs with “physiological” salt concentrations has traditionally been more difficult as salt can have a significant effect on the rehydration/swelling step of GUV formation Several lipid compositions were also tested. Both DPhPC and EPC∶EPA (9∶1 by mole) proteo-SUVs allowed the production of a high yield of GUVs. Confocal images of labeled KvAP in these vesicles , showed Although the yield of GUVs depended on the exact parameters used, GUV formation was generally quite efficient with hundreds of GUVs per electro-formation chamber. The average GUV diameter also depended strongly on the exact parameters used, but was typically of the order of 10 µm see . To deteThe following sections described in detail studies of the incorporation and activity of the protein.The channel density in GUVs was determined by measuring the fluorescence intensity of the membrane in confocal images of the vesicle equatorial plane. The fluorescence signal depends upon the protein density, intensity per fluorophore and the effective area of the GUV membrane within the confocal volume. Following the approach of Galush et. al. refM, could be used to convert membrane fluorescence intensity to fluorophore density.To determine membrane intensity, confocal images of GUVs containing known densities of Bodipy-HPC were analyzed using Matlab, as shown in F, the fluorescence intensity of a molecule of Alexa-488 linked to KvAP relative to a molecule of Bodipy-HPC, was determined by measuring the fluorescence intensity of bulk solutions (SUVs and detergent micelles) with the confocal microscope. Following this calibration, the protein density in GUVs, D, could be deduced from the intensity of proteo-GUVs membrane, I, using the equation,fN is the number of Alexa 488 fluorophores per protein. This calibration method is described in detail in These measurements of the reference fluorophore were then related to the protein through the relative intensity of the two fluorophores. 2 , while allowing the diffusion of K+ into the pipette . Thus, even when the voltage across the membrane is zero, current should flow through a potassium channel as K+ ions diffuse into the pipette. As shown in + gradient drove current through the channels even when opposed by a small negative voltage (−4 mV). The dependence of mean single-channel current on applied voltage is shown in To characterize ionic selectivity, experiments were conducted in which 90% of the KCl in the pipette solution was substituted with NaCl. A potassium channel would block the diffusion of NaThe voltage-dependent gating of channels was studied by measuring the current induced by a transient step in voltage. To characterize the ensemble behavior, these experiments were performed using larger patch pipettes (diameter of ∼3–4 µm) so as to obtain membrane patches containing more channels. Patch and bath solutions both contained 100 mM KCl, and the patch was held at −100 mV between voltage steps. As shown in While it is tempting to compare the number of active channels in a membrane patch to the density of channels in the GUVs, studies have shown that the composition of the cell and patch membrane can differ Despite the great interest in the reconstitution of transmembrane proteins into GUVs, progress has been challenging because many protocols that have a high GUV yield use conditions that could damage proteins. To date, two main strategies have been employed. In the first approach, lipid-only GUVs are first prepared and the protein is then incorporated afterwards. Efforts have been made to introduce the protein by mixing GUVs with protein-detergent micelles In the second approach, GUVs are formed directly from a membrane film that already contains the protein The results presented in this paper indicate that the SUV dehydration/electro-formation method is an effective method to reconstitute KvAP into GUVs. Starting with a modest amount of material (∼10 µg of SUVs), the method yielded hundreds of GUVs per chamber with a typical GUV diameter of ∼10 µm. Like many GUV preparation methods, the protocol produced a fraction of multi-lamellar objects. However, most GUV experiments manipulate or at least analyze GUVs individually so multi-lamellar objects can be readily detected (and excluded) by fluorescence microscopy. Importantly, the protocol does not require any specific membrane components , and GUVs were grown with several different membrane compositions simply by changing the composition of the initial SUV solution. This ability to vary membrane composition is very important for studies of protein-membrane interactions. Finally, even though electro-formation has traditionally been applied using low-salt buffers, GUVs could be effectively prepared in buffers containing physiological salt concentrations by applying a voltage with a higher frequency (∼500 Hz) 2. The density of voltage-gated ion channels in cell membranes varies widely from close to zero (∼1 channel/µm2) to several thousand channels per square micron in specialized regions such as the nodes of Ranvier in the axon To study the incorporation of protein in GUVs, a calibration method was developed to allow quantitative measurements of the protein density in GUVs using only a confocal fluorescence microscope. Images of the labeled channel in GUVs showed that the protein was homogeneously distributed at optical length-scales, while measurements of protein diffusion . Although membrane proteins reconstituted into SUVs can have a net orientation, it seems reasonable that this would be scrambled during the SUV fusion and that GUVs would contain approximately equal amounts of both insertions To conclude, we have presented a method to reconstitute functional Kv channels at a relatively high density in cell-sized, defect-free unilamellar vesicles. These GUVs should be of great help in unraveling important open questions concerning the biophysics of Kv channels. For example, the interaction between channels and lipids can be directly studied by observing the lateral distribution of ion channels in GUVs containing co-existing domains. Measurements of diffusion (for example using FCS or single particle tracking) should also give insight into the effects of membrane parameters on Kv channel mobility and clustering state Figure S1Size Exclusion Column Profile of purified KvAP. The peak at 11 ml corresponds to KvAP.(TIF)Click here for additional data file.Figure S2SDS-PAGE of purified KvAP. The left image shows the gel stained with Coomassie Blue while the image on the right image shows fluorescence from the same gel (prior to staining). . Samples were run on a 12% SDS-PAGE gel and approximately 50 mM of beta-mercaptoethanol was added to samples to reduce disulfide bonds. Under these conditions KvAP runs as a monomer with an apparent molecular weight of ∼28 kDa.(TIF)Click here for additional data file.Figure S3Schematic of the electro-formation process. Droplets containing SUVs are deposited on the electrode. Partial dehydration of the solution causes the SUVs to fuse to form a stack of membranes. Buffer is then added and an AC electric field applied. As the film swells, individual membranes detach from the stack to form GUVs.(TIF)Click here for additional data file.Figure S4GUV electroformation chamber. The two platinum electrodes are mounted in a teflon block with 3 wells. The top and bottom of the wells are sealed with microscope coverslips, and the two platinum wires are connected to a signal generator via alligator clips.(TIF)Click here for additional data file.Figure S5Phase contrast image showing GUVs growing on the platinum electrode. GUVs were grown from an EPC∶EPA proteo-SUV solution . The growth buffer was 100 mM KCl, 10 mM HEPES (pH 7.4), 2 mM EDTA, and 200 mM sucrose. GUVs were formed by growth overnight with an applied voltage of 0.8 VRMS, f = 500 Hz. The scale bar is 20 µm.(TIF)Click here for additional data file.Figure S6Histogram of Proteo-GUV Diameters. GUVs were formed from EPC∶EPA SUVs using a low salt buffer , and a histogram of GUV protein density for the same population is show in (TIF)Click here for additional data file.Figure S7Representative image of objects harvested from the GUV growth chamber. A) Confocal image of GUVs containing the red fluorescent lipid, TR-DHPE (0.5% by mole). B) Corresponding signal for KvAP . The white arrows indicate examples of round vesicles with a single membrane. The fluorescence intensity of these “apparent GUVs” was then analyzed for unilamellarity. Note that the fluorescence intensity is brighter in the center of this image because of the extremely large field of view. Scale bar: 20 µm.(TIF)Click here for additional data file.Figure S8Vesicle unilamellarity. Histograms of membrane fluorescence intensity for GUV prepared with lipids only (Reference), proteins in low-salt buffer, and proteins in high-salt buffer. Each distribution was fitted to a gaussian function to determine the mean and standard deviation .(TIF)Click here for additional data file.Figure S9Dependence of protein density on GUV preparation method. Histograms of protein density for batches of GUVs prepared in low salt buffer  and high salt buffer . Note these batches represent extreme cases showing the most homogenous and heterogeneous distributions. Low-salt GUVs were not always so uniform (e.g. orm e.g.  while hiorm e.g. .(TIF)Click here for additional data file.Figure S10Variability of protein density in “high salt” GUVs is not due to multilamellarity. Protein density histogram (N = 75) of the population of high salt proteo-GUVs presented in (TIF)Click here for additional data file.Figure S11Activity of Membrane Patch from a GUV formed with low-salt buffer. A) GUV membrane patch current in response to an applied voltage step. Patch and bath solutions were both 100 mM KCl, 4 mM HEPES, pH 7.2, and the patch was formed from a EPC∶EPA (9∶1 by mole) GUV formed using low-salt buffer (5 mM) B) Section of the trace showing distinct jumps in conductance that are consistent with the opening and closing of individual channels.(TIF)Click here for additional data file.Text S1KvAP Purification Protocol. Procedures for the expression and solubilization, purification, fluorescent labeling and reconstitution into proteoliposomes of KvAP.(PDF)Click here for additional data file.Text S2Proteo-GUV formation Protocol. Methods used to produce GUVs with reconstituted KvAP, GUVs containing co-existing liquid domains and pure lipid GUVs.(PDF)Click here for additional data file.Text S3Characterization of the Proteo-GUVs. Description of methods used to characterized vesicle unilamellarity, size distribution and protein incorporation.(PDF)Click here for additional data file.Text S4Measurement of Protein Density. Explanation of the fluorescence calibration method, quantification of membrane fluorescence intensity, area of the GUV membrane in the confocal volume and fluorophores per protein.(PDF)Click here for additional data file.


There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy.In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand.A robust reference comparator set comprising following ‘scrub typhus infection criteria’ (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the ‘gold standard’ indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays.Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96–99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%.The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus. Orientia tsutsugamushi PCR result despite negative serology. These findings could reflect potential benefits of the PCR assay in detecting rickettsaemia before antibody responses set in and/or a diagnostic advantage in endemic areas with high background levels of antibody in the population. Serology is complicated by the heterogeneity of strains present in Southeast Asia, but high resource costs and training make realtime PCR assays impractical for many areas where scrub typhus is endemic. This is where the new LAMP methodology has potential: it is inexpensive, simple to perform and requires only a waterbath or simple heating block instead of a thermocycler. In the context of a prospective fever study in a scrub typhus-endemic area in Thailand, the results support the validity of LAMP methodology for the diagnosis of scrub typhus, highlight the difficulties in comparing antibody- with DNA-based methods and also contribute towards understanding the dynamics of bacteraemia in this under recognised and under studied disease.There is an urgent need for alternative diagnostic methods for scrub typhus, but evaluation of these is hampered because the current serological gold standard (IFA) is imperfect. In a study from Thailand, 3 of 20 (15%) patients with fever had a positive  Orientia tsutsugamushi, is an important acute febrile illness in the Asia-Pacific region and is endemic in Thailand and Laos O. tsutsugamushi antibodies during the acute and convalescent phases of the disease and the gold standard serological assay is the indirect immunofluorescence antibody assay (IFA) Scrub typhus, caused by However many of these criteria have limitations in scrub typhus: serology is complicated by the significant observed antigenic heterogeneity of disease-causing strains; positivity cut-off titers for single admission IgM are unreliable for different areas of endemicity and demonstrate wide ranges; dynamic serology requires a follow-up sample and cannot guide patient management, bacterial isolation while very specific is hampered by low sensitivity and requires even more time. Further the ‘diagnostic window’ for antigen- or DNA-based assays is limited and remains to be determined in scrub typhus patients. While these methods allow for a retrospective diagnosis with high confidence, to date no single diagnostic test is capable of reliably detecting all scrub typhus cases on admission. There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy groEL gene, encoding the 60 kDa Heat-Shock-Protein of Orientia tsutsugamushiIn this study we aimed to evaluate the diagnostic accuracy for a loop-mediated isothermal PCR assay (LAMP) in the diagnosis of acute scrub typhus infection, targeting the The evaluation of this assay was performed in the context of a prospective fever study in the scrub typhus-endemic environment of Chiang Rai, northern Thailand.Ethical approval for this study was granted by following local ethical committees: Chiang Rai Hospital, the Thai Ministry of Public Health, the National Ethics Committee for Health Research and the Oxford Tropical Research Ethics Committee, UK. All patients provided provided written informed consent prior to sample collection.A total of 161 patients were prospectively recruited during a fever study conducted in a scrub typhus endemic area, at Prachanukhao Hospital in Chiang Rai, Northern Thailand. Over one calendar year (08/2007-08//2008) the study recruited in-patients over 15 years old with acute fever of less than 2 weeks duration, no evidence of primary focus of infection, three negative malaria blood smears and who provided written informed consent.Full blood admission samples in EDTA and serum were collected from patients following written informed consent. Convalescent specimens were tested for serology only (defined as follow-up serum to be paired with admission sera) and were available for 138/161 patients, representing a follow-up rate of 86%. Serum samples were collected and stored at −80°C until testing. For DNA templates, buffy coat samples were collected at the clinical study sites and stored at −80°C until DNA-extraction using the Qiagen Mini Blood kit .O. tsutsugamushi pooled Karp, Kato, Gilliam antigens) and murine typhus (R. typhi Wilmington strain) IgM antibodies were detected using the gold standard indirect micro immunofluorescence assay (IFA) Scrub typhus  (ST ICT) was performed on 159/161 (99%) admission specimens according to the manufacturers instructions. Briefly, 5 µl of serum was applied to the reagent pad of the ICT strip followed by 2 drops of buffer. Results were read visually exactly 10 minutes later and recorded as positive, equivocal or negative for the presence of anti-In vitro isolation of O. tsutsugamushi was performed at a biocontainment level 3 laboratory in Bangkok using a previously described method O. tsutsugamushi was ascertained by IFA every 7 days.A previously described nested conventional PCR assay targeting the 56 kDa gene was performed, with reduction of the reaction end volume from 100 µL to 25 µL This assay used previously published primers groEL gene with a specific product of 160 bp length and melt temperature of 84.5°C. A serial dilution of plasmids served as external controls for quantitation assays and melt curve analysis was performed for product differentiation Recently we designed a highly sensitive real-time PCR primer set based on the 4, 20 mM [NH]4SO4, 0.2% Tween 20, 1.6 M betaine and deoxynucleotide triphosphates 2.8 mM each), 1 µL Bst DNA polymerase, 4 µL template DNA and distilled water to a total reaction volume of 25 µL. The reaction mixture was incubated in a realtime turbidometer  at 65°C for 120 min. Positivity, defined as detection of exponential increase of turbidity was recorded at the first time point at which the change in turbidity increased by 0.1 optical density (OD) units/sec. Buffy coat derived DNA templates were used for the prospective evaluation of LAMP.All LAMP assay reactions were performed in triplicate, each in separate runs, using the methods previously described In order for the admission diagnosis to be sufficiently robust, a panel of reference criteria was defined which included parameters of high specificity, that define scrub typhus with a high level of confidence .O. tsutsugamushi; ii. an admission IgM titer ≥1∶12,800; iii. a 4-fold rising IgM titer in paired serum samples; and iv. a positive result in at least two out of the three PCR assays described above.One or more of the following criteria had to be fulfilled for the diagnosis of ‘scrub typhus infection’: i. positive cell culture isolation of While isolation and dynamic serology criteria are retrospective by nature, they provide the strongest evidence of active disease. Similarly the second-highest possible IgM titer measured upon admission  and two positive PCR results targeting two different genes provide high specificity and confidence of diagnosis. While single admission IgM titers in endemic areas are varied, and the various cut-offs used are notoriously unreliable, we opted to choose a ‘no-compromise’ titer for criterion to achieve maximal confidence in the diagnosis. The second-highest and highest measurable titers  cannot provide a 4-fold rise, hence a cut-off titer of 1∶12,800 was chosen as the highest single-titer for positivity upon admission.in vitro isolation of O. tsutsugamushi or by a 4-fold rising IgM or IgG antibody titer in paired serum samples using the gold standard reference Diagnostic accuracy was calculated by comparing the LAMP assay results with a diagnostic reference comparator for each patient sample. Conventionally, scrub typhus infection is confirmed by Analysis included cross-tabulation of reference comparators and LAMP assay results. Probability values for the comparison of positivity rates for LAMP with other assays were determined using McNemar’s test. Clinical and demographic results are reported as medians with interquartile range (IQR) and groups were compared using the Kruskal-Wallis test. Standard diagnostic accuracy indices were calculated using Stata/SE 10.0 .Of the prospectively recruited patients, 57/158 (36%) consisted of females and 99/158 (63%) were male. The median (IQR) age for females was 44 (34–55) and for males 39 (28–48). The median (IQR) fever days prior admission of all patients was 5 (3–7), and for patients fulfilling the STIC was 6 (4–7) days of fever. All recruited patients were in-patients and as such presented with symptoms at the more severe end of the disease spectrum – to date no reliable markers or panel of parameters to define disease severity has been evaluated for scrub typhus.In vitro isolation was successful in 9/161 (6%), a ≥4-fold rise of IgM titer in paired sera was seen in 26/138 (19%), an admission IgM titer ≥1∶12,800 was present in 20/161 (12%) and two of the three PCR assays performed were positive in 27/161 (17%) of the patients (In this study of patients with ‘typhus-like illness’ 55/161 (34%) cases fulfilled the robust STIC reference criteria . In vitrpatients . The LAMpatients . The medpatients .The standard IgG-based IFA used in non-endemic areas, was performed in addition to the IgM-based IFA, but the results not used diagnostically in this manuscript as the patient samples are derived from a highly endemic area. High endemicity, presumably, resulted in a high number of patients with ‘diagnostically high’ admission IgG levels (n = 61/161) based on a 1∶400 cutoff titer groEL realtime PCR assays detected 35/161 (22%) samples, followed by the PanBio ICT 31/161 (19%) and the 47 kDa realtime PCR assay 28/161 (17%). Using the STIC criteria as reference comparator, the LAMP assay demonstrated high diagnostic specificity (95% CI) of 94% (88–98) and a sensitivity of 53% (39–66), and the IgM-based rapidtest PanBio ICT performed with a sensitivity of 47% (34–61) and a specificity of 95% (89–98). When the LAMP and PanBio ICT IgM rapid test were combined, an increased sensitivity of 67% (53–79) was achieved with a specificity of 91% (83–95). Comparisons of the groEL, 47 kD and 56 kD PCR were not performed as they are constituents of the STIC criteria.The currently most practical diagnostic assays for use in the setting of acute illness (‘point-of-care’) are ICT rapidtests and, with more limitations, realtime PCR assays. LAMP was assessed against these assays in this analysis. LAMP and To understand the relative (dis)advantages of a molecular test when compared against the broad diagnostic panel, the discrepancies in positivity agreement between the assays were dissected at the individual diagnosis level .Discordant results with STIC criteria: In the ‘admission IgM ≥1∶12,800’ group, the number of LAMP negative cases was 6/20 (30%). Despite high admission titers, these cases had low support from the other PCR assays  and in the ‘≥2/3 PCR positive’ group there were 5/27 (19%) LAMP negative cases, which had strong support through very high positivity rates in the other PCR modalities. In the ‘isolation positive’ group 2/9 (22%) LAMP negative cases were found, one of which was realtime PCR positive. The largest proportion of STIC sub-criterion positive results that were discrepant to LAMP results was found in the ‘dynamic serology’ group, which included 17/26 (65%) cases. However these LAMP negative cases demonstrated low serological and low PCR support by the other modalities .groEL and 47 kD realtime assays (both 17%), and the antibody-based rapidtest PanBio ICT was lowest with 8%  markedly dropped to approximately 20% at 10 ‘days of fever’. The PanBio ICT assay, after low positivity rates in the early phase of illness, demonstrated a steady increase of sensitivity and surpassed the diagnostic efficacy of DNA based assays at day 10 with a 50% positivity rate  diagnostic criteria in the acute setting, but also in relative terms, compared to available diagnostic assays used upon admission, thus supporting clinical management. This form of evaluation was challenging for LAMP, as it was based on a prospective cohort in an endemic area using broad robust diagnostic criteria across different methodologies.This study aimed to assess the true diagnostic utility of LAMP in a clinical setting, by evaluating its diagnostic accuracy, in O. tsutsugamushiin vitro isolation methods to determine a broad final diagnosis with high specificity. The perfect diagnostic tool for the diagnosis of scrub typhus would have to cover the same (or an even greater) patient spectrum in a single assay, as the STIC criteria do comprising multiple assays.The increased use of molecular diagnostic methods in the diagnosis of scrub typhus, has highlighted the difficulties of comparing DNA-based with antibody-based assays Unfortunately the absolute sensitivities of all individual assays that are currently useful in the acute setting  were lower or equal to 56% for the diagnosis of scrub typhus, when compared to the retrospective and definitive STIC criteria . The low sensitivities for these individual assays reflect the current inability of a single assay to cover the spectrum of diagnostic positivity for all ligands included in the STIC criteria. On the other hand, it underlines the usefulness of these criteria for evaluation of different diagnostic methods, and also demonstrates how severely under-diagnosed this disease is, as almost every second case may go undiagnosed upon admission, even if the currently most sensitive methods are applied.On dissection of the STIC criteria by subgroup analysis, major discrepancies between the LAMP assay and the criterion ‘dynamic serology’ were revealed. The highest level of disparity occurred when admission sample IgM antibody titers determined by IFA were low (i.e. median titers of 1∶50 rising to 1∶400). Only a very small proportion of patients with a dynamic rise in IgM antibody titer that exhibited low IgM antibody titers had additional supportive evidence of acute scrub typhus . AlternaO. tsutsugamushi in vivo) but patient IgM levels continue to rise, allowing the PanBio ICT to outperform the LAMP assay at this timepoint. This finding supports the potential benefit of combining the LAMP  and PanBio IgM ICT  as the two most practical POC assays; in an ‘OR’ Boolean operator manner. The benefit of combining these tests is reflected by an increase of the overall diagnostic sensitivity to 67% (95% CI: 53–79), when compared to STIC criteria. With a total of 47/161 (29%) patients diagnosed upon admission and a specificity of 91% (95% CI: 83–95) this modality demonstrated the best option for diagnostic use in the acute setting and approached the diagnostic accuracy of the retrospective STIC reference criteria.The intended ideal role for the LAMP assay is in the acute point-of-care (POC) setting where there is limited availability of sophisticated molecular diagnostics. Comparison of the relative positivity rates of LAMP and the easy-to-use PanBio ICT against the STIC criteria revealed slightly better sensitivity for the LAMP assay 53% vs. 47% for the ICT, but similar specificities of 96%. While the overall agreement of positive and negative results for both assays was 82% (131/159), the agreement of positive results was low at 68% (19/28). This finding could be explained in a dynamic context as in the early stages of infection high LAMP positivity paralleled low PanBio ICT positivity rates . After 1Further, the combination of these two assays also expanded the temporal spectrum of diagnosis in the acute setting, as it utilises DNA and/or antibody positivity endpoints. This approach has the potential for future improvement if advances in antibody-based ICT technology and simplification of DNA extraction methods are made available in the future. As an analogy, the combination of Dengue NS1 antigen and IgM antibody assays for the diagnosis of acute dengue infections has dramatically increased the sensitivity of admission diagnosis It is notable, that the median number of ‘fever days’ prior to admission for all patients from N-Thailand was 5 with an IQR of 3–7 and this diagnostic window is sufficiently covered by the LAMP assay.In conclusion, in a clinical evaluation against a broad and robust panel of diagnostic criteria the LAMP assay performance was similar to that of the realtime and conventional nested PCR assays. The diagnostic discrepancies between PCR assays and ‘dynamic serology’ were associated with low admission IgM antibody titers (i.e. low antibody titers and PCR negativity) and warrant further investigation into endemic background antibody levels, which should include consideration of potential immune cross-reactivity or non-specific polyclonal activation due to other diseases.The LAMP methodology was unsurprisingly unable to cover the whole spectrum of diagnostic capabilities as defined by our STIC criteria. However the combination of a DNA- and antibody-based detection method increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute POC phase of scrub typhus. The LAMP assay is simple and inexpensive, performs like a realtime PCR and can be considered a valid molecular method for the early diagnosis of scrub typhus.


Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence. Since the proposal of the Free Radical Theory of Ageing The role of ROS in replicative senescence, the permanent arrest of the cell cycle in normally proliferating cells such as fibroblasts, is not well understood.in vitro until senescence underwent time-dependent accumulation of damage. Senescent cell populations were found to have increased production of ROS Early evidence suggested that cells which were continuously passaged More importantly, interventions which decreased ROS in cells were found to impact on replicative senescence. Exposure to low ambient oxygen concentrations However, evidence has emerged suggesting a novel role for ROS in replicative senescence. It has been reported that the activation of key players in the senescence pathway contributes to ROS generation, without cells undergoing time-dependent damage accumulation. Signalling via Ras However, one question remains: how can we reconcile data suggesting a gradual ROS increase preceding induction of senescence with the observation that ROS increases in a rapid, step-wise manner as a consequence of signalling events, similar to apoptosis?Cross-sectional observations of ROS and ROS-derived products in populations of cells at different Population Doublings (PDs) are consistent with a hypothesis of a gradual, cumulative increase in ROS generation. However, cell populations are highly heterogeneous hTERT) passaged 90 PDs beyond the Hayflick limit, which is not consistent with a hypothesis of gradual accumulation of damage in cells, but is consistent with our alternative hypothesis and our model.To test our alternative hypothesis we developed a Stochastic Step Model of Replicative Senescence, describing discrete, stochastic changes in cellular ROS levels upon transition from proliferation to replicative senescence. We present a dataset describing increasing rates of Mitochondrial ROS production with increasing PD in populations of human MRC5 fibroblasts, repeatedly passaged until they reach replicative senescence. By comparing experimental observations with model predictions we demonstrate that the gradual, continuous ROS dynamics observed in these populations could also be explained by stochastic, discrete transitions between proliferative and senescent states at the cellular level without any recourse to gradual accumulation of damage in individual cells. In other words, the increase of ROS observed during long-term cultivation of primary cells could be a consequence of the senescent phenotype rather than a gradual accumulation of damage as earlier proposed. We demonstrate that, in mixed populations of senescent and proliferating cells, those cells which are not expressing senescence markers  and the fraction of senescent cells (s):s(PD), from an observed growth curve for that population as described previously s(PD) from hundreds of parametric estimates generated previously between PD25 and PD47 and constructed an estimate for Mixϕ as a function of population doubling number:ϕ subject to assumptions about the covariance between phenotype distributions for proliferating and senescent cells.We can then describe MSMM) rate as our phenotype ϕ, expressing it as a function of population doubling number as in Eq. 5. To compare model predictions with observed data, we estimated Pϕ as the mean normalised ROS production rate observed in a population of cells which are known to be >95% proliferating (e.g. a population with low doubling number (<30), Sϕ/Pϕ, where Sϕ is similarly estimated from a population of cells known to be ∼100% senescent (e.g. a population with doubling number close to the Hayflick limit which has stopped dividing). Model predictions for intermediate, mixed populations were plotted and compared with experimental observations  and fully senescent cells (PD45) using GraphExtract2.5 for Windows. We converted these to probability density distributions and sampled from these repeatedly to simulate mixed populations of senescent and proliferating cells consistent with the step model and tested simulated mixed populations for bimodality as above , 41, PD44 and PD45) we simulated 10,000 cells, assigning a probability that cells were proliferating or senescent, based on the uncertainty in s(PD), Pϕ. Similarly, for each cell classified as senescent, we sampled a MitoSOX fluorescence observation from Sϕ. We then plotted the simulated mixed populations alongside observed intermediate distributions and observed good agreement .We also investigated whether predicted population distribution changes could be adequately predicted by the SSMRS. To do this, we considered MitoSOX fluorescence alone as our phenotype Human embryonic lung MRC5 fibroblasts were obtained from ECACC  and cultured until reaching senescence. Cells were considered senescent when positive for Sen-β-Gal and negative for proliferating markers (BrdU and Ki67).30 with the human catalytic subunit (hTERT) of the enzyme telomerase as described in 100. No cytogenetic abnormalities were observed.MRC5 cells were transfected retro-virally at PDMouse ear fibroblasts (MEFs) were obtained from C57Bl6 wild-type mice at 3 months of age.2) in a 3-gas incubator. Cells were always passaged before confluency.All cells were grown in DMEM supplemented with 10% foetal calf serum, 2 mM glutamine and 1% pen/strept under controlled conditions  for 10 min at 37°C. Cells were washed with PBS and fixed with 2% paraformaldehyde for 5 min.Immunofluorescence was then conducted as described in http://rsb.info.nih.gov/ij/). For each experiment a minimum of 50 cells were analysed.All fluorescence microscopy was performed using a Leica DM5500B fluorescence microscope, using a 40×1.3 NA oil objective. Fluorescent images were always taken under identical excitation and emission condition. MitoSOX intensity was quantified using ImageJ (18 until replicative senescence (PD47) and measured average population MitoSOX fluorescence by flow cytometry. We observed an exponential increase in population MitoSOX fluorescence with PD (MSMM) we approximated each of the fluorescence time courses from distinct, replicate cell populations by linearly interpolating between observations (27), giving estimates of fluorescence kinetics across the full range of observed PDs. We constructed MSMM from separately normalised MitoSOX and mitochondrial mass observations. We modelled MSMM dynamics with the stochastic step model described above, setting P = MSMMϕ observed at PD27 and Sϕ = MSMM observed at PD47. We repeated this analysis for normalised MitoSOX and mitochondrial mass separately. Modelled population mean MitoSOX fluorescence, mitochondrial mass and MSMM increased in an approximately exponential manner with PD  and from populations of senescent cells (PD47) and analysing the resultant mixture distributions.To test if this observation was consistent with our step-model we simulated MitoSOX distributions of a population containing a mixture of proliferating (50%) and senescent (50%) cells by mixing experimental observations from cell populations containing predominantly proliferating cells  was a quantitative marker of senescence in both human and mouse fibroblasts Firstly, we performed MitoSOX staining on MRC5 fibroblasts at various PDs e.g.  and quanSecondly, we combined MitoSOX staining and immunofluorescence against Ki67 and γH2A.X . We obseThe same exponential increase in MitoSOX fluorescence, consistent with the step model, was confirmed in fibroblasts isolated from C57Bl6 wild-type mice ears. During the proliferative stage, no differences in MitoSOX staining were observed. Once the fibroblasts reached senescence a two-fold increase in MitoSOX fluorescence was observed .hTERT in human MRC5 fibroblasts at PD30We ectopically overexpressed in vitro studies, cross-sectional samples of passaged cell populations are taken after various population doublings. Cell age is typically classified on a population-level by PD, indicating the mean number of divisions undergone since an initial progenitor cell. However, it has been extensively described that cell populations are heterogeneous in terms of their division potential, even in clonal populations derived from single progenitors During in vitro senescence. The inability of cells to deal with atmospheric oxygen concentration has been proposed as a major contributor to the time-dependent accumulation of several types of damage, such as lipofuscin, mitochondrial dysfunction, protein oxidation and DNA damage As cells reach senescence, a progressive increase in ROS generation can be observed in vitro cultivation does not lead to time-dependent ROS generation in proliferation-competent cells and that the majority of ROS generated is a consequence of processes which occur secondarily to cells becoming irreversibly arrested.Recently, it has been demonstrated that ROS were necessary for the long-term maintenance of DDR and for the stability of growth arrest which characterises the senescent phenotype Our observations are consistent with such a concept. Firstly, proliferating cells  appear to be unchanged in terms of time-dependent ROS accumulation. Secondly, a model simulating a step-like ROS increase in senescent cells is able to explain population kinetics of ROS generation during serial cultivation of populations of human fibroblasts.Other factors linked to senescence will also contribute to this ROS increase. The fact that senescent cells are larger and contain more mitochondria Although our model can explain the major changes occurring during cultivation, we cannot invalidate damage accumulation as a contributing factor to the induction of senescence, since it has been well demonstrated that interventions reducing oxidative damage can extend replicative lifespan and reduce fractions of cells bearing senescent markers The model we present here is distinct from the hypothesis that ROS increase is a consequence of time-dependent damage accumulation in proliferating cultured fibroblasts. While both hypotheses are consistent with cross-sectional, population average experimental observations from fibroblasts undergoing the transition to senescence, new experimental observations presented here, particularly ROS immutability in proliferation-competent cells in old cell populations and in hTERT-overexpressing cells dividing well past the Hayflick limit, strongly support our stochastic step model.Further experimental dissection of these alternate hypotheses might greatly improve our understanding of the ageing process since cells expressing various senescence markers, including DNA damage foci, do accumulate in human in vitro models to the in vivo context, as further experiments need to be conducted to ascertain the role of ROS produced by senescent cells in vivo.The association of senescence with a sudden, large increase in ROS production might be responsible for the negative impact of senescent cells on their tissue environment and contribute to organismal ageing. However, one needs to be cautious when extrapolating results from Nonetheless, supporting this hypothesis is the finding that mice with critically short telomeres, the late generation TERC−/−, exhibit increased fraction of senescent cells To fully discriminate between a hypothesis of gradual accumulation of ROS during senescence and our alternate hypothesis of discrete stochastic changes in heterogeneous populations, and to develop our understanding of the causes and consequences of replicative senescence, longitudinal studies of single cells and their lineages are now required.Figure S1The stochastic step model of replicative senescence predicts unimodal phenotype distributions for mixed populations of cells undergoing transition to senescence. A) MitoSOX fluorescence per cell quantified from microscopy images for a proliferating population  and a wholly senescent population  of MRC5 fibroblasts. MitoSOX levels for both populations were modelled as following gamma distributions (red and blue curves respectively). A mixed population of 500 cells (50% senescent) was simulated by sampling from the two distribution models and the kernel density estimate for the mixed population is shown here (black dashed line). 500 is a reasonable emulation of a cell population which can practically be observed. B) Frequency distributions from 19 and PD45 were digitised and replotted here after conversion to probability density distributions (red and blue solid lines respectively). Assuming the step model describes the change in cell volume in these cells, simulating mixed populations of proliferating and senescent cells by weighted random sampling from distributions from PD19 and PD45 does not produce bimodal cell volume distributions .(EPS)Click here for additional data file.Figure S2The stochastic step model of replicative senesence predicts the shape of MitoSOX distributions for mixed populations of cells undergoing transition to senescence. A) MitoSOX fluorescence density distribution obtained by flow cytometry of MRC5 fibroblasts at PD37.9. Approximately 90% of cells at this PD were proliferation competent (see panel F); B–D) For PD41.1 (B) PD44.1 (C) and PD45.1 (D) we simulated mixtures of 10,000 cells, assigning a probability that cells were proliferating or senescent by analysis of cell growth curve (see panel F) and randomly sampling from distributions at PD37.9 and PD46.4 depending on whether cells were classified as proliferating or senesecent. Density distributions show that the simulated mixed populations (red line) and experimental data obtained by flow cytometry (black line) are in good agreement; E) MitoSOX fluorescence distributions obtained by flow cytometry of MRC5 fibroblasts at PD46.4. The majority of these cells at this PD were senescent. F) Senescent cell fraction dynamics estimated from analysis of growth curves. Solid black line is mean cell fraction estimate and dashed lines represent 95% confidence range. Vertical blue lines represent PDs for model calibration distributions . Vertical red lines represent PDs for intermediate model validation distributions (B–D).(PDF)Click here for additional data file.


Mus musculus  and Homo sapiens  tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties.Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both  The traditional view of tumorigenesis has long been considered in the perspective of oncogenes and tumor suppressor genes governing diverse cellular processes such as cell proliferation, survival, migration, and metastasis, with a prominent focus on understanding the cellular imbalance existing among these two populations of nuclear encoded genes. However, the importance of the surrounding tumor environment has begun to emerge as an active participant in orchestrating many aspects of tumor growth and progression including invasion, metastasis, and angiogenesis DCN) located on chromosome 12q23 which contains two alternatively-spliced leader exons DCN transcriptional activity is induced by hypomethylation of its promoter Borrelia burgdorferi, the spirochete responsible for Lyme disease in humans The stromal-specific decorin is encoded by a large and complex gene  Under growth conditions, β-catenin and Myc are stabilized by activation of Wnt and various RTK signaling pathways, which are often hyperactive in cancer. Thus, upstream activation of these signaling pathways leads to inactivating phosphorylation of GSK-3β by PI3K/Akt to allow for translocation of active β-catenin and Myc into the nucleus where, through association with various nuclear co-activator complexes, enable induction of a large number of genes including Myc itself Decorin is also involved in the regulation of angiogenesis Thus, we wished to deepen our molecular understanding of its potential role in modulating the tumor stroma by directly investigating, at high resolution, the gene expression signature of a triple negative breast carcinoma microenvironment. Therefore, for the first time, we were able to dissect the complex inter-relationship present between the tumor and the stroma by utilizing a novel high-density microarray platform capable of simultaneously measuring expressed transcripts derived from both the mouse (i.e. the stroma) and the neoplasm  via interrogation of orthotopic tumor xenografts treated systemically with decorin.Mrgpra2, Siglech, Irg1 and Il1b to be potently suppressed concurrent with Zc3hav1, Peg3, and Bmp2k induction. These data posit for decorin protein core as a potent tumor repressor by attenuating inflammation and metastasis, which constitute several hallmarks of cancer.In this study, we report the stromal gene expression signature obtained with systemic delivery of decorin protein core and discover a total of 374 genes, which showed differential expression profiles. Further, and unexpectedly, we found by DAVID gene ontology analysis, a predominant inhibition of inflammatory and immune response genes with concurrent induction of various tumor suppressors and cellular adhesion molecules. Moreover, independent verification of the top 12 genes demonstrating >2-fold up or down regulatory changes identified in vitro, enhanced apoptosis and blocked EGF- and FGF-mediated evasion from Matrigel (not shown), indicating that decorin exerts similar inhibitory activity on EGFR and Met dependent pathways as shown for other carcinoma cells We performed experiments utilizing a very aggressive MDA-231(GFP+) triple-negative breast carcinoma cell line. We discovered that decorin evoked growth inhibition P-value, and then by fold change. Genes with P<0.05 were placed into upregulated and downregulated groups as illustrated with the resultant heat maps. We have deposited the results of the microarray data sets in the publically-available GEO System with accession number GSE37937. Global expression profiles comparing human breast carcinoma in decorin treated when compared to vehicle control showed that most of the genes in two independent experiments (n = 6 each) showed close grouping with only minor changes (P<0.05) changes in any of the human-derived genes. In contrast, the number of genes derived from the tumor stroma of decorin-treated animals showed significant changes, with both upregulated and downregulated genes . The mixed gene expression chip allows three arrays per slide with each array exhibiting 720,000 probes representative of transcripts originating from both human neoplastic cells and mouse stroma components. Appropriate sample labeling of the validated cDNA was followed by hybridization to the 3×720 K array followed by scanning using the Roche NimbleScan software, which normalizes the expression data using quantile normalization as determined by the Robust Multichip Average algorithm. Expression data were tested for significance using a mixed effects model ANOVA in R. Genes were sorted by  changes . Indeed,ed genes . To furtery well .These findings show that systemic delivery of decorin in this triple-negative orthotopic breast carcinoma xenograft predominantly affects the global gene signature of the tumor microenvironment.P<0.05. Utilization of the Database for Annotation, Visualization, and Integrated Discovery (DAVID v6.7) Gene ontology (GO) analysis was performed on the microarray data set comprising the 374 differentially expressed genes exhibiting P = 0.0014) among the differentially regulated stromal genes followed by contractile fiber genes and those responsible for orchestrating the general immune response , Lipg , and Il1b . Siglech is a member of the Ig gene superfamily, which binds avidly to sugars and is the mouse homologue of SIGLEC-15, the only known family member to maintain complete conservation throughout vertebrate evolution Other suppressed immunomodulatory genes of interest included Siglech  followed by cytoskeletal components , and cell cycle regulatory genes   . The celThe top stromal upregulated gene was a kinase induced by Bmp2, known as Bmp2 inducible kinase (Bmp2k). Bmp2k is a largely unknown and poorly investigated nuclear-localized serine/threonine kinase that represents another candidate gene not yet implicated in human breast cancer and might represent an important target for decorin bioactivity as well as a potentially novel biomarker for tumor progression.P = 0.0011), a transmembrane glycoprotein containing Ig-like C2 modules in the ectodomain Another decorin-induced stromal gene was cell adhesion molecule 1  and it encodes a zinc finger transcription factor (Cys2His2 variety) of the Krüpple-type family. Similarly to Cadm1, this gene is also frequently silenced by promoter hypermethylation and/or loss of heterozygosity in several tumors including ovarian cancer An additional decorin-induced target classified as a tumor suppressor gene within the stroma, was Peg3  we therefore designed primers using the Universal Probe Library  stromal genes. Thereby, we have rigorously authenticated our primers in both M. musculus cDNA, harvested from NIH3T3 cells (positive control), and Homo sapiens cDNA as derived from HeLa cells (negative control for primer specificity). Our primers were capable of differentiating and recognizing only M. musculus mRNA species, in contrast to H. sapiens mRNA as validated by qPCR under identical thermal cycling conditions  and for Bmp2k, Cadm1, Hey1, Peg3, Brd4, Zc3hav1 (upregulated genes). We were able to recapitulate and confirm the gene expression signatures for four (∼67%) of the downregulated genes  and for three (50%) of the upregulated genes   could not be faithfully reproduced due primarily to unfavorably high thermal cycle value or lack of amplification and detection under the cycling conditions employed, suggesting low transcript number.Utilizing mRNA extracted from the same tumor xenografts (systemic decorin treatment for 23 days at 10 mg/Kg) and that used for the microarray experiments, we performed qPCR as a method to validate stromal gene expression for P<0.001) . The remPeg3) within the stromal compartment as well as implicating a novel role for Bmp2k in breast cancer samples.These data suggest a modulation of immunological genes, consistent with the aforementioned DAVID gene ontology analysis. Importantly, we demonstrate a reproducible induction of a presumed tumor suppressor gene  as the host for the tumor xenografts and added human mammary carcinoma MDA-231 cells to this feeder layer of primary mammary fibroblasts. Once attached, co-cultures were serum starved for 24 hours followed by chronic treatment with decorin (100 nM) for 3 days whereupon RNA was harvested and prepared as a mixed cDNA population. We focused on the expression pattern of the seven previously verified genes as reported above. Analysis of these stromal genes in context of the co-culture via qPCR revealed a recapitulation of the expression patterns of Peg3, Bmp2k, and Zc3hav1 (P<0.001), the same stromal genes verified as induced by decorin within the tumor xenografts. However, the extents of induction among these genes were not as dramatic as shown for the verification in vivo .It is of note to mention that expression patterns of the immunomodulatory genes could not be reconstituted since our cultures lacked immune system components and/or fibroblasts that do not normally express these genes. Finally, and although pression ; P>0.05.Collectively, these findings indicate that decorin is capable of targeting the tumor stroma in addition to the well-established anti-oncogenic activity targeting the carcinoma cells.in vivo relevance of our data, we determined the expression of Peg3 and Bmp2k in the same orthotopic mammary tumor xenografts as used above. Systemic treatment with decorin resulted in an induction of Peg3  or decorin (200 nM) for up to 3 days. We found a marked increase of both Peg3 and Bmp2k expression in treated cells compared to controls as detected by immunoblot . To furt of Peg3  as well  of Peg3  and Zc3h of Peg3  within tPEG3 expression in different publically available breast cancer microarray datasets using the ONCOMINE database and gene microarray data analysis tool PEG3 mRNA levels detected in various ductal breast carcinoma samples. Two independent studies PEG3 mRNA expression when compared to normal, non-neoplastic controls. Precisely, the dataset by Karnoub et al. reported a 3.1-fold decrease (P = 3.6×10−6) of PEG3 mRNA within invasive ductal breast carcinoma  of PEG3 expression was found in ductal breast carcinoma  to invasive ducal carcinoma (IDC), providing evidence that these cell types all participate in tumorigenesis. Park and colleagues more recently obtained gene expression profiles of both epithelial and stromal compartments from the same tumor biopsy using laser capture microdissection (LCM) and described a stromal gene expression profile capable of predicting clinical outcome in situ-to-invasive growth transition A current prevailing view is that tumor-associated stroma is activated by cancer cells to foster tumor growth by secreting growth factors, enhancing angiogenesis, and facilitating cell migration, ultimately culminating in metastasis to remote organ sites Similarly, analysis of the tumor microenvironment by the same authors revealed, in analogy to the epithelium, that the tumor stroma undergoes extensive gene expression alterations even at the pre-invasive stage of DCIS, supporting the view that paracrine mechanisms play an important role The anti-tumorigenic properties of decorin have been presented as a stromally derived tumor repressor that functions by directly binding to receptor tyrosine kinases situated on the tumor cell membrane We found that soluble decorin protein core, acting in a paracrine capacity, is capable of substantially altering the stromal gene signature as evidenced by the differential modulation of 374 stromal genes. Gene ontology analysis revealed unexpected enrichments for suppressed genes constituting immune responses while concomitantly inducing cell adhesion and tumor suppressor genes.Mrgpra2, Siglech, Irg1, and Il1b (downregulated subset) and Peg3, Bmp2k, and Zc3hav1 (induced subset). Establishment of co-cultures (primary mouse mammary fibroblasts and MDA-231 cells) allowed recapitulation of the mRNA expression pattern of the upregulated genes only; our co-culture models lacked immune-competent cells, thus the downregulated subset was not detected. Seemingly, our data presented above seem to contradict a recently published article indicating that decorin advocates for a more pro-inflammatory tumor microenvironment Peg3, Bmp2k, and Zc3hav1 were independently corroborated via immunofluorescence and immunoblotting in not only tumor xenografts but also in co-culture conditions, respectively, and revealed, for the first time, a novel role for Peg3, Bmp2k, and Zc3hav1 in breast cancer progression.Subsequently, the array yielded reproducible signatures for Peg3 represents a unique and distinctive class among the repertoire of inherited genes by virtue of its genomically imprinted status, where in this case, the paternal allele is exclusively responsible for expressing a molecule which harbors 12 Krüppel-type zinc finger domains with two proline-rich periodic repeat domains, and is frequently rendered inactive in several malignancies BMP2K variant, was reported to be strongly correlated with the development of high myopia Our second candidate gene is Bmp2k, a poorly understood serine/threonine kinase that plays a role in skeletal development where activation of Bmp2k is able to attenuate osteocalcin expression concomitant with reduced osteoblast differentiation Lastly, we were able to confirm induction of Zc3hav1 , a gene hypothesized to encode CCCH-type zinc finger that is largely thought to prevent viral infection by retroviruses Moreover, the data obtained from our co-culture suggests a potent modulatory effect on cancer-associated fibroblasts that will have a broad impact on tumor progression. Therefore, the changes in differential expression, as reported above, reflect gene signatures operative within the tumor microenvironment and thus makes assessing whether or not these changes are the result of direct effects on the tumor proper difficult. Thus, excluding the possibility of decorin protein core signaling via the fibroblast α2β1 integrin receptor, it is plausible that decorin is targeting the tumor cells directly by suppressing EGFR and Met signaling , and thereby functions as a paracrine agent to elicit changes within the surrounding tumor stroma. However, this model cannot exclude decorin binding directly to receptor tyrosine kinases within the stroma . Importantly, we have reported that decorin severely inhibits Myc function through targeted proteasomal degradation in vitro and in vivo revealed and confirmed a novel involvement for Peg3, Bmp2k, and Zc3hav1, for the first time, in breast carcinomas where, future studies may reveal direct functions related to modulating the surrounding tumor environment as dictated by decorin. These findings provide a new paradigm for decorin protein core in controlling the tumor microenvironment as a fundamental biological mechanism with great implications for curbing tumorigenic growth by the induction of novel tumor suppressor genes within the stroma and for the discovery of novel gene signatures that could eventually help clinical assessment and prognosis.In conclusion, validation of the mixed microarray data as it pertains to stromal gene changes via several experimental methodologies performed 2 in accordance with institutional guidelines. (Approved Protocol # 196 G).All animal studies were approved by the Institutional Review Board of Thomas Jefferson University. At the end of each experiment, mice were anesthetized with isoflurane and euthanized with COMDA-MB-231 triple-negative breast carcinoma cells were obtained from American Type Culture Collection . MDA-231(GFP+) were previously described 6 within a Celligen Plus bioreactor Decorin protein core was purified as described elsewhere 6 MDA-231(GFP+) cells into the upper left mammary fat pad. Two weeks later, once tumors were established, mice were randomized into two groups. Half the mice received a daily intraperitoneal injection of recombinant protein core (10 mg/Kg). The controls received 100 µl of PBS. Three independent experiments were performed. Tumor growth was measured every day with a micro-caliper according to the following formula: V = a(b2/2), where a and b represent the larger and smaller diameters of the tumor, respectively. At the end of the experiment (day 23) mice were taken to the Small Animal Imaging Facility of Thomas Jefferson University and in vivo expression of GFP within the xenografts was analyzed with a Kodak In-Vivo Multispectral Imaging System FX. Soon after all animals were sacrificed and all major organs were dissected and fixed in formalin, whereas tumors were cut in half and one half was snap-frozen in liquid nitrogen subsequent to RNA extraction whereas the other half was embedded in OCT compound (x) and frozen at −20°C. 10 µm thick cryostat sections were cut from the blocks and mount on superfrost slides which were then subjected to immunofluorescence staining.All the animal studies performed were approved by the Institutional Review Board of Thomas Jefferson University. Twelve severe-combined immunodeficient (SCID) hairless female mice  were injected with 2×10Immunofluorescence studies were performed as described before 2 dishes coated with gelatin and cultured in growth media containing fungizone. After a week, the fibroblast culture was switched to growth media and passaged accordingly.Female wild type C57BL/6J mice were used to isolate primary mammary fibroblasts. Briefly, the fourth and fifth mammary glands were removed aseptically and minced with surgical blades. All the fragments were collected and diluted in 10 ml of digestion media  and incubated in a shaker (2 hours at 37°C) with gentle shaking. The solution was then centrifuged 10 minutes at 1000 rpm in order to eliminate floating fat cells. The obtained pellet was washed twice in 10 ml growth media  containing fungizone. The cell pellet was then disaggregated by gently pipetting up and down with a 1 ml pipette tip. The cell resuspension was plated in 6-cm5 MDA-231 mammary carcinoma cells. The co-cultures were allowed to incubate for several hours for engagement of the fibroblast feeder layer. Co-cultures were then serum starved overnight whereupon chronic decorin treatment (100 nM decorin protein core) began for a total of three days. Arrival of the end-point resulted in lysing of the co-cultures in either a sufficient volume of RIPA buffer for immunoblot analyses or TRIzol (Invitrogen) to isolate total RNA and subsequent cDNA synthesis for gene expression analysis.For co-culture experiments, sub-confluent primary cultures of mouse mammary fibroblasts were seeded with 1.0–1.5×10in vivo samples, MDA-231 tumor xenografts tissue samples treated systemically with decorin protein core (10 mg/kg) for 23 days were snap-frozen with liquid nitrogen and homogenized with a mortar and pestle prior to solubilization in 2 ml of TRIzol , or alternatively, at the end point of the co-cultures (following three days of chronic 100 nM decorin protein core exposure), 6-cm2 dishes were lysed in 2 ml of TRIzol. Subsequently, isolated total RNA (1 µg) was annealed with oligo(dT) primers, and cDNA was synthesized utilizing the SuperScript Reverse Transcriptase II (Invitrogen) according to the manufacturer’s instructions. Gene-specific primer sets for Mus musculus mRNA . All samples were run in triplicate with quadruplicate plate replicates on the Roche LightCycler 480-II Real Time PCR platform  and the cycle number (Ct) was obtained for each independent amplicon reaction followed fold change determination via the Comparative Ct method for gene expression data analysis. Delta Ct (ΔCt) values are representative of normalized gene expression levels with respect to Actb. The delta delta Ct (ΔΔCt) values represent experimental cDNA (samples treated with decorin protein core) minus the corresponding ΔCt of the calibrator sample (control samples). Finally, the reported fold change represents an average of the fold changes as calculated using the double ΔCt method (2−ΔΔCt).Independent authentication of gene expression analysis was carried out by quantitative real-time polymerase chain reaction (qPCR) to confirm gene changes. Briefly, for refer to  were rigFigure S1SDS-PAGE gel demonstrating the purity of the decorin protein core preparation. SDS-PAGE gel representing increasing amounts  of decorin protein core run in parallel with increasing amounts of BSA (2 and 5 µg), as indicated. The gel was subsequently stained with colloidal Coomassie blue for highly sensitive detection (as low as 5 ng) of any co-purifying bands in the decorin protein core preparation used. The left lane indicates the migration of the molecular mass (kDa) of standard proteins.(TIF)Click here for additional data file.Figure S2Systemic administration of decorin protein coreinduces Zc3hav1 levels in MDA-231(GFP+) xenografts.A–B: Immunofluorescence images of control and decorin-treated MDA-231(GFP+) tumor xenografts, reacted with an anti-Zc3hav1 antibody. Mice bearing MDA-231(GFP+) tumor xenografts were treated with intraperitoneal injection of human recombinant decorin core protein (10 mg/kg) every other day for 23 days. All the micrographs were taken using the same exposure and gain. Three-dimensional surface plots, on the right of each panel, were generated utilizing ImageJ software and represent Zc3hav1 expression which directly corresponds to the signal intensity obtained by the immunofluorescence. The scale bars for signal intensity are included on the right of each surface plot. Bar = 20 µm.(TIF)Click here for additional data file.Table S1Primer pairs specific for the exclusive detection of Mus Musculus genes with accompanying gene symbol and NCBI accession number.(DOCX)Click here for additional data file.