EGFR gene promoter methylation and clinical outcome in metastatic colorectal cancer patients receiving chemotherapy with irinotecan and cetuximab.Epidermal growth factor receptor (EGFR) promoter methylation may be responsible for the loss of EGFR expression in neoplastic cells. The primary aim of our study was to verify a possible correlation between Colorectal samples from patients treated with irinotecan–cetuximab were analysed for EGFR promoter methylation and EGFR immunohistochemistry.EGFR promoter hypermethylation. In EGFR promoter methylated and EGFR promoter unmethylated patients, we observed a partial response in 3 (10%) and 13 (59%) patients, respectively (P=0.03), progressive disease was obtained in 19 (63%) and 2 (9%) patients, respectively, with EGFR promoter methylated and EGFR promoter unmethylated tumours (P=0.0001). Median progression-free survival was 2.4 months in patients showing EGFR promoter methylated tumours and 7.4 months for those who had EGFR promoter unmethylated tumours  showed  EGFR gene amplification, EGFR mutations, and markers of EGFR downstream signalling  overexpressing colorectal tumours to anti-EGFR compounds are still largely unknown. However, economic costs and toxicity risks deriving from the use of anti-EGFR therapeutic options made increasingly essential the identification of molecular or clinical predictive factors of response (or resistance) for a better, more accurate, actually targeted, selection of patients more likely to benefit from such a treatment approach. The main research areas in this setting have been focusing on the role of EGFR protein expression, K-RAS gene  , on the other hand we are still incapable to accurately select responding patients among those without K-RAS mutations. In fact clinical observations suggested that a non-negligible proportion of patients, usually ranging from 40 to 70%, does not seem to benefit from the use of anti-EGFR-targeted antibodies although in the absence of a mutation of the We now know that under normal circumstances, EGFR expression is primarily regulated by the abundance of its m-RNA  normal colon tissue  in this area. The aim of our study was then to verify a possible correlation between EGFR gene promoter methylation and clinical outcome in metastatic colorectal cancer patients receiving chemotherapy with irinotecan and cetuximab. The possible correlation between EGFR promoter methylation status and EGFR protein expression was also tested.Based on these considerations, we then hypothesised that Patients with histologically proven EGFR-positive, K-RAS wild-type, metastatic, colorectal cancer receiving a combination of cetuximab and irinotecan after at least one line of previous chemotherapy were eligible for our analysis. To be eligible, patients must also have received an irinotecan-based chemotherapy regimen for at least 6 weeks and must have presented progression of disease during receipt of this regimen or within 3 months thereafter. All patients received cetuximab at an initial dose of 400 mg per square metre followed by weekly infusions of 250 mg per square metre. Irinotecan was administered at a dose of 180 mg per square metre every 2 weeks either alone or in combination with five fluorouracil and leucovorin. Tumour response was evaluated every 8 weeks by clinicians’ assessment and according to the Response Evaluation Criteria in Solid Tumours (RECIST).Formalin-fixed and paraffin-embedded tumour samples (either primary site or metastasis or both when available) of colorectal cancer patients were analysed for EGFR protein expression (immunohistochemistry) and for EGFR promoter methylation.Analysis of EGFR promoter methylation was performed following a DNA Extraction Protocol from paraffin-embedded tissue and a methylation-specific PCR (MSP). The tumour samples were processed according to the QIAamp DNA mini Tissue Protocol, using QIAamp DNA Mini Kit . Before PCR amplification, the DNA extract was treated with sodium bisulphite as described in the handbook of the ‘EpiTect Bisulfite Kit’ (QIAGEN GmbH). Bisulphite modification of DNA to convert all unmethylated cytosines to uracil and then to thymidine during the subsequent PCR step while leaving the methylated cytosines unaffected was performed as described by M dNTP mixture (each); 1.5 mM MgCl2; 0.2 μM Primer mix (each); 1.0 unit Platinum Taq DNA Polymerase ; and bisulphite-modified DNA (of 1 ng–2 μg) in a final volume of 50 μl. Controls without DNA were performed for each set of PCRs. Each PCR product (30 μl) was directly visualised on 10% acrylamide gels. The gel was stained with ethidium bromide and photographed under UV illumination. An enzymatically methylated human male genomic DNA  was used as a methylation-positive control for gene methylation studies and was processed as above mentioned.The primer sequences used were 5′-TGTTTTGTTTTTTTGTGTTTTGGTTTGTGT-3′ (sense) and 5′-CATCCAATCTAAACAACAACAACCACCA-3′ (antisense) for unmethylated DNA and 5′-TGTTTTTTCGCGTTTCGGTTCGCGC-3′ (sense) and 5′-CGTCTAAACGACGACGACCGCCG-3′(antisense) for methylated DNA, both of which amplify ∼150 bp products (μm-thick tissue section obtained from paraffin-embedded specimens fixed in 10% (v/v) neutral buffered formalin, and was performed using kit EGFR PharmaDx (DakoCytomation) according to the manufacturer's instructions as previously described  was evaluated with an immunohistochemistry technique on 5 Statistical analysis was performed with MedCalc package .χ2-test. Survival distribution was estimated by the Kaplan–Meier method. Significant differences in probability of relapsing between the strata were evaluated by log-rank test.The association between categorical variables was analysed by A significant level of 0.05 was chosen to assess the statistical significance.For statistical analysis, overall survival (OS) and progression-free survival (PFS) were defined, respectively, as the interval between the start of cetuximab and irinotecan therapy to death or last follow-up visit and as the interval between the start of cetuximab and irinotecan therapy to clinical progression or death or last follow-up visit if not progressed.Fifty-two metastatic colorectal cancer patients were eligible for our analysis: 33 patients were males (63%) and 19 females (37%), median age at diagnosis was 62 years (range 35–80) .EGFR gene promoter methylation and clinical outcome in patients treated with cetuximab for metastatic disease. Therefore, we can hypothesise that the methylation status in metastases is more relevant for response/resistance to such treatment approach.In 27 patients (52%), the EGFR promoter methylation study was conducted on primary colorectal tumours, in 21 cases (40%) both primary tumours and corresponding metastasis  were available for analysis, while in 4 cases (8%) only the metastatic site  was investigated. For study purposes when results for EGFR methylation status in primary colorectal tumours and corresponding metastases resulted conflicting, only the methylation status in metastases was considered biologically relevant. Although data guiding this choice are lacking the purpose of the study was to assess the correlation between EGFR promoter hypermethylation either in primary colorectal cancer or in metastasis. In 12 cases (40%), EGFR promoter methylation resulted biallelic, whereas in the remaining 18 tumours (60%) only one allele resulted methylated. This two groups of patients  comparable for all main clinical characteristics and experienced a similar clinical outcome during treatment with cetuximab : 4 patients with EGFR promoter hypermethylation and 12 patients with unmethylated tumours. In the remaining five patients (24%), there was not concordance in EGFR promoter methylation status between primary tumour and metastasis. In particular, two patients with EGFR hypermethylation in the primary tumour showed unmethylated EGFR in metastasis and three metastases showing EGFR promoter hypermethylation derived from unmethylated EGFR primary tumours.Globally, 30 patients (58%) showed etuximab . Among tEGFR promoter methylated and EGFR promoter unmethylated groups of patients. In particular, no differences were noticed for sex, age at diagnosis, and previous lines of chemotherapy  and 13 (59%) patients, respectively (P=0.03), progressive disease was obtained in 19 (63%) and 2 (9%) patients, respectively, with EGFR promoter methylated and EGFR promoter unmethylated tumours (P=0.0001). No statistically significant differences were noticed for stable disease .EGFR promoter methylated tumours (30%) and in seven EGFR promoter unmethylated tumours (32%).Lack of EGFR protein expression was observed in nine The expanding role of anti-EGFR therapeutic modalities for the treatment of colorectal cancer patients, along with the growing number of cases potentially requiring such a treatment approach, made the need for a correct and reliable identification of responding tumours increasingly crucial.Unfortunately beside K-RAS mutational status and although the intense ongoing translational research no clear indications for further predictive molecular markers are available at the moment.EGFR gene silencing by promoter methylation are substantially lacking and may help clarifying the role of this epigenetic mechanism on colorectal cancer biology  of important growth control genes . HoweverEGFR promoter methylation was evident in 30 patients (58%), thus indicating that this biological phenomenon should not be considered an infrequent event in colorectal tumours and confirming our previous report. Moreover, patients presenting an EGFR promoter methylated tumour experienced in fact a worse clinical outcome thus confirming our hypothesis of a role for EGFR promoter methylation in determining the efficacy of anti-EGFR-targeted monoclonal antibodies.In our analysis, EGFR promoter methylation and loss of EGFR immunohistochemical expression was not evident. It is then unlikely that loss of EGFR expression as determined with immunohistochemistry may be related to EGFR promoter hypermethylation. When we consider that EGFR promoter methylation represents a predictive factor for cetuximab treatment in our series, the observation of a lack of concordance of this biological phenomenon with EGFR immunohistochemistry expression seems clinically sound and in accordance to previous data, indicating that loss of EGFR immunohistochemical expression should not be considered a predictive factor for anti-EGFR monoclonal antibodies activity . Therefore, we can speculate that other relevant biological mechanisms, such as loss of heterozygosity, may contribute to loss of gene function along with monoallelic promoter methylation in tumour tissues.It is also important to note that only in 12 patients (40%) EGFR methylation status between primary tumour and metastases (76%). This observation is however in line with our previous observation of a concordance for EGFR methylation status in 69% of patients with metastatic colorectal cancer.However, the relatively small number of patients analysed in our series represents a clear limitation for a definitive conclusion about the role of EGFR promoter methylation in determining response to anti-EGFR treatments, and our findings should be considered speculative. A further possible limiting factor is represented by the proportion of patients with a concordant EGFR promoter methylation in EGFR gene silencing could provide new clues in anti-EGFR biological agents use optimisation. It has been in fact reported that treatment with the demethylating agent decitabine may result in the re-expression of EGFR in two breast cancer cell lines, in which EGFR transcription was abolished by EGFR promoter hypermethylation. Both cell lines are relatively resistant to killing by the EGFR inhibitor gefitinib; however, after co-treatment with decitabine and gefitinib, a significant effect on the induction of apoptosis was observed  with secondary agents that target the re-expressed genes and/or reconstituted signal transduction pathways (EGFR promoter methylated tumours.Combined approaches targeting pathways . This trEGFR promoter hypermethylation, after confirmation in larger data set, may represent a valuable and important asset to be considered in further studies investigating the role of EGFR as a therapeutic target in colorectal cancer patients.We believe that


Elevated low density lipoprotein (LDL) and lipoprotein(a) are independent risk factors for the development of atherosclerosis. Using a proteomic approach we aimed to determine early changes in arterial protein expression in transgenic mice containing both human LDL and lipoprotein(a) in circulation.P<0.05) in lipoprotein(a) transgenic mice compared to wildtype including 17 that showed a ≥2 fold difference. Some proteins of interest showed a similarly altered abundance at the transcript level. These changes collectively indicated an initial metabolic response that included a down regulation in energy, redox and lipid metabolism proteins and changes in structural proteins at a stage when atherosclerosis had not yet developed.Plasma lipid analyses showed the lipoprotein(a) transgenic mice had significantly higher lipid levels than wildtype, including a much increased LDL and high density lipoprotein (HDL) cholesterol. Analysis of aortae from lipoprotein(a) mice showed lipoprotein(a) accumulation but no lipid accumulation or foam cells, leaving the arteries essentially atherosclerosis free. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 34 arterial proteins with significantly altered abundance (Our study shows that human LDL and lipoprotein(a) promote changes in the expression of a unique set of arterial proteins which may be early indicators of the metabolic disturbances preceding atherosclerosis. However, whether the same changes occur in mouse models with a different pattern of lipid perturbation is unknown.A number of transgenic mouse models have been generated to study atherosclerosis Large clinical trials have established that elevated low density lipoprotein (LDL) levels are a common risk factor for the development of atherosclerosis bona fide Lp(a) in circulation A mouse model for Lp(a) was initially developed by expressing a human apo(a) transgene Here we applied a proteomic approach to the artery of the human Lp(a) mouse to identify early changes that occur in response to the perturbed lipoprotein profile present in these animals without the influence of a high fat diet.All animal protocols were reviewed and approved by the University of Otago Animal Ethics Committee (AEC protocol No. 25/03).Wildtype C57BL/6 mice were purchased from Jackson Laboratories . Lp(a) transgenic mice were generated by breeding transgenic mice expressing a human apo(a) cDNA encoding seventeen KIV domains in situ with phosphate-buffered formalin, removed and further fixed in formalin. The aortic arch from the aortic root to the first intercostal branch was processed for histology. Arteries were embedded in paraffin and 4 μm serial sections taken through the longitudinal axis of the aortic arch. Sections were stained with either Verhoeff's elastic stain and Curtis' modified van Gieson stain or haematoxylin and eosin. Sections were also immunostained with a polyclonal rabbit anti-human Lp(a) antibody  followed by a goat anti-rabbit hrp-conjugated antibody .For histological analysis, the aortae from eight Lp(a) transgenic and eight wildtype mice were perfusion fixed Whole blood was collected via cardiac puncture before perfusion of aortae and placed in EDTA microtubes. Plasma was isolated and stored at −80°C. Plasma levels of cholesterol and triglyceride were measured using enzymatic reagents from Roche Diagnostics  and phospholipids measured using an enzymatic reagent from Wako . Plasma levels of human Lp(a) and human apoB were measured using apo(a)-specific and apoB-specific enzyme-linked immunosorbent assays (ELISAs) as previously described Lipids were extracted from a pool of six aortic sections adjacent to that used for proteomics with a modification of the method by Bartels et al. in situ with PBS and removed. The aortic arch from the aortic root to the first intercostal branch pair was sectioned and this and the remaining artery rinsed thoroughly in PBS and immediately frozen in liquid nitrogen. The aortic arches were used for proteomic analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and the remaining sections for lipid analysis. Detailed methodology of the proteomic analysis and mass spectrometry  transgenic and 12 wildtype mice were perfused trometry  used to Quantitative RT-PCR was used to quantify the level of mRNA transcript for proteins of interest showing a ≥2 fold difference in protein abundance. Total RNA was isolated from 6 pooled aorta samples from either wildtype or Lp(a) mice using Trizol . One μg of RNA was reverse transcribed to cDNA using Transcriptor Reverse Transcriptase and random pd(N)6 primers (Roche). Quantitative PCR was performed on a LightCycler 480 instrument (Roche) using the SYBR Green I detection system. The primer sequences for each transcript are available from the authors on request. The cDNA was amplified with the following conditions: 95°C for 5 min, 50 cycles of 95°C for 5 sec, 59°C for 5 sec and 72°C for 8 sec. Melt curve analyses were performed at the end of cycling. The level of each transcript was quantified relative to 18S rRNA using the Fit Points and 2nd Derivative Maximum calculation for each sample.t-test was used to test for significant differences in mean lipid levels and the mean normalized spot volumes obtained from 2-D PAGE analysis of the Lp(a) transgenic versus wildtype mice. A difference with P<0.05 was considered significant. The AmiGO search tool was used to screen the gene ontology (GO) database to annotate the functional relevance of proteins identified as being significantly different in relative abundance between Lp(a) and wildtype mice.Statistical analyses were performed using GraphPad Prism . A non-paired Student's P<0.001), 98 versus 65 mg/dL triglyceride (P<0.05) and 116 versus 45 mg/dL phospholipids (P<0.001) (Lp(a) transgenic mice had significantly higher concentrations of cholesterol, triglyceride and phospholipids in plasma than wildtype mice (99 versus 40 mg/dL cholesterol (P<0.001) . SeparatP<0.001)  showed tP<0.001) . HoweverP<0.001) .The elevated levels of plasma lipids in the Lp(a) mice were not associated with lipid accumulation in the arteries. Indeed, cholesterol and triglyceride concentrations in the aorta of Lp(a) transgenic mice were significantly reduced compared to wildtype mice . InteresHistological analysis showed no evidence of atherosclerosis in the arteries of Lp(a) transgenic mice or wildtype mice including no evidence of foam cells in the aortic arch  or aortiChanges in the proteome of the aortic arch between Lp(a) and wildtype mice was assessed by 2-D PAGE. Several energy metabolism enzymes were significantly decreased by ≥ 2 fold . These iTwo subunits required for the electron transfer from FADH into complex II of the electron transport chain (electron transfer flavoprotein dehydrogenase and electron transfer flavoprotein) were significantly decreased, as was a subunit of complex III (cytochrome b-cl subunit 1).There was a major reduction in glucose-3-phosphate dehydrogenase, a key enzyme for lipid synthesis that converts dihydroxyacetone to glycerol-3 phosphate for triglyceride and phospholipid synthesis . A majorAs well as alterations in energy metabolism proteins involved in NADH and NADPH production which could be expected to alter the redox status of arterial cells, a number of proteins involved in oxidative stress responses were altered including the heat shock proteins, Hsp60 and 70, carbonic anhydrase 3 and peroxiredoxin 4, an antioxidant enzyme that reduces lipid peroxides .A number of structural proteins were ≥2 fold increased in the arteries of Lp(a) mice . These iQuantitative RT-PCR was performed to investigate if proteins of interest showing a ≥2 fold change at the protein level were also regulated at the mRNA transcript level between the Lp(a) and wildtype mice . With re–/– mice with no apparent lesions which also revealed alterations in energy, lipid and redox metabolism The aim of our study was to investigate the response of the mouse artery to the atherogenic lipoprotein profile of elevated human LDL and Lp(a). The study presents the first proteomic assessment of a mouse model containing a lipoprotein profile that more closely resembles that in humans with a focus on early changes within the artery and without the influence of diet. The results indicate changes in energy, lipid and redox metabolism with some vascular remodelling collectively pointing to an early response to the elevated lipid profile before any atherosclerotic lesions are evident. The Lp(a) mice did accumulate Lp(a) and lipid oxidation products in their arteries both of which are likely to have contributed to this response. Our results are in line with a previous proteomic study of young apoE–/– mice –/– mice A downregulation in energy metabolism was evident with a number of enzyme subunits from the glycolysis, TCA cycle and electron transport pathways showing ≥2 fold decrease in Lp(a) mice. Collectively these changes would be expected to impair vascular ATP and NADH/NADPH production. Three enzymes involved in NADH generation in the mitochondria, dihydrolipoyllysine dehydrogenase, dihydolipoyllysine succinyltransferase, both part of the oxoglutarate dehydrogenase complex, and isocitrate dehydrogenase, were downregulated at the protein level. Dihydrolipoyllysine succinyltransferase also showed a significant (2.9 fold) downregulation at the transcript level. Two of only three enzymes capable of generating NADPH i.e. glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase were significantly downregulated at the protein level, although neither showed a reduced transcript level suggesting a regulation at a post-transcriptional level. A reduction in NADPH could induce oxidative stress by decreasing the regeneration of major cellular antioxidants, glutathione and thioredoxin, which work in conjunction with glutathione peroxidase and peroxiredoxins respectively to reduce lipid peroxides The downregulation in glycolysis might be expected to impact on lipid metabolism by ultimately reducing the amount of acetyl CoA available for fatty acid synthesis. Moreover, there was a significant decrease at both protein and transcript level in Fabp4, a fatty acid binding protein that traffics fatty acids for synthesis and glycerol-3 phosphate dehydrogenase, a key regulatory enzyme for triglyceride synthesis. Indeed, Fabp4 was the most significantly downregulated protein in the Lp(a) mice at both the protein (6.3 fold) and transcript level (3.1 fold). All of these changes would be expected to reduce arterial triglyceride synthesis. The downregulation of enzymes producing NADPH would be expected to reduce cholesterol synthesis which utilizes significant quantities of NADPH. The proteomic data supports the lipid data which showed a significant decrease in both triglyceride and cholesterol levels in the arteries of Lp(a) mice, despite a significant increase in plasma lipids and arterial Lp(a) accumulation.–/– background show a striking protection from atherosclerosis The large decrease in Fapb4 is of particular interest in that Fabp4 deficient mice on an apoE–/– mice Also of interest was a 2.2 fold increase in the cytoskeletal protein, transgelin and a 2-fold increase in the cell adhesion protein, Mfap4. Interestingly, transgelin, an actin-binding protein abundant in smooth muscle cells is downregulated in atherosclerosis and a deficiency accelerates atherosclerosis in apoEOur study shows that human LDL and moderate Lp(a) levels promote an early metabolic response in the arteries of mice independent of lesion development. A longer term study would be required to extend this observation as the response is likely to change with age. Indeed, older apo(a) transgenic animals do develop atherosclerosis on a chow diet Our study has not shown that Lp(a) is specifically responsible for the proteomic changes seen in our mice since the mice also had elevated LDL levels as a result of the human apoB transgene. Although the main aim of our study was to investigate the combined effect of elevated LDL and the presence of Lp(a), it would be of interest to compare human apoB mice with human Lp(a) mice to dissect the response of the artery to human LDL versus Lp(a). It would also be of interest to compare the response of the Lp(a) transgenic mice used here to those with higher Lp(a) levels as generated by Schneider et al. Our study does not discriminate between different arterial cell types, which is a common restriction for many proteomic studies particularly in mice Here we show that human LDL and Lp(a) promote an initial response in mouse arteries free from atherosclerosis involving changes in energy, redox and lipid metabolism and structural remodeling. We have identified a set of proteins underlying this response some of which have independently been shown to be atheroprotective and others which may be early indicators of atherosclerosis development.Text S1Proteomics Methods (DOC).(DOC)Click here for additional data file.Figure S1RT-PCR analysis of the liver Fabp4 transcript (TIF). Total RNA was isolated from liver samples (n = 6) from wildtype and Lp(a) mice. RNA was reverse transcribed to cDNA and quantitative PCR for Fabp4 relative to 18S rRNA was performed. Fatty acid-binding protein 4 (Fabp4) transcript was decreased in Lp(a) mice liver compared to wildtype. Results are presented as relative levels of transcript normalized to wildtype. **P<0.01 versus wildtype.(TIFF)Click here for additional data file.Table S1P<0.05) differential expression in the aortic arches of Lp(a) versus wildtype mice on a normal chow diet (DOC). * Fold change between Lp(a) versus wildtype mice. Positive number indicates an increased expression in the Lp(a) mice. Negative number indicates a decreased expression in the Lp(a) mice.Proteins showing significant ((DOC)Click here for additional data file.Table S21 Raw spot volumes were log transformed and normalized by zero-centering through median subtraction.Proteins identified by MALDI-TOF MS/MS from the 2D-PAGE of aortic arches from wildtype versus Lp(a) mice (DOC). (DOC)Click here for additional data file.


Given that a Na+ currents were followed during treatment and were investigated in relation to oxaliplatin dose level. Eighteen patients were assessed before and after a single oxaliplatin infusion with motor and sensory axonal excitability studies performed on the median nerve at the wrist. While refractoriness (associated with Na+ channel inactivation) was significantly altered post-oxaliplatin infusion in both motor  and sensory axons , strength-duration time constant (marker of persistent Na+ conductances) was not significantly altered post-infusion . However, changes in strength-duration time constant were significantly correlated with changes in refractoriness in motor and sensory axons .Specifically the function of transient and persistent Na+ conductances. These findings are likely to have implications for the design and trial of neuroprotective strategies.It is concluded that the predominant effect of acute oxaliplatin exposure in human motor and sensory axons is mediated through changes in transient rather than persistent Na Oxaliplatin is a third generation platinum-based chemotherapy effective in the treatment of colorectal cancer + channels, specifically by slowing Na+ channel inactivation kinetics + current + channelopathy” in oxaliplatin treated patients, with in vitro studies inconsistent to date concerning the relative involvement of transient and persistent Na+ conductances Recent experimental studies across a variety of models have suggested that oxaliplatin modulates axonal voltage-gated Na+ channel related parameters + conductance subtypes in both motor and sensory axons in the development of acute oxaliplatin-induced neurotoxicity. As Na+ channel modulation remains a potential neuroprotective strategy for oxaliplatin-induced neurotoxicity, it is important to investigate the specific types of Na+ conductances targeted by oxaliplatin. The present study utilised novel functional and specialised neurophysiological approaches to assess the effects of acute oxaliplatin administration on markers of axonal transient and persistent Na+ conductances and determined the dose-response relationship of oxaliplatin on Na+ currents in vivo.In previous studies, our group has identified striking changes in Na2 given every 2 to 3 weeks for a total of 6–12 treatment cycles. Oxaliplatin  was infused every 2 weeks in conjunction with leucovorin (200 mg/m2) and followed by bolus 5-fluorouracil . For the next 48 hours, a continuous infusion of 5-FU (600 mg/m2) was given, followed by leucovorin (200 mg/m2) and 5-FU bolus (400 mg/m2) on the 2nd day 2) was given intravenously every 3 weeks with oral capecitabine (1000 mg/m2) twice daily for 2 weeks Eighteen patients treated with oxaliplatin for colorectal cancer were prospectively assessed before and immediately following oxaliplatin infusion. Three of these patients were included in a prior study The study was approved by the South Eastern Area Health Service (Eastern Section) Human Research Ethics Committee and University of New South Wales Human Research Ethics Committee. Participants provided written informed consent in accordance with the declaration of Helsinki.Each patient was assessed once immediately prior to oxaliplatin infusion and once immediately following the same infusion to compare the changes pre- and post-infusion.nd treatment to the 6th treatment, corresponding a cumulative dose range of 200–650 mg/m2 oxaliplatin. For analysis, patients were divided into groups based on cumulative oxaliplatin dose (less than or greater than 400 mg/m2) as prior studies highlighted the changes in acute excitability parameters after this dose level Patients were tested in treatment cycles from the 2To assess the extent of acute neuropathic symptoms, the Oxaliplatin-Specific Neurotoxicity Scale (OSNS) was utilized with the following grades: Grade 1 – dysesthesia or paresthesia that completely regresses before the next cycle of therapy; Grade 2 – dysesthesia or paresthesia persisting between courses of therapy; and Grade 3 - dysesthesia or paresthesia causing functional impairment Multiple excitability studies were undertaken, utilising previously described protocols + channel activity + currents as SDTC measurements may be affected by other factors + channels Stimulus-response curves were recorded by increasing the stimulus intensity until the maximal compound response amplitude was obtained. The stimulus threshold was defined as the stimulus intensity (mA) required to produce a compound response 50% of maximal amplitude. The stimulus-response slope was derived from the range between 25% and 75% of maximal amplitude All statistics were performed in SPSS . Results were expressed as mean ± standard error of the mean and statistical significance was defined as P≤0.05. Recordings were paired for each patient pre- and post-infusion. Wilcoxon sign rank tests (two-tailed) were utilised to compare paired recordings pre- and post-oxaliplatin within individual patients. Mann Whitney U tests (two-tailed) were used to compare findings within groups divided by dose level. Single oxaliplatin dose was defined as the oxaliplatin dose infused at the time of testing. Cumulative oxaliplatin dose was calculated as the sum total oxaliplatin dose received in all treatment cycles including the tested cycle. Correlations were performed using Spearman's rank correlation coefficients.2). The most commonly reported symptom was cold-induced paresthesia in distal upper limbs, lasting for 2 to 10 days post infusion. The majority of patients displayed mild neurotoxicity (89%), with an NCI score of 1 and an OSNS neurotoxicity score of 1, reflecting the resolution of acute neuropathic symptoms within two weeks. 11% of patients had an OSNS score of 2, reflecting the persistence of neuropathic symptoms for greater than two weeks. Further patient characteristics are detailed in All patients reported the development of acute neuropathic symptoms following oxaliplatin infusion . However, there were no significant differences in SDTC . In patients assessed at a cumulative dose of less than 400 mg/m2, there was also a significant correlation between strength-duration properties and refractoriness. Change in refractoriness post-oxaliplatin was significantly correlated with change in SDTC , although again the extent of change in refractoriness far exceeded the change in SDTC (In sensory axons, the extent of change in pre-oxaliplatin refractoriness was determined by the cumulative oxaliplatin dose (Correlation coefficient = −.72;  in SDTC .in vivo. Markers of transient and persistent Na+ conductances were investigated in patients receiving neurotoxic chemotherapy with oxaliplatin, to clarify existing in vitro data which suggested tremendous variability between experimental preparations. Importantly, the present study is the first to directly compare the relationship between oxaliplatin dose and transient and persistent Na+ conductances, in both motor and sensory axons. While there were significant changes in markers of Na+ channel inactivation in both motor and sensory axons post-oxaliplatin infusion, changes in markers of non-inactivating Na+ conductances were relatively minor. There were significant correlations between oxaliplatin dose level and Na+ channel-associated parameters, suggesting a dose-response relationship between Na+ channel dysfunction and oxaliplatin dose. Taken in total, the present findings suggest that acute oxaliplatin administration preferentially affects markers of transient Na+ conductances, at least in the clinical setting.The present study represents the first assessment of the dose-response properties of acute and cumulative oxaliplatin exposure in human axons + currents have been functionally identified + current contributes an estimated 98% of the total nodal Na+ current + current accounts for only 1.0–2.5% of the total Na+ current + conductances active at threshold + channels v1.6 channels In human peripheral nerves, both transient and persistent Na+ channel blocker tetrodotoxin As in previous studies, a different pattern of excitability change was identified in sensory axons acutely following oxaliplatin administration In motor axons, refractoriness depends on the transmission of impulses through the neuromuscular junction In vitro studies have suggested that oxaliplatin modulates axonal Na+ channel inactivation properties via slowing of inactivation kinetics or changing the voltage dependence of activation or inactivation + current, with no effect on the persistent current elicited by long ramp pulses + current + channels and vertebrate Na+ channels to tetrodotoxin and other toxins which may underlie these apparent differences + currents with transient inactivation kinetics. What then are the implications of these findings for the design of neuroprotective strategies? Many local anesthestics and antiepileptic drugs target Na+ channel properties. While the effects of oxaliplatin have been antagonised in vitro by the antiepileptic drug carbamazepine + currents and is thought to modulate channel inactivation, with a much higher affinity for inactivated channels + channel antagonism will be required to avert the development of neuropathy, such as enhancement of slow channel inactivation rather than interaction with fast inactivation properties, as may be observed with the novel antiepileptic drug lacosamide + channel modulators should inevitably provide improved selective subtype and current type modulation of channel function, which may prove useful in treating oxaliplatin-induced neurotoxicity, as well as a variety of inherited and acquired Na+ channelopathies.In total, results from the present study suggest that oxaliplatin preferentially affects voltage-gated Na


Two such fusions were obtained, corresponding to a translational yfp insertion in iscR and iolR, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.In this study we adapted a Mu E. coli nucleoid is accomplished by large DNA regions (around 1 Mb in size), termed macrodomains, which restrict certain rearrangements in the linear-order sequence of the chromosome The intracellular organization of prokaryotes is proving to be increasingly complex, often constituting a structural and functional prelude to eukaryotic counterparts and emphasizing that bacteria are useful models for studying universal cellular mechanisms. The nucleoid is one of the most important structures inside the bacterial cell, and although it has been first described 50 years ago, only very recently more insight was gained about its organization and mechanism of compaction Although it is clear that more and more valuable information is obtained about the standing architecture of the nucleoid, studies involving global chromosome dynamics in response to environmental stress are generally lacking. Moreover, the use of specific DNA binding dyes  and fixing procedures to fluorescently stain and image the bacterial nucleoid are often genotoxic and prone to produce artefacts. Such approaches are therefore not suitable to properly follow-up live nucleoid dynamics in cells, especially if the latter are already suffering DNA damage and/or affected in their DNA repairing capabilities.Salmonella Typhimurium LT2 for chromosome based nucleoid reporters, using a customized transposon. Two nucleoid reporters were found of which one was further validated and subsequently used to track the nucleoid dynamics of LT2 after stressful growth under high hydrostatic pressure (HP), a cryptic but environmentally relevant stress that causes excessive filamentation in mesophilic bacteria In this study, we screened a random translational YFP library in 630) of 0.4–0.6 was reached. When appropriate the following chemicals  were added to the growth medium at the indicated final concentrations: ampicillin , chloramphenicol , kanamycin , tetracycline , oxytetracycline , glucose (0.2%) and L-arabinose (0.2%).Bacterial strains, phages and plasmids used throughout this study are listed in 630 nm measurements every 15 min. The resulting OD630 nm values were averaged across three replicate cultures and standard deviations were<11%.For obtaining growth curves, stationary phase cultures were diluted 1/1000 in 300 μl of LB medium, placed in a honeycomb well and incubated in the Bioscreen C system  for a 24 h period at 37°C, with regular shaking and automatic ODS. Typhimurium LT2 as plaques in LB soft-agar or as lysates in LB broth as described previously HT105/1 int-201 as described previously http://rothlab.ucdavis.edu/Recipes/mintgreen.html) were used to indicate cell lysis. The latter medium contains glucose as a carbon source, and a pH indicator dye that turns dark green at sites where phage infection causes cell lysis and the concomitant release of organic acids.Phages were propagated on dY was done by modifying the well characterized MudK transposon S. Typhimurium strain . Next, this pool of plasmid DNA was transformed by electroporation to E. coli DH5α and transformants were first selected on OxyTc10 and then individually screened for loss of ampicillin resistance. As such, a pKD46 derivative could be isolated in which the bla gene was knocked-out by the mini-Tn10 transposon (designated pKD46 bla::Tn10).Construction of Mu TH2145,  adapted dK donor strain TH2145 was equipped with pKD46 bla::Tn10 and recombineering lacZYA-npt part of MudK with a yfp-frt-cat-frt cassette. For this, the yfp-frt-cat-frt cassette was PCR amplified  from plasmid pAC dY transposon, was purified and then grown in LB broth at 30°C to suppress transposase expression 10 to cure the pKD46 bla::Tn10 plasmid. The resulting strain was named TH2145 MudY  and was used as the donor strain for constructing a random MudY insertion library.Subsequently, the MudY donor strain was still capable of producing random transposon insertion mutants, a high-copy plasmid of around 6 kb containing a PBAD promotor and bla resistance cassette was mutagenized with MudY. First, a phage P22 HT105/1 int-201 lysate was made from the MudY donor strain and subsequently mixed with a stationary phase culture of S. Typhimurium LT2 containing the high copy plasmid and incubated for 30 min at 37°C to permit phage adsorption. The mixture was subsequently plated out on LB Cm30 Ap100 agar plates and incubated overnight. The resulting library of around 10,000 mutants was scraped off the plates, pooled and subjected to plasmid DNA extraction . Next, this pool of plasmid DNA was transformed by electroporation to S. Typhimurium LT2 and transformants were selected on Cm30 and Amp100. 10 transformants were picked up and the exact location of MudY in the plasmid was determined using the protocol described below. YFP fluorescence could be induced in one transformant by adding 0.2% arabinose, indicating the yfp gene became inserted in frame and downstream of the PBAD promoter.To ensure that the MuHT105/1 int-201 lysate of TH2145 hsiD1284::MudY hisA9944::MudI was mixed with a stationary culture of S. Typhimurium LT2 acceptor strain and incubated for 30 min at 37°C to permit phage adsorption. Next, the mixture was diluted in LB broth complemented with 10 mM EGTA and plated out on LB Cm30 agar plates containing 1 mM EGTA and incubated at 37°C. EGTA was added to block adsorption of P22 and thereby prevent subsequent cycles of infection on the plate dY transposon, which lacks natural transposition activity, is packed into phage P22 together with the transposase of the adjacent MudI element, it is able to randomly transpose into the bacterial genome. Due to the packaging constraints of P22 (±44 kb) MudY and MudI can never transpose simultaneously, as a consequence the transposase is lost by degradation and segregation yielding single insertion mutants with Cm30 resistance.The P22 dY insertion library constituted of approximately 25.000 mutants and was scraped off the plates using LB broth containing 10 mM EGTA. This large pool was subjected to one round of fluorescence activated cell sorting (FACS).The resulting MudY pool in LT2 was sorted by a Fluorescence Activated Cell Sorter  to enrich for YFP expressing mutants. A 488 nm excitation laser in combination with a 530/40 nm emission filter was used, and the 0.1% most YFP-fluorescent clones were sorted and plated out on MG agar plates containing Cm30 to discern P22 infected from phage free MudY mutants.The MuFluorescence microscopy was used to screen 200 mutants for nucleoid reporter activity. All fluorescence microscopy and time-lapse fluorescence microscopy experiments were performed with a temperature controlled  Ti-Eclipse inverted microscope  equipped with a TI-CT-E motorized condenser, a YFP filter , a DAPI filter , and a CoolSnap HQ2 FireWire CCD-camera. For imaging, cells were grown to mid-log phase and placed between LB agar pads and a cover glass, essentially as described previously http://rsbweb.nih.gov/ij/).Images were acquired using NIS-Elements (Nikon) and resulting pictures were further handled with open source software ImageJ  dY mutant was extracted via phenol∶chloroform extraction dY (MudY_out_up), a primer specific to the Y linker (Y linker primer) and a Taq polymerase . The PCR products were purified, sequenced and the exact position of the MudY transposon was determined using BLAST search. Initially, five clones were retained as nucleoid reporter candidates but after mapping the MudY transposon it was found that this number could be reduced to two different clones (iscR::MudY and iolR::MudY). This clonal enrichment was likely due to the pooling of the library before sorting the most fluorescent cells.Mapping of the MudY insertions in iscR and iolR, yfp translational fusions were de novo constructed at the 3′ end of the same genes. For this, the yfp-frt-cat-frt cassette was PCR amplified  from plasmid pAC iscR::yfp and LT2 iolR::yfp via recombineering in LT2 and flipping out the cat cassette, using pKD46 iscR::yfp recA1 strain was subsequently constructed by cotransducing the recA1 and srl-202::Tn10 alleles from TT521  with P22 HT105/1 int-201iscR::yfp.Based on the MuiscR::yfp strain constructed via pAC, an LT2 iscR::yfp-frt-cat-frt strain was constructed by recombineering an yfp-frt-cat-frt amplicon obtained from plasmid pGKBD  by PCR with primers iscR_yfp_C-term_Fw and iscR_yfp_C-term_Rev_pGKBD. In contrast to pAC, pGKBD contains an yfp-frt-cat-frt cassette in which the cat locus is oriented in the same direction as the yfp gene. In the latter strain, the YFP moiety is C-terminally fused to the IscR protein with a SGGGG linker.To mitigate the apparent growth defect of the LT2 For treatment with HP, stationary phase cultures were 1/100 diluted in LB broth, after which 200 μl of this suspension was heat sealed in a sterile polyethylene bag after exclusion of the air bubbles, and subjected for 16-20 h to a pressure of 40–45 MPa in an 8 ml pressure vessel , held at 37°C. After pressure release, cells were 1/10 diluted in fresh LB and quickly prepared for imaging as described above.dK transposon and its delivery system have been described previously lacZ translational fusions in Salmonella Typhimurium. For our application, we used recombineering lacZYA-npt part of MudK with a yfp-frt-cat-frt module, while making sure not to introduce stop codons upstream of the yfp open reading frame . The MudY transposon is thus able to randomly transpose and can result in YFP fluorescence when the transposon hops in a gene in the correct orientation and reading frame.In order to validate random transposition and YFP fluorescence, a 6 kb plasmid containing a PdY transposon and delivery system, a random MudY transposition library was constructed in the S. Typhimurium LT2 chromosome in order to search for a useful nucleoid reporter. For this, the library of ca. 25.000 clones was first enriched in those clones displaying a clear YFP fluorescence through fluorescence activated cell sorting . Subsequently, after screening ca. 200 individual clones of this sub-library with fluorescence microscopy, two clones were retained in which YFP fluorescence clearly coincided with the nucleoid as stained with DAPI  and iolR  gene, respectively  to the penultimate codon of the iscR or iolR open reading frame on the chromosome, yielding LT2 iscR::yfp and LT2 iolR::yfp, respectively. Interestingly, the localization of this reconstructed IolR::YFP fusion protein differed from the original MudY mediated one, yielding small nucleoid associated foci instead of the typical “cloudy” appearance of the DAPI stained DNA . The latter condition has been documented to cause excessive filamentation in mesophilic bacteria Through monitoring the IscR::YFP nucleoid reporter, it could be demonstrated that immediately after HP release the nucleoids appeared as an unsegregated mass that nevertheless quickly started to segregate during cell elongation . SubsequPlease note that in contrast to the IscR::YFP reporter, DAPI staining and corresponding UV excitation caused HP treated cells to quickly stop growing, leading to completely inactive cells after 60 min . The latiscR::yfp, a small growth was defected for this strain (and LT2 iscR::MudY as well) in comparison with LT2 wild-type  and a subsequent screen based on FACS and fluorescence microscopy yielded IolR159::YFP and IscR144::YFP fusion proteins to be specifically located on the nucleoid  operon involved in Fe-S biogenesis S. Typhimurium has a very high sequence identity (97%) with its E. coli homolog and is thus expected to have similar properties. Recently, crystallography data showed that IscR binds as a homodimer to DNA and comprises of two major domains: a DNA binding domain (winged HTH motif) and a dimerization helix The IscR protein has been studied extensively in While the IscR-YFP protein could be further validated as a nucleoid reporter through a number of colocalization experiments with the well-established DNA binding dye DAPI , it cleaSalmonella proteome, although insertions in some NAPs that are essential in nucleoid organization and regulation might compromise the viability of the corresponding clones and escape detection. Please note that during the preparation of this manuscript other nucleoid reporters such as GFP-Fis And HupA-mCherry have been validated as well, and used to study chromosome organization and dynamics at high resolution Please note that our screen for nucleoid reporters was not exhaustive but could in principle be employed to map functional DNA binding domains  throughout the d-based random transposon delivery system to generate translational fusions to the yfp reporter gene throughout the S. Typhimurium chromosome, and have screened a library of such clones for a suitable nucleoid reporter. The resulting IscR-YFP nucleoid reporter was subsequently used to initiate studies on bacterial nucleoid dynamics resulting from HP stress. More generally, non-invasive fluorescent nucleoid reporters will allow proper nucleoid dynamics to be examined under DNA stressing physiology.In summary, we have adopted and validated a MuFigure S1Representative images showing the localization of the IolR::YFP protein on the DAPI stained nucleoid in LT2 iolR::yfp cells. Phase-contrast (A), DAPI (B), YFP (C), and merged (D) images are shown. Scale bar corresponds to 5 μm.(TIF)Click here for additional data file.Figure S2Growth curves of LT2 wild-type, LT2 iscR::MudY and LT2 iscR::yfp630 nm) in time.., with growth monitored as an increase in optical density Click here for additional data file.Figure S3Growth curves of LT2 wild-type and LT2 iscR::yfp-frt-cat-frt630 nm) in time., with growth monitored as an increase in optical density Click here for additional data file.


Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders. © 2010 Wiley-Liss, Inc.Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the  Demyelination is a cardinal neuropathological feature of several central nervous system (CNS) disorders including multiple sclerosis (MS), white matter strokes (WMS), progressive multifocal leukoencephalopathy (PML), periventricular leukomalacia (PVL), and certain primary mitochondrial disorders due to mitochondrial DNA (mtDNA) mutations  was greater than in OPCs (identified by PDGFRA or NG2 expression). Mitochondria were abundant and mitochondrial membrane potential (MMP), an electrochemical gradient generated and maintained across the inner mitochondrial membrane by proton pumps including complex IV, was significantly greater in processes of differentiated oligodendrocytes compared to OPCs. The formation of oligodendrocyte processes was notably impaired when complex IV was inhibited throughout or briefly at onset of OPC differentiation. Thus, mitochondrial respiratory chain dysfunction both injures mature oligodendrocytes and impairs differentiation of OPCs from P0 rat. These affects have potentially important implications for demyelinating disorders, including PVL and MS.In this study, we investigated the impact of complex IV inhibition on oligodendrocyte lineage cells from P0 rat and on the capacity of OPCs to differentiateL-cystein. Dissociated cells were plated on poly-D-lysine coated tissue culture flasks and grown at 37°C with 7.5% CO2 for 10 days in DMEM +10% FCS +1%Pen/Strep. OPCs were derived from mixed glial cultures containing OPCs and microglia grown on astrocyte monolayer by shaking the flask first for 1 h at 250 rpm and then, after replacing cell culture medium, overnight for a minimum of 18 h at 450 rpm. Microglia were removed by differential adhesion method. OPCs were seeded into PDL-coated 8-well chamber slides at a density of 60,000 cells/mL  and maintained in SATO medium. For the first 2 days, 10 ng/mL PDGF and 10 ng/mL FGF were added for proliferation and afterwards the medium was replaced by SATO with 0.5% FCS for differentiation. Cells remained in SATO with 0.5% FCS for the 5-day-period of differentiation with replacement of 25% of the medium (100 μL) on the 4th day. Cell viability was determined using Trypan blue and ethidium homodimer extrusion methods as well as TUNEL staining. The sublethal range of sodium azide and potassium cyanide concentrations (1–100 μM) was determined based on greater than 90% of oligodendrocyte lineage that remained attached in the chamber slides being viable judged by Trypan blue and ethidium homodimer extrusion and TUNEL staining.Mixed glial cultures were prepared according to the method of McCarthy and DeVillis  and gently disrupted with a Teflon-coated tissue homogenizer. The homogenate was centrifuged for 10 min at 2200 rpm at 4°C. The pellet was resuspended before spectrophotometric analysis of complex IV and citrate synthase activity as previously described , and routine respiratory oxygen flux was established over 10 min with automatic deduction of background flux using the online DatLab software . Cell counts were made using a small aliquot of cells at the end of the experiment, and rates were then normalized to pmol of oxygen consumed per 106 cells per unit of time.For respirometry, differentiated cells with or without exposure to complex IV inhibitor were washed briefly with PBS, dissociated from plastic using TripLE™ Express and resuspended in SATO with 0.5% FCS. High-resolution respirometry was performed at 37°C using an Oroboros Oxygraph-2K . Approximately 0.5 × 10MMP of P0 rat oligodendrocyte lineage cells was measured using monomeric JC-1 , which aggregates in mitochondria depending on the MMP, at a concentration of 10 μg/mL for 20 min. The ratio of densitometric values for red (590 nm) and green (510–527 nm) fluorescence was used as an indicator of MMP as previously described  following brief fixation in 4% paraformaldehyde in PBS pH 7.4 and immunocytochemical labeling of oligodendrocyte lineage cells. Following immunocytochemistry, the cells on slides were washed three times for 5 min in PBS and incubated in permeabilization solution [freshly prepared 0.1% sodium citrate (BDH) and 0.1% Triton X-100 (Sigma)] for 3 min on ice. Following three washing steps, the TUNEL reaction mixture with enzyme and label solutions was applied to the slides according to manufacturer's instructions. Nuclear staining was performed using DAPI. Three independent experiments, each in triplicate chambers, were performed for controls and different concentrations of complex IV inhibitors. Following differentiation of OPCs the morphology of mature oligodendrocytes expressing MBP were graded. Cells without processes (category I), with primary or simple processes (category II), with secondary or complex processes (category III) and with myelin membranes based on the presence of continuous myelin staining between processes (category IV) were identified.To assess cell viability we identified cells that failed to extrude Trypan blue and ethidium homodimer. After removing media and washing with PBS, the cells were incubated for 5 min in 50% Trypan blue or ethidium homodimer followed by brief wash with PBS. All cells that were stained blue or contained red fluorescence were counted in 20 adjacent ×40 fields of view per chamber. The assessment of TUNEL positive cells was performed using Immunocytochemical and immunohistochemical stains were analyzed using Zeiss Axiovert and Zeiss Axioplan microscopes (Germany), respectively. For ICC (attached cells in slides and detached cells in cytospins), 10 fields (×20) were randomly chosen per slide or cytospin and FITC, Rhodamine and DAPI channels were imaged using an Axiocam mRM (Zeiss). The cells of interest were manually counted per field (10 fields per chamber in triplicates) and an average obtained per chamber or cytospin for each experiment.t test.Significant differences between multiple groups were analyzed using one-way ANOVA and differences between two groups was determined using Student's The exposure of oligodendrocyte lineage cells from P0 rat to 1 μM, 10 μM, and 100 μM of sodium azide for 3 h decreased complex IV activity of mitochondria isolated from oligodendrocytes by 38.6% (mild), 68.7% (moderate), and 83.9% (severe), respectively, compared with control samples when normalized using citrate synthase. In differentiated cultures, 93.7% of DAPI positive nuclei, on average, expressed Olig2 and oligodendrocyte lineage cells were then characterized based on MBP or PDGFRA and NG2 labeling a–f. LessComplex IV inhibition led to a striking loss of processes in P0 rat oligodendrocyte lineage cells a–f. The To confirm that the cell loss following mitochondrial inhibition occurred prior to immunostaining we determined the cell density in supernatant following complex IV inhibition, which reflected oligodendrocyte lineage cell loss a. The dep-trifluoromethoxyphenylhydrazone (FCCP), caused a dramatic reduction in MMP within differentiated oligodendrocytes and OPCs  in the vulnerability of oligodendrocyte lineage cells to a number of insults (see Supp. Info. Table 1), we measured mitochondrial superoxide in differentiated and then resuspended live oligodendrocytes as well as undifferentiated OPCs. Interestingly, superoxide levels in differentiated oligodendrocytes and OPCs exposed to sodium azide, at variable concentrations  and exposure times , did not change compared with cells not exposed to inhibitors .As OPCs from P0 rat withstand complex IV dysfunction better than their mature counterparts we investigated whether complex IV inhibition affected OPC proliferation. Interestingly, the exposure of OPCs to sodium azide did not affect PDGF and FGF mediated OPC proliferation over a 48 h period, judged by the absolute number of Olig2 expressing cells (Supp. Info. Table 2).As the next step we determine the impact of mitochondrial injury on differentiation capacity of P0 rat OPCs using complex IV inhibitors at concentrations (up to 10 μM of inhibitor) that did not reduce OPC density . The comTo establish whether the reduction in percentage of MBP expressing cells that differentiated following constant exposure to a complex IV inhibitor was due to a reduction in the capacity to differentiate or differentiation into a more susceptible phenotype, we briefly exposed OPCs in suspension to inhibitors, prior to differentiation . InhibitIn this study, we identified mature oligodendrocytes differentiated from P0 rat as more prone to mitochondrial respiratory chain complex IV inhibitor mediated injury than OPCs. Furthermore, complex IV inhibition throughout and briefly prior to differentiation of OPCs impaired formation of oligodendrocyte processes.Surprisingly inhibitors of mitochondrial respiratory chain complex IV did not significantly change the viability of cells that remained attached. Instead cells, mostly mature oligodendrocytes, detached from the slides following inhibition of complex IV in a concentration dependent manner. Energy requirements of mature oligodendrocyte processes appear to be much greater than processes of OPC. MMP in attached mature oligodendrocyte processes decreased significantly at lower levels of complex IV inhibition and to a greater extent as a proportion of controls than MMP in OPC processes. Furthermore, injury to processes as a prerequisite to cell detachment is suggested by the differentiation experiments with persistent inhibitors where process formation was impaired and cell detachment was modest. Imbalance of calcium downstream of mitochondrial injury may be important in the differential susceptibility of oligodendrocytes to complex IV inhibition (Marino et al., in vivo, will address some of the limitations of in vitro studies. Furthermore, as the outcome of in vitro studies is influenced by differences in experimental paradigm, age of animals, species from which OPCs were derived and culture techniques our findings may not be applicable to oligodendrocytes differentiated in vivo and neonatal OPC from other species.Maturation dependent susceptibility of oligodendrocyte lineage cells has been the focus of a number of studies. Several studies have reported OPCs to be more vulnerable than MBP+ oligodendrocytes. Although mitochondria have been implicated in oligodendrocyte death in a number of studies complex IV activity was not determined in previous studies and mitochondrial activity and cell viability were assessed using MTT assay, which is based mitochondrial dehydrogenases. Superoxide, implicated in a number of previous studies, was not involved in the differential susceptibility in this study, which is not unexpected as MMP dissipation may prevent ROS upregulation and, unlike complex I, complex IV defects need to be severe to upregulate ROS (Adam-Vizi, in vitro (Druzhyna et al., The most notable feature to be affected by complex IV inhibition during OPC differentiation was formation and structure of oligodendrocyte processes. Differentiation of OPCs into myelinating oligodendrocytes is a metabolically active process (Bauer et al., in vitro findings recapitulate some of the features of WMS, PVL, and MS: distal oligodendrogliopathy involving first the processes and then leading to oligodendrocyte loss, partial preservation of OPCs, and differentiation block with limited remyelination. We propose complex IV defects as an important cause of oligodendrocyte injury and differentiation block of OPCs in demyelinating disorders.Mitochondrial dysfunction is now established in WMS and MS. Our


Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.Strains from a collection of CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins. Drosophila insulator proteins are present at several thousand sites throughout the genome where they may play a variety of functions The three-dimensional organization of the chromatin fiber in the nucleus is important for the spatial and temporal regulation of transcription during development Drosophila but additional factors may be required to explain all the properties of these sequences The use of screens based on yeast two hybrid or classical genetics has allowed the identification of a number of proteins that interact with insulator DNA sequences or other insulator components in Drosophila alleles with the goal of identifying proteins with interesting nuclear localization patterns Drosophila cells. We focus our analysis on four of these proteins, Drosophila Nucleoplasmin (dNlp), Decondensation factor 31 (Df31), Stonewall (Stwl) and CG11138, that show a punctate distribution similar to that of insulator or Pc bodies. The results give a glimpse into the function of these proteins in nuclear biology and set the stage for future investigation of the role of these proteins in chromatin structure and transcription.We have used immunofluorescence microscopy of live tissues to screen a collection of GFP-tagged protein-trap Drosophila proteins Drosophila Nucleoplasmin (dNlp).In order to identify proteins present in a distinct pattern in the nucleus, we dissected third instar larvae from strains expressing GFP fused to different CG11138 gene. The GFP-tagged CG11138 protein is distributed in punctate bodies present throughout the nucleus , CG11138-PC (60.0 kDa) and CG11138-PD (61.6 kDa). In order to study the role of these proteins, we prepared antibodies against a region common to all three isoforms. Western analysis using these antibodies suggests that the different isoforms are differentially expressed during Drosophila development, with the smallest isoform being absent in third instar larvae . The disruption of CG11138 distribution in the nucleus may be a consequence of its direct interaction with Lamin proteins or a secondary consequence from the disruption of the nuclear lamina and corresponding alteration in nuclear architecture that occurs when Lamins are mutated.The protein trap line CA06844 contains an insertion of GFP in the  nucleus . This par larvae . A majorp allele . CG11138p allele . Colocalp allele  and Lami insulator The CG11138 protein is present at the band/interband border of polytene chromosomes . This paXenopus sperm DNA in vitroDrosophila life cycle and appears to be closely associated with DNA Drosophila embryos results in an abnormal number of nuclei and clumping or disorganization of the chromatin and eventual lethality In vitro studies suggest that Df31 can associate with histone H3 tails, can bind oligonucleosomes and can facilitate inter-strand bridging between chromatin substrates in vitro, the in vivo function of Df31 in the nucleus has yet to be clearly defined.A second protein identified in the GFP trap screen is Decondensation factor 31 (Df31). Previous studies suggest that Df31 is a highly disordered protein with the potential for interaction with different binding partners Analysis of GFP fluorescence of protein trap alleles of Df31 suggests that this protein is distributed around the polytene chromosomes of salivary gland nuclei rather than at the chromosomes; its localization in diploid cells is rather diffuse throughout the nucleus but there are distinct regions of localization –S1D. FurDrosophila contains four chromosome pairs that occupy four chromosome territories in diploid cells The distribution pattern of Df31 is of particular interest as Drosophila Nucleoplasmin (dNlp). Published work suggests that dNlp can bind core histones, facilitating nucleosome assembly, and is capable of decondensing Xenopus sperm in vitroDrosophila, we analyzed its distribution in the female ovary. dNlp is highly expressed in nurse cells during oogenesis and is also present in the egg  has been shown to be essential to maintain germline stem cells in the adult ovary Drosophila insulators, colocalization studies were done in female ovaries using antibodies to Stwl and CP190. The results from these experiments suggest that Stwl and CP190 appear to colocalize both in the terminal filament cells, which are known to regulate germline stem cells Analysis of Stwl-GFP protein trap lines shows that this protein is present at many sites on polytene chromosomes, suggesting that it may bind DNA at multiple genomic locations in somatic cells . In addiFurther analyses confirm that Stwl is strongly expressed in diploid cells of third instar imaginal tissue as well as binding to several regions on polytene chromosomes . It is iDrosophila cells Drosophila proteins were identified by co-affinity purification followed by mass spectrometry analysis of epitope-tagged proteins. Since Stwl and dNlp are present in a punctate pattern in the cell and the former partially co-localizes with insulator bodies, we used the published Drosophila protein interactome to examine the possibility that these proteins interact and perhaps gain information on the nature of other interaction partners that could shed light on the role of these proteins in nuclear biology. We could not examine possible interactions of CG11138 because this protein was not among those analyzed in the global interactome study. The results of this analysis indicate that Stwl, Df31 and dNlp interact with each other and form part of a large network of interacting proteins , Stwl also associates with several proteins that have been extensively studied in the context of the role of chromatin in transcription. This includes the transcription factor tramtrack; Otefin, Boksbeutel and Man1, two LEM-domain proteins present in the nuclear lamina; the Iswi, and Nurf38 components of chromatin remodeling complexes; and Prod, a protein required for mitotic chromosome condensation. These results, together with findings showing their subnuclear localization, suggest that Df31, dNlp and Stwl may participate in gene expression processes that require the localization of the chromatin fiber to specific nuclear compartments. Although the understanding of the details of these processes will require further studies, these findings pave the way for future work in the field.The last protein we chose to analyze is Stwl, which was initially characterized as a germline stem cell maintenance factor in Antibodies used in this study were prepared by Pocono Rabbit Farm & Laboratory (PRF&L). Protocols used were approved by the Animal Care and Use Committee (IACUC) of PRF&L under numbers PRF2A and PRF2B. PRF&L is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and by the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW.) The assurance number is A3886-01 with an expiration date of January 31, 2013.http://flytrap.med.yale.edu/), or from Dr. Allan C. Spradling. These fly lines contain the coding region of Green Fluorescent Protein (GFP) randomly inserted throughout the Drosophila genome. Drosophila proteins tagged with GFP are created by P element-mediated insertion into genes of an artificial exon encoding GFP flanked by splice acceptor and splice donor sites. Expression of GFP requires splicing into mature mRNAs and in-frame fusion Ex296lamin C 04643laminSZ18laminAll fly stocks were maintained on standard yeast-agar media and kept at 18°C or 25°C. Flies used for screening were obtained from either Flytrap, the GFP protein trap database at Yale University  was then used to obtain information on the location of the GFP insertion site; this information was confirmed using inverse PCR analysis of the PEP P-element as detailed by the Berkeley Drosophila Genome Project Collections of GFP trap lines were screened for the nuclear distribution of the GFP signal; salivary glands and imaginal tissue from third instar larvae were dissected in 1 X Phosphate Buffered Saline (PBS) and mounted on clean slides using Vectashield containing the DNA stain DAPI to mark the nuclei. When mounting, the tissue was gently squashed or spread for improved visualization of individual cells. Adult ovaries from specific lines were dissected in a similar manner. Once prepared, samples were visualized using a Zeiss Axioplan fluorescence microscope and IP Lab software. Samples were screened to identify the cellular location of the GFP fluorescence. Those with strong nuclear localization, particularly those in which GFP was distributed in unique patterns, were selected for further analysis. The Flytrap database  and fixed for 15 min with 4% paraformaldehyde in 100 mM sodium phosphate at room temperature Immunostaining of Kc cells was done as described above. For immunostaining of polytene chromosomes, third instar larvae were dissected in 0.7% NaCl and the salivary glands were removed 2PO4, 7 mM Na2HPO4, 130 mM NaCl, and.125% Tween 20 (PBT) and once for 10 min in PBT plus 1.5% BSA (PBTA) again using solutions described in McKearin and Ohlstein. Samples were then incubated in secondary antibodies in PBTA for 2 hr at room temperature and then washed three times for 10 min each in PBT Immunostaining of adult ovaries was done as described The following primary antibodies were used: rabbit α-GFP (Molecular Probes) 1∶400, rat α-Stwl (gift from Dr. Dennis McKearin) 1∶20, guinea pig α-Stwl 1∶800, rabbit α-CG11138 1∶1000, rabbit α-Df31 1∶400, rabbit α-dNlp (gift from Dr. James Kadonaga) 1∶200, mouse α-Lamin Dm0  1∶200, mouse α-Lamin C  1∶200, rabbit α-CP190 1∶4000, rat α-Vasa  1∶200, rat α-Mod(mdg4)2.2 1∶1000, rabbit α-Su(Hw) 1∶2000, mouse α-MSL1 (gift from Dr. John Lucchesi), rabbit α-histone H3S10 ph (Millipore), rabbit α- phospho-Pol IIser5  and guinea pig α-CTCF 1∶2000. Antibodies complexed to Alexafluor 488 and Alexafluor 594 targeting the appropriate primary antibodies were used as secondary antibodies.Escherichia coli on nickel-agarose columns (Quiagen) and were used to immunize rabbits using standard procedures. Protein fragments include amino acids 358–615 for CG11138, 600–1025 for Stwl and 1–185 for Df31. The specificity of the resulting antibodies was validated by western analysis and immunostaining of polytene chromosomes and imaginal tissue from wild type and mutant larvae. In all three cases, antibodies gave the same results as antibodies to GFP used in similar analyses with strains carrying GFP protein traps in each of the genes.For preparation of antibodies against the CG11138, Stwl and Df31 proteins, fragments from each protein containing N-terminal GST-tags and C-terminal His-tags were purified from Extracts from third instar larva imaginal tissue and Kc cells were prepared using standard protocols and run on tris-glycine-SDS gels Figure S1GFP autofluorescence in protein-trap alleles depicting both nuclear and nonnuclear GFP localization. (A) Protein-trap allele CA07367 showing nonnuclear GFP localization in diploid cells. (B) Protein-trap allele CC006263 with nonnuclear GFP in salivary gland nuclei containing polytene chromosomes. (C) Df31 protein-trap allele G00196 with nuclear GFP in diploid cells showing distinct foci of localization amidst more diffuse GFP fluorescence. (D) Salivary gland nuclei of the Df31 protein-trap allele G00196 showing GFP fluorescence predominantly surrounding the polytene chromosomes. In all panels DNA is labeled blue using DAPI and the GFP is green.(TIF)Click here for additional data file.Figure S2Western blot analysis of CG11138 protein. Lanes 1, 2, and 3 contain increasing amounts of extract from wild type OR third instar larvae imaginal tissue. Lanes 4, 5 and 6 contain increasing amounts of extract from CG11138 protein-trap allele third instar larvae imaginal tissue. Lanes 7, 8 and 9 contain increasing amounts of extract from Df31 protein-trap allele third instar larvae imaginal tissue. As can be seen in lanes 1, 2 and 3 the predominant isoform of CG11138 has a molecular weight of ca. 65 kDa, similar to that predicted for isoforms CG11138-PC and CG11138-PD. In addition, there are two larger bands of about 100 kDa and 150 kDa, respectively. The nature of these bands is unclear but they appear to be specific for CG11138, as their size shifts in rows 4, 5 and 6, which contain the CG11138-GFP fusion protein. Lanes 7, 8 and 9 suggest that this shift is specific for CG11138-GFP, as flies expressing a Df31-GFP protein show bands matching the wild type OR extracts when probed for CG11138.(TIF)Click here for additional data file.Table S1Summary of protein-trap screen results.(XLSX)Click here for additional data file.


Because few murine models share with HypoE mice these cardinal, human-like, features of CHD, HypoE mice represent a novel, small animal, diet-inducible and genetically tractable model for CHD. To better describe the properties of this model, we have explored the effects of varying the composition and timing of administration of atherogenic diets, as well as social isolation vs. group housing, on these animals.Mice with a deficiency in the HDL receptor SR-BI and low expression of a modified apolipoprotein E gene (SR-BI KO/ApoeR61HypoE mice were maintained on a standard lab chow diet (control) until two months of age. Subsequently they received one of three atherogenic diets  or control diet for varying times and were housed in groups or singly, and we determined the plasma cholesterol levels, extent of cardiomegaly and/or survival. The rate of disease progression could be reduced by lowering the severity of the atherogenic diet and accelerated by social isolation. Disease could be induced by Paigen diets either containing or free of cholate. We also established conditions under which CHD could be initiated by an atherogenic diet and then subsequently, by replacing this diet with standard lab chow, hypercholesterolemia could be reduced and progression to early death prevented.HypoE mice provide a powerful, surgery-free, diet-‘titratable’ small animal model that can be used to study the onset of recovery from occlusive, atherosclerotic CHD and heart failure due to MI. HypoE mice can be used for the analysis of the effects of environment  on a variety of features of cardiovascular disease. ApoeR61h/h mice developed by Raffai and colleagues h/h mice  can develop hypercholesterolemia and aortic atherosclerosis when fed an atherogenic diet and that replacement of the diet with lab chow reduces the hypercholesterolemia and results in regression of the atherosclerosis Mice with homozygous null mutations in the gene encoding the HDL receptor , SR-BI KO) exhibit hypercholesterolemia with abnormally large, unesterified cholesterol-rich HDL particles Here we have further characterized the HypoE model by exploring the effects of varying the composition and timing of administration of atherogenic diets, as well as social isolation vs. group housing, on the plasma cholesterol levels, cardiomegaly and/or survival. We found that the rate of disease progression could be adjusted by modifying the severity of the atherogenic diet and could be accelerated by social isolation. We also established conditions under which CHD could be initiated by feeding atherogenic diet and then reduction in hypercholesterolemia achieved and progression to early death prevented by replacing this diet with standard lab chow. This protocol may generate a powerful murine model for the study of heart remodeling and heart failure due to MI. Thus, HypoE mice appear to be an attractive model for studying the effects of environment  on a variety of features of cardiovascular disease.We examined the effects of atherogenic diets with differing severities on the plasma lipids, heart-to-body weight ratios and survival of HypoE mice. The animals were weaned onto a normal chow diet  and at two months of age we began continuous ad lib feeding for the indicated times with one of the four following diets: normal chow, Western diet , Paigen diet without cholate , or a standard Paigen diet . Except where indicated otherwise, animals were housed in groups of 4–5 per cage at the beginning of the ad lib feeding of the indicated diets. As indicated below, mortality reduced the population density in some cages.After administration of one of the four experimental diets for one month, we collected plasma samples and determined both plasma total cholesterol (TC) and unesterified cholesterol (UC) levels  and, forBecause cardiomegaly  is associated with abnormal heart function and MI/premature death in Paigen-diet fed HypoE mice Social isolation increases CHD mortality and morbidity in the general human population and can influence disease in animals as well As in the experiments described above, HypoE mice were weaned onto a normal chow diet until two months of age. At two months of age the diets of the mice were switched from chow to an atherogenic diet (Paigen or Paigen NC) and they were placed in cages at population densities of either 1, 2–3, or 4–5 mice per cage  with a uniform HypoE genotype. +/−ApoeR61h/h (mixed genotypes). As expected, none of these atherogenic diet-fed companion animals (SR-BI+/−ApoeR61h/h) died during the experiments reported here. +/−ApoeR61h/h mice).A potentially confounding feature of the experiment in The data in To determine if more rapid onset of cardiomegaly was associated with the shorter median survival times observed in singly housed mice, we measured the heart-to-body weight ratios of singly and group housed male and female HypoE mice fed the Paigen diet for 19 days . Indeed, the heart-to-body weight ratios were higher in the singly housed mice. The ratios (mg/g) for males were: group housing, 6.9±0.2 (n = 19); single housing, 11.0±0.5 (n = 16) (p<0.0001) and for females: group housing, 8.3±0.4 (n = 24); single housing 11.2±0.5 (n = 13) (p<0.0001). Thus, it seems likely that more rapid onset of CHD in the singly housed mice was responsible for the shorter median survival times.The principle goal of the current studies was to describe the characteristics of HypoE mice. However, we have begun to explore potential mechanisms underlying the differences in the rate of development of cardiomegaly and median survival times of singly and group housed HypoE mice fed the Paigen diet. First, we measured the cholesterol (TC and UC) concentrations determined from plasma sampled at two times after initiation of the atherogenic diet: 19 days  and 10 days (prior to any deaths or evidence of reduction in weight gain or loss of weight associated with severe CHD). The results were similar for male HypoE mice fed the Paigen diet for only 10 days. Compared to group housed animals, singly housed male mice exhibited 41% higher TC  and 39% higher UC , n = 6/set). There were no significant differences in the UC/TC ratios . Thus, the population density effect on plasma cholesterol preceded any gross, outward signs of pathology and may have contributed to the dependence of the rate of disease progression on population density.+/−ApoeR61h/h, or SR-BI−/− mice with wild-type apoE genes). They crumbled the food pellets, generating substantial amounts of crumbs in the bedding of the cage that were difficult to quantify reliably with our experimental apparatus , or group housed mice with mixed genotype (HypoE and SR-BI+/−ApoeR61h/h), even though there were differences in plasma cholesterol levels at 10 days between singly and group housed HypoE mice (see above). Differences in the endocrine systems  Plasma oxytocin levels  in femalThe ability to induce fatal CHD in HypoE mice by administering an atherogenic diet  raised the possibility that exposure to this diet for only a relatively brief period followed by feeding a normal chow diet might be sufficient to induce CHD  but not premature death. Such conditions might permit the HypoE mice to be used as a model for cardiac remodeling and recovery from atherosclerosis associated heart damage. Therefore, we initiated Paigen diet feeding of singly housed mice at two months of age as described above, but subsequently replaced the Paigen diet with a normal chow diet 10, 12 or 14 days later, or continued to feed the Paigen diet for the remainder of the study (control).The heart-to-body weight ratio 10 days after initiation of the Paigen diet feeding was 8.9±1.2 (n = 5), 1.8–times larger than for chow diet fed animals (∼5.0), indicating substantial cardiomegaly. Cardiomegaly (heart-to-body weight ratio 8.2±1.4 (n = 13)) in 10 day transiently Paigen-diet fed mice also was observed after an additional 60 days of chow feeding. Although substantial, these heart-to-body weight ratios were significantly smaller than that observed in mice continuously fed the Paigen diet for 19 days (11.1±1.8 (n = 29), p<0.05), indicating that an upper limit of cardiomegaly had not been reached. There was a very sharp temporal threshold of approximately 10 days of Paigen diet feeding, two days beyond which the progression to death could not readily be prevented simply by withdrawal of the atherogenic diet. Time-course analysis of plasma TC levels revealed that there were no obvious differences between 10 and 12 days of Paigen diet feeding on the maximal plasma TC level attained nor in the shape of the TC decay curves after withdrawal from the Paigen diet . It seemh/h) mice that represent a novel, diet-induced model of atherosclerotic CHD. We have explored the effects of varying the composition and timing of administration of atherogenic diets, as well as social isolation vs. group housing, on the plasma cholesterol levels, cardiomegaly and/or survival of HypoE mice. These mice do not express the HDL receptor SR-BI and they express no endogenous apoE and only very low levels of a human E4-like isoform of murine apoE (Thr61→Arg61). The Thr61→Arg61 substitution was initially designed to convert the murine apoE from a protein expected to resemble the human E3 isoform of apoE to one more like that of the human E4 isoform In the current studies, we provide additional phenotypic characterization of HypoE  and atherogenic diets of increasing severity: Western diet , Paigen diet without cholate , and a standard Paigen diet . The most sever atherogentic diet (Paigen) caused the greatest increase in plasma cholesterol carried in large (VLDL- and IDL-size) lipoprotein particles, most rapid development of cardiomegaly, and shortest survival times . The intermediate severity diet (Paigen NC) induced a less dramatic increase in large lipoproteins, a more gradual onset of cardiomegaly and somewhat longer survival times . Thus, as in previous studies , the current study shows that the extent of cardiomegaly is correlated with the median age of death. The results using the Paigen NC diet show that disease development in HypoE mice did not require cholate, whose inclusion in atherogenic diets is associated with hepatic fibrosis/inflammation The rapid onset and progression of atherosclerosis, CHD, and early death in hypercholesterolemic SR-BI/apoE dKO mice fed a normal chow diet The deleterious effects on health of stress and social isolation have been recognized for decades Although we have seen no differences in the survival of SR-BI/apoE dKO mice housed singly or in groups, we observed a profound reduction in longevity in singly housed HypoE mice when fed either a Paigen- (by 7 days) or Paigen NC- (by 32 days) diet. Strikingly, although social isolation did not appear to affect food intake or body weight, it resulted in significantly higher plasma TC and UC levels during a 10-day course of Paigen diet feeding. It seems likely that the increased exposure to higher plasma cholesterol levels could promote more rapid atherosclerosis development and thus shorter survival. We have observed that the qualitative nature of the occlusive atheroscleorosis and MI at the median age of death for singly housed HypoE mice are similar to those seen at the median age of death for the group housed mice (unpublished), indicating that occlusive coronary arterial atherosclerosis is the likely pathophysiological mechanism responsible for the premature death.To explore the potential relationship of alterations in the endocrine system with the effects of social isolation, we measured plasma levels of two hormones: corticosterone and oxytocin. Corticosterone is an adrenocortical steroid whose plasma levels are often used as a quantitative measure of stress, although SR-BI KO mice exhibit defective plasma corticosterone responses to stress Oxytocin, a hypothalamic neuropeptide produced in the posterior pituitary gland and other tissues (including the heart), can regulate behavioral responses to stressors and the reactivity of the hypothalamic-pituitary-adrenal (HPA) axis A particularly intriguing feature of the HypoE mouse is the potential of initiating the development of CHD by feeding an atherogenic diet and then replacing that diet with a normal chow diet to stop additional atherosclerosis progression. Indeed, we found that the hypercholesterolemia in HypoE mice that reaches a steady state after five days of Paigen diet feeding can be substantially reversed after returning the mice to a chow diet for four days. When HypoE mice were fed the Paigen diet for 10 days, which causes profound cardiomegaly (and presumably cardiac damage due to MI) and then returned thereafter to a chow diet, most of the animals (87%) were able to survive for at least 60 days. At that time, the mice exhibited cardiomegaly and preliminary histology studies raise the possibility that their hearts may have undergone remodeling during the 60 day post-atherogenic diet period. Thus, HypoE mice might be a promising novel model for the study of heart remodeling, reversion of occlusive coronary arterial atherosclerosis , and the pathophysiology of heart failure without the need for complicating surgical procedures such as coronary ligation.h/h) mice are infertile h/h mice with heterozygous null mutations in SR-BI were mated to generate the HypoE mice. Mice were fed a normal chow (low fat) diet  from weaning to 2 months of age. Then they were fed either Western , Paigen without cholate (Paigen NC) , or Paigen  diets for the indicated periods. In some cases animals fed the Paigen diet were switched back to a standard chow diet, as indicated. After weaning (3 weeks of age) mice were housed with the same sex littermates until 2 months of age. Animals from multiple, independent litters were distributed among the different experimental groups. Number of animals used in each experiment are indicated (‘n’).All mice were on mixed C57BL/6x129 backgrounds  All experiments using animals described here were performed in strict accordance with NIH and Massachusetts Institute of Technology guidelines and approval of the Committee on Animal Care at the Massachusetts Institute of Technology (approved protocol numbers 0209-015 and 0212-015).Mice were anesthetized by intraperitoneal injection of 1.25% Avertin, weighed, and perfused with heparinized PBS and intact hearts were dissected and weighed.Levels of plasma total cholesterol (TC) and unesterified cholesterol (UC) were determined using standard assay kits from Wako Chemicals , according to the vendor’s instructions. FPLC plasma fractionation was performed as previously described +/−ApoeR61h/h) were housed in groups of 4 to 5 per cage, and (c) “isolated” or “single” housing: mice were individually housed in cages. All of the cages were equal in size.The experimental housing conditions from 2 months of age were as follows: (a) “group housing”: mice with the same genotype  and sex were housed in groups of 2 to 5 per cage, (b) “group housing with mixed genotype”: mice of the same sex but different genotypes (HypoE and SRBI+/−ApoeR61h/h) and group housed mice with mixed genotypes (HypoE with SRBI+/−ApoeR61h/h). For group housed mice, the intake per mouse is the total divided by the number of mice in the cage (n = 4 or 5) and thus represents an average contributed to by mice with both genotypes.Apparent daily food intake  was recorded daily from day 2 to day10 after initiation of Paigen diet feeding of singly housed mice . The genotypes of mice used for plasma hormone measurements were all HypoE, and group housed SR-BISingly housed HypoE mice were fed the Paigen diet for 10, 12, or 14 days from the age of 2 months, then were fed a normal chow diet thereafter. For some animals, small amounts of blood were drawn serially from the retro-orbital plexus with heparinized capillary tubes for determination of plasma TC after 0, 5, 10, 12, 14, 16, 21 or 28 days after initiating feeding the Paigen diet, as indicated. Controls included mice fed only a chow diet throughout the experiment or those fed a Paigen diet without changing back to a chow diet.p<0.05 was considered significant.Data are shown as the means ± S.D. Statistically significant differences were determined by Kruskal-Wallis tests followed by the Dunn’s multiple comparison post-test for comparison of three or more groups, two-tailed, unpaired Student’s t or Mann-Whitney test for comparison of two samples, and log-rank test to compare survival curves. Correlation between heart-to-body weight ratio and plasma hormone levels was evaluated by Pearson’s correlation or Spearman’s rank correlation. A value of Figure S1Effects of population density on apparent daily food intake of Paigen diet-fed HypoE female mice. Female mice were housed in mixed genotype groups  or singly  or SRBI+/−ApoeR61h/h mice, dashed line, n = 4). Beginning at two months of age the animals were switched from a normal chow diet to the Paigen diet and then the food reservoir was weighed daily for each cage. The estimated food intake per day per mouse was calculated as the difference between reservoir weights on sequential days. The per mouse values for the group housed, mixed genotypes are averages from all of the animals in each cage.(TIF)Click here for additional data file.Figure S2Effects of population density on apparent daily food intake of Paigen diet-fed HypoE male mice. Male mice were housed in mixed genotype groups  or singly  or SRBI+/−ApoeR61h/h mice, square symbols) dashed line, n = 6). Beginning at two months of age the animals were switched from a normal chow diet to the Paigen diet and then the food reservoir was weighed daily for each cage. A The apparent food intake per day per mouse was calculated as the difference between reservoir weights on sequential days. The per mouse values for the group housed, mixed genotypes are averages from all of the animals in each cage. B The apparent average food intake was determined over the 2–10 day period for each condition. There were no statistically significant differences.(TIF)Click here for additional data file.Figure S3Relationships between plasma levels of oxytocin and corticosterone. HypoE mice were housed in mixed genotype groups  or singly  Beginning at two months of age the animals were switched from a normal chow diet to the Paigen diet. After 19 days of Paigen diet feeding, the mice were weighed, plasma samples were harvested, and subsequently plasma levels of corticosterone and oxytocin were determined. Statistics were evaluated using Spearman’s rank correlation. The genotypes of mice were all HypoE, and group housed SR-BI+/−ApoeR61h/h were not included in the analysis.(TIF)Click here for additional data file.