Herpes simplex viruses (HSV) are significant human pathogens that provide one of the best-described examples of viral latency and reactivation. HSV latency occurs in sensory neurons, being characterized by the absence of virus replication and only fragmentary evidence of protein production. In mouse models, HSV latency is especially stable but the detection of some lytic gene transcription and the ongoing presence of activated immune cells in latent ganglia have been used to suggest that this state is not entirely quiescent. Alternatively, these findings can be interpreted as signs of a low, but constant level of abortive reactivation punctuating otherwise silent latency. Using single cell analysis of transcription in mouse dorsal root ganglia, we reveal that HSV-1 latency is highly dynamic in the majority of neurons. Specifically, transcription from areas of the HSV genome associated with at least one viral lytic gene occurs in nearly two thirds of latently-infected neurons and more than half of these have RNA from more than one lytic gene locus. Further, bioinformatics analyses of host transcription showed that progressive appearance of these lytic transcripts correlated with alterations in expression of cellular genes. These data show for the first time that transcription consistent with lytic gene expression is a frequent event, taking place in the majority of HSV latently-infected neurons. Furthermore, this transcription is of biological significance in that it influences host gene expression. We suggest that the maintenance of HSV latency involves an active host response to frequent viral activity. Primary herpes simplex virus (HSV) infections are characterized by acute disease that resolves rapidly, but the virus persists in a latent form in sensory neurons that can be a source of renewed disease. Analyzing gene expression in single mouse neurons harboring latent HSV, we show directly that HSV latency is dynamic and heterogeneous. HSV lytic gene transcripts were frequently detected in latently infected neurons and often in combinations. Expression of selected cellular anti-viral and survival genes showed that transcriptional profiles differed between latently infected and uninfected neurons from the same ganglia. The pattern of host gene expression also differed between latently infected neurons that were and were not experiencing HSV lytic gene expression. Our study suggests that HSV latency is characterized by very frequent switching on of lytic genes and a rapid response by the host, presumably to halt progression to reactivation. Herpes simplex virus (HSV) is a ubiquitous human pathogen that initially replicates in skin and mucosal surfaces prior to establishing an infection in sensory neurons The key to understanding the meaning of non-LAT transcription in HSV latency lies in the ability to examine events at the level of individual neurons. Recent advances in single-cell approaches to transcriptional analysis increase the sensitivity of detecting gene expression and allow the filtering out of samples containing transcripts from irrelevant cell types UL54 (ICP27), UL29 (ICP8) and US6 (gD) (Mice were inoculated by flank scarification using HSV type 1 (HSV-1) strain KOS, which results in the infection of the skin and innervating dorsal root ganglia (DRG) US6 (gD) .LacZ) genes driven by the ubiquitous ROSA26 promoter interrupted with a floxed neomycin cassette were infected with HSVs expressing Cre recombinase under the CMV IE promoter  In order to move the analysis to single neurons, we employed a transgenic mouse model in which all infected neurons are marked so that they can be distinguished from other cells + neurons in DRG from uninfected mice. While the method worked well for the identification of latently-infected neurons, it had limitations; including the likelihood of partial cell capture, variations in capture volume, and inter-cell contamination. We reasoned that the impact of such limitations would be minimized if we examined many neuronal sections (hereafter referred to simply as neurons for brevity) in each analysis. In addition, variations associated with partial cell capture were expected to be consistent across all groups, allowing valid comparisons that would be similar to what could be found if whole neurons were to be used.Our approach to obtaining single neuron samples was to use laser capture microscopy (LCM) to identify by YFP expression and cut relevant cells from 8 µm sections of pooled latent DRG . Marking+ and YFP− neurons. Most individual YFP+ cells contained HSV DNA, whereas more than 80% of those with no detectable YFP did not  followed by a multiplexed pre-amplification step before splitting into 61 qPCR (Taqman) reactions. This method has some caveats, for example we cannot verify the strand of transcripts see . This wo+ and 256 YFP− neurons from DRG of mice taken 50 days after infection (combined from four independent infections) and 136 neurons from DRG of uninfected mice (from three independent experiments). Each of these single cell RNA samples was analyzed with 61 primer/probe sets, targeting 51 cellular and 10 viral transcripts. Of the cellular transcripts, 47 of the 51 primers/probes spanned exon-exon boundaries. We included markers that allowed application of stringent selection criteria to neurons for further analyses. These included Gfap and Rbfox3 as markers for satellite cells and neurons with adequate RNA, respectively. To eliminate the impact of possible errors in capturing neurons by LCM and spurious detections, only −Gfap, Rbfox3+ neurons in which more than 10% of the host genes could be detected were analysed further. In addition, uninfected neurons (YFP− or from uninfected DRG) in which any viral transcripts were detected were also excluded. Numbers of neurons excluded for any of these reasons and those remaining in analyses are noted in For the main analysis of transcription our study samples initially comprised RNA from 205 YFPNtrk1, Ntrk2, Ntrk3, Runx1, Runx3 and Ret) characterized different neuronal lineages. Analysis of these 6 genes showed that infection was heavily biased towards Ntrk1+ neurons (Ntrk1 gene (encoding TrkA protein) expression was significantly over-represented in YFP+ neurons: 86% of the YFP+ neurons in the infected DRG were Ntrk1+ compared to only 34% YFP− neurons  neurons that did not express detectable lytic genes (non-lytic); ii) those that expressed lytic genes from 1 or 2, but not all 3, classes ; iii) those that expressed multiple gene transcripts with at least 1 member of each lytic class (full-lytic). +YFP+ neurons were roughly divided into thirds among the 3 categories. So there were roughly as many latently-infected neurons with transcripts consistent with the expression of lytic genes from all kinetic classes as there were that had none of these RNAs. Therefore even at times when latency appears to be highly stable, HSV-infected neurons express non-LAT transcripts at a frequency that has previously only been demonstrated to occur in lytic infection.Differences in sensitivity might also underlie variable accounts of lytic gene expression in latency e.g. . We were neurons . When the to 64% . Also ofNtrk1+ neurons. PC1, 2 and 3 captured 44.27%, 6.73% and 5.22% of the variation in the gene expression data, respectively. Most YFP+ neurons segregated from those that were YFP− and neurons from uninfected DRG on PC2 and PC3, with some overlap captured on PC1  and the proportion of individual cells distributed along a given expression value (x-axis). The value of this representation can be seen in that most genes showed a bimodal distribution, indicating that the population contained individual cells with either high or low levels of a given gene transcript. We also calculated differences in mean transcript levels  Transcripts of Tnfrsf10b, Bcl2 and Tnfrsf14 were over-represented in full-lytic when compared to both of the other groups. 3) The single gene that differed between non-lytic and the groups that expressed lytic genes was Pou2f1. These data revealed heterogeneity in host gene expression in latently infected neurons that correlated with the number of classes of viral lytic genes detected. We conclude that transcription of HSV lytic genes during HSV latency is biologically relevant and suggest that even in the absence of overt reactivation, there is a progressive neuronal response against increasing viral activity.We reasoned that if the HSV lytic gene expression observed in latently-infected neurons was biologically relevant, it might influence the expression of host genes. For this reason, we compared host gene expression across the three groups of latently-infected neurons as identified by expression of HSV lytic genes . To do this, we visualized the transcriptional data as violin plots  and S4. t levels  and whetGenes involved in the same biological processes or pathways are often expressed with correlated profiles. We wondered whether we could identify such sets of genes that were co-regulated in response to viral lytic gene transcription in latently-infected neurons. Finding these sets would provide evidence of changes to broader transcriptional circuits. To do this, we examined the correlations in expression profiles between pairs of cellular genes from full- and non-lytic neurons. In the full-lytic group, strong gene correlations (τ>0.6) were found between 75 pairs of genes  and S7. Long-term HSV infection in mice is generally viewed as a model of profound latency, with few, if any, of the spontaneous reactivation episodes seen in other species and virtually no active production of virus once the lytic phase of infection is extinguished. In the context of this model of stable latency, the frequency with which transcripts consistent with lytic gene expression were found here is striking. Whilst our data does not break ground in terms of the detection of viral transcripts from outside the LAT region during latency, that this might happen so often has previously been only a matter of conjecture bona fide mRNAs from the various lytic genes. Some regions of the HSV genome are transcribed to produce co-linear messages for a set of genes and further, especially at late times of lytic infection, the termination of transcription is not always accurate. For this reason, in some cases we may not have detected only the HSV transcript intended. In addition, our reverse transcription method used random primers, being chosen to allow multiple transcripts to be detected at high sensitivity, but this also sacrifices strand-specificity. However, the nine HSV genes targeted are spread across the entire genome and different patterns of host gene expression are associated with increasing detection of the virus transcripts when ordered by expected kinetic classes. Furthermore, in some cases others have demonstrated elegantly that transcripts genuinely originating from specific HSV lytic genes are found in latently-infected ganglia Some caveats remain in our study with regard to the nature of the viral transcripts detected. The most important of these is that we were unable to be certain that the transcripts detected were + T cells Serpinb9, which encodes a protein that can protect cells from CD8+ T cell cytotoxicity, was up-regulated in the neurons showing the most viral transcription The relative roles of virus and host in maintaining latency remains an area of some contention, as is the extent to which detection of HSV transcripts other than LAT is of any significance. Much work has focused on mechanisms by which transcription from the majority of the genome is inhibited or regulated during latency, either through the silencing effect of heterochromatin or expression of miRNAs Pou2f1 (Oct1), which is important for viral reactivation Zbp1  is more frequently and significantly expressed in latently-infected, compared with uninfected neurons is of interest because this DNA sensor is associated with the response of fibroblasts to HSV Mx1, Oasl2, Eif2ak2 (PKR), Ifitm3 and Samhd1). Thus, we propose that there is a barrier to productive infection downstream of the initiation of lytic gene transcription and this is a threshold that is rarely exceeded in mice These difficulties and our results could be used to argue for a third model in which the host acts to maintain latency in a cell-intrinsic manner. The viral transcription detected here may represent abortive reactivation events All experiments were done according to Australian NHMRC guidelines contained within the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and under approvals given by The University of Melbourne Animal Ethics Committee (1112345.7) and The Australian National University Animal Experimentation Ethics Committee (A2011/015).tm1So/J, also referred to as ROSA26R mice, were from the Australian Phenomics Facility .C57BL/6 mice were bred at the Department of Microbiology and Immunology of the University of Melbourne. The gzmBCreERT2/ROSA26EYFP transgenic mice have been described previously UL43 gene of HSV-1. Similarly, KOS/pCMV/eGC contains HCMV IE promoter, but driving an EGFP-Cre fusion from the intergenic region beween UL3 and UL4. Mice were infected with 1×106 PFU via flank scarification as previously described The HSV-1 strains KOS0152, KOS/pCMV/eGC and wild-type parental strain KOS (WT HSV) were grown and titrated on Vero cells  as described in in situ by perfusing the mice from the left ventricle with 0.5% paraformaldehyde and 20% sucrose made in nuclease free PBS (fixative). DRG (T8–T12) were harvested, immersed in the fixative, and allowed to equilibrate on ice. Fixed DRG from two mice were pooled and then embedded in OCT  and frozen in liquid nitrogen. Frozen DRG were sectioned into 8 µm slices on a cryostat (Leica CM3050S) and consecutive DRG sections were placed on each subsequent slide (LOMB) that has been pre-treated with RNaseAway (Life Technologies). Freshly prepared tissue sections were dehydrated with 50%, 75% and 95% ethanol sequentially and air-dried just before laser capture. YFP− and YFP+ cells were identified from 8–24 non-consecutive tissue sections and laser microdissected using a PALM MicroBeam instrument (Carl Zeiss). Laser capture was performed at the Biological Optical Microscopy Platform, University of Melbourne, Australia. Cells were then captured into collection tubes containing lysis buffer. We avoided collecting cells from 2–9 consecutive sections to prevent repeated sampling from the same cell. In total, 205 YFP+ and 256 YFP− cells from HSV infected DRG and 136 cells from uninfected DRG were collected from 3–4 independent experiments with two mice per OCT block.Mice were culled and tissues were fixed Total RNA from LCM-captured single neurons was made into amplified cDNA using the Single Cell-to-CT kit (Life Technologies) according to manufacturer's instructions. Briefly, LCM-captured samples were lysed and treated with DNAse; and cell lysates were then reverse transcribed. In some samples, single cell lysates were split into halves whereby one half was reverse transcribed and the other was not. Products of RT (or sham RT) were then pre-amplified in a gene specific manner with a pool of 61 Taqman gene expression assays for 14 cycles; then diluted 1∶15 to enable use in the same number of Taqman assays  and S10.Total RNA from whole DRG was extracted and DNAse-treated using RNAqueous microkit (Life Technologies) and reverse transcribed into cDNA using Superscript VILO (Life Technologies) that was then diluted 1∶40. Aliquots of some samples were not reverse transcribed and served as ‘no RT’ controls. In some whole DRG samples, pre-amplification was carried out as described for single cell samples, but these pre-amplified products were not diluted See .DNA from LCM-captured neurons and whole DRG were extracted using the Picopure DNA extraction kit (Life Technologies). Briefly, single cells and DRG were incubated in proteinase K at 65°C for 3 h before enzyme inactivation at 95°C for 10 min.Pgk1. Relative expression calculated using the 2−ΔCt method Quantitative PCR assays were performed using Taqman gene expression assays  and S10 UL23) gene, are as follows: forward primer 5′-TTGTCTCCTTCCGTGTTTCAGTT-3′, reverse primer 5′-GGCTCCATACCGACGATCTG-3′, and Taqman probe 5′-FAM-CCATCTCCCGGGCAAACGTGC-MGB-NFQ-3′. DNA extracted from uninfected DRG was used as negative control. Average viral copy number was calculated using a standard curve generated using WT HSV or KOS0152 viral DNA (extracted as described for DRG), which was serially diluted such that 5 µl contained 100 copies to 107 HSV-1 DNA. Our limit of detection for DNA was 10 copies, as we could not detect below 10 copies consistently. In single cell analysis, a duplex reaction containing the primers/FAM-probe for HSV TK gene, and the primers/VIC-probe for β-actin (Actb) gene  that detects genomic DNA was used to filter out LCM samples that did not contain any cells. In all single cell quantitative PCR, mock LCM samples, which contain lysis buffer but no cells, were used as negative controls.HSV DNA detection in whole DRG and single neurons were performed as previously described in situ with 2% paraformaldehyde and 20% sucrose, harvested, secured onto a 10 mm dish with Vetbond tissue adhesive (3M), and immersed in PBS. Images were acquired with an upright LSM710 NLO multiphoton microscope (Carl Zeiss) as described previously DRG were fixed + cells was counted from the images with the aid of ImageJ software.DRG from HSV infected ROSA26 mice were fixed and stained with X-Gal  as previously described in situ with and equilibrated in 2% paraformaldehyde and 20% sucrose before embedding in OCT and frozen in liquid nitrogen. DRG were sectioned at 8 µm thickness. Sections were fixed in −20°C acetone for 5 min, rehydrated with PBS for 5 min, stained with NeuroTrace  at 1∶200 v/v in PBS for 20 min, rinsed in PBS as per manufacturer's instructions, incubated with Hoeschst nuclear stain  for 3 min, and then mounted with ProLongGold . Images were acquired with a Zeiss LSM710 microscope and processed using Imaris 7.1 software (Bitplane).DRG were fixed Rbfox3 (neuron marker), were positive for Gfap (satellite cell marker), and expressed less than 10% of the 51 cellular genes tested were removed from analysis. We consider the remaining Rbfox3+Gfap− cells as neurons. As noted above, transcription of genes follows intermittent bursts of biosynthesis Rbfox3 at the time of fixation and these would be wrongly excluded by our approach, but we felt that it was better to set conservative criteria for keeping samples in the final analyses. In a small fraction of YFP− neurons from infected DRG (27.4%) and uninfected neurons from uninfected DRG (8.5%), HSV transcripts were detected and these cells were excluded from subsequent analysis.All Ct values obtained were converted into relative expression threshold (Et) by subtracting the values from the maximum cycle value of 40. When the value of the gene expression is undetermined, we treat this gene as not expressed and assigned an Et value ‘0’ to the gene Rbfox3, Gfap and Ntrk1 were excluded because single cells were categorized based on these genes. Eigenvectors were then visualized with XLSTAT-3Dplot . Singular analysis toolset  was used to generate violin plots Principal components analysis was performed on 48 cellular genes using XLSTAT . Viral genes, Categorical variables were summarized using frequency and percentage. Continuous variables were first assessed for skew using a Shapiro-Wilk test and summarized using mean and standard deviation (SD) or median and inter-quartile range (IQR) as appropriate. Comparison of gene expression levels by neuron and lytic groups were undertaken using a Kruskal-Wallis test with a post-hoc Dunn test to correct for multiple simultaneous comparisons. As a sensitivity analysis, gene expression variables were dichotomized into expression recorded/no expression recorded quantities and compared across neuron or lytic groups using a chi-square test or a Fisher's exact test as appropriate. All analyses were conducted using Stata version 12 .k-means or complete-linkage. For k-means clustering, 4 clusters were chosen from the full-lytic YFP+ neurons subset based on the elbow method by comparing the sum of squared error (SSE) of a number of clusters. k-means clustering, complete-linkage clustering and SSE were performed in R statistical program.Correlation coefficients were calculated based on gene expression levels using Kendall rank correlation in XLSTAT . The correlation coefficients were then clustered using Figure S1+ neurons.Single cells are free of contaminating viral DNA. Heatmap showing detection of 2 kb LAT in standard (+RT) and sham (No RT) reverse transcribed RNA from a set of 16 YFP(TIF)Click here for additional data file.Figure S2Rbfox3+Gfap−) determined using quantitative RT-PCR. Data from main single cell experiments.Heatmap showing expression of neuronal-related genes in single neurons ((TIF)Click here for additional data file.Figure S3Ntrk1+ neurons determined using quantitative RT-PCR. Data from main single cell experiments.Heatmap showing 48 cellular genes in single (TIF)Click here for additional data file.Figure S4+Ntrk1+YFP+ neurons, categorized based on their lytic gene expression profiles. Data from main single cell experiments.Violin plot representation of 48 cellular transcripts in LAT(TIF)Click here for additional data file.Table S1Numbers of neurons excluded from gene transcripts analysis.(DOCX)Click here for additional data file.Table S2Ntrk1+ and LAT+Ntrk1+YFP+ neurons.Ratio of mean gene expression levels (Et) from comparisons within all, (DOCX)Click here for additional data file.Table S3p values for the gene expression levels from comparisons within all, Ntrk1+ and LAT+Ntrk1+YFP+ neurons.(DOCX)Click here for additional data file.Table S4p values for the proportion of cells expressing the gene from comparisons within all, Ntrk1+ and LAT+Ntrk1+YFP+ neurons.(DOCX)Click here for additional data file.Table S5+Ntrk1+YFP+ neurons: Complete-linkage clustering of gene expression correlation coefficients matrix.Full-lytic LAT(XLSX)Click here for additional data file.Table S6+Ntrk1+YFP+ neurons: Complete-linkage clustering of gene expression correlation coefficients matrix.Non-lytic LAT(XLSX)Click here for additional data file.Table S7+Ntrk1+YFP+ neurons: p values of complete-linkage clustering of gene expression correlation coefficients matrix.Full-lytic LAT(XLSX)Click here for additional data file.Table S8+Ntrk1+YFP+ neurons: p values of complete-linkage clustering of gene expression correlation coefficients matrix.Non-lytic LAT(XLSX)Click here for additional data file.Table S9Taqman gene expression assays for cellular genes (Life Technologies).(DOCX)Click here for additional data file.Table S10Taqman probe and primers sequences for HSV-1 KOS genes.(DOCX)Click here for additional data file.


Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. Cancer has long been regarded a clonal disease. While this view still applies to certain haematological malignancies, the majority of cancers, and in particular solid tumors, follow another structure and are characterized by tremendous cellular complexity. This diIn search of a common signature for ovarian CSC, we recently analysed the expression of several CSC markers in various ovarian cancer cell lines n=13). This screen revealed that of all markers tested ), only the SP marker detecting dye-extrusion by drug transporters robustly identified minor populations in virtually all cell lines investigated  , breast (n=4), lung (n=3), colorectum (n=2) and adrenal gland n=1). These experiments revealed that the SP phenotype was again a robust marker, defining distinct populations in 4/4 cell lines of breast cancer, 2/3 cell lines of lung cancer, 2/2 cell lines of colorectal cancer, 1/1 cell line of adrenal gland cancer, and still 1/5 cell lines of prostate cancer (Table . These ede novo resistance’ might therefore often be a misinterpretation of what actually happens, that is Darwinian selection of small, pre-existing cancer clones inherently showing resistance. In this respect, cancer cell lines are therefore very similar to antibiotic-treated bacterial cultures[The high conservation of SP cells in human cancer cell lines indicates a functional role of this cell compartment. In ovarian and several other cancers for instance, we and others have shown that SP cells exhibit functional SC characteristics,4,5. Thu cultures.ex vivo tumor tissue and whether these primary cells exhibit functional SC traits as well. This could not only further our understanding of in vivo tumor evolution and biology, but could also provide rationale for developing treatment strategies against a specific SC pool conserved within the tremendous complexity of solid tumors.Altogether, we propose that phenotypic and functional heterogeneity is a hallmark of most human cancer cell lines that needs to be considered for the correct interpretation of findings obtained with these model systems. Especially for observations related to SC characteristics and drug resistance, the SP minority compartment might often be responsible. Henceforth, it will be important to elucidate whether SP cells can also be found at such high frequency in


Megaloceros giganteus is among the most fascinating Late Pleistocene Eurasian megafauna that became extinct at the end of the last ice age. Important questions persist regarding its phylogenetic relationship to contemporary taxa and the reasons for its extinction. We analyzed two large ancient cervid bone fragments recovered from cave sites in the Swabian Jura  dated to 12,000 years ago. Using hybridization capture in combination with next generation sequencing, we were able to reconstruct nearly complete mitochondrial genomes from both specimens. Both mtDNAs cluster phylogenetically with fallow deer and show high similarity to previously studied partial Megaloceros giganteus DNA from Kamyshlov in western Siberia and Killavullen in Ireland. The unexpected presence of Megaloceros giganteus in Southern Germany after the Ice Age suggests a later survival in Central Europe than previously proposed. The complete mtDNAs provide strong phylogenetic support for a Dama-Megaloceros clade. Furthermore, isotope analyses support an increasing competition between giant deer, red deer, and reindeer after the Last Glacial Maximum, which might have contributed to the extinction of Megaloceros in Central Europe.The giant deer  Megaloceros giganteus , first described by Blumenbach in 1799Megaloceros recolonized northwestern Europe in the Late Glacial InterstadialThe extinct giant deer Megaloceros within the family Cervidae is still debated. The presence of large palmate antlers in extant fallow deer (Dama dama) and Megaloceros giganteus suggests a close relationship between the two species6Megaloceros into a group comprising red deer (Cervus elaphus), cheetal (Axis), and bush antlered deer (Eucladoceros)9Dama - Megaloceros clade is low, likely due to the partial mtDNA regions studied and the limited availability of modern cervid mtDNA for comparison11Megaloceros giganteus. Comparison against mtDNAs from 44 extant deer species provides furthermore strong support for a Dama - Megaloceros mtDNA clade.The phylogenetic position of During an excavation at the Hohlenstein Stadel cave at the Lone Valley , an accumulation of large cervid bones was discovered including an almost complete atlas, two scapulae and two pelvic fragments, two ribs, a tooth (M3) and six fragments of a tibia. Apart from these finds, a metatarsus shaft fragment from a large cervid was obtained from the Hohle Fels cave at Schelklingen . We genetically analyzed one of the six tibia fragments  was chosen as an outgroup. Both ML and MP topologies place the mtDNA sequences reconstructed from our two ancient cervid bones (Hohlenstein Stadel and Hohle Fels) in a completely resolved clade with both extant fallow deer subspecies (Dama dama and Dama mesopotamica), and exclude them from the group comprising Pere David’s Deer (Elaphurus davidianus), Rusa sp. and red deer (Cervus elaphus sp). The other large cervid present in Europe during the Pleistocene, the European elk, Alces alces, can also be excluded as the source of our ancient cervid bones.The reconstructed mtDNAs from both ancient cervid bones were aligned with 44 publicly available full mitochondrial genomes of extant cervids. The hypervariable D-loop region was excluded from the cervid mtDNA alignment due to its fast evolutionary rate that may decrease the phylogenetic resolution. We used MEGA 6.0.6 to construct both a maximum-likelihood (ML) tree  and a maDama species within the Dama clade is, however, not completely resolved. As Dama was only introduced to Europe in the Medieval periodAlces alces) based on phylogenetic evidence, we conclude that both specimens originate from Megaloceros giganteus. To test this hypothesis we further compared our reconstructed ancient cervid mtDNAs to cytochrome b (cytb) regions of Megaloceros mtDNA previously published The small nucleotide distance of our ancient large cervids and both previously determined giant deer 13C) and nitrogen (15N) were measured and compared to large cervids present in Central Europe during the Pleistocene such as red deer, reindeer, and giant deer1819Megaloceros samples from Southern France and Belgium typically show isotopic signatures of collagen comparable to those of red deer (Cervus elaphus), while reindeer (Rangifer tarandus) provides systematically higher δ13Ccoll values likely due to the consumption of lichen13 C-based distinction among larger cervids from the Swabian, French, and Swiss Jura decreases. Stable isotope signatures of our both ancient cervid bones from the Hohle Fels cave and the Holenstein Stadel cave fall inside the red deer-reindeer cluster reflecting a potential overlap in diet and habitat . Based on the phylogenetic position of our reconstructed ancient mtDNAs, their close genetic relationship to the previously determined partial cytb sequence from a complete giant deer skeleton from western Siberiacytb sequence from a giant deer astralagus from IrelandMegaloceros giganteus. We find strong support for a close maternal relationship to both Dama species. The maternal relationship within the Megaloceros-Dama clade however could not be resolved in our phylogeny suggesting an almost equal genetic distance of the two fallow deer species and giant deer. Our results disagree with the morphological conclusions that Megaloceros is closer related with a group comprising Cervus, Axis, and Eucladoceros8Megaloceros and Dama must be the result of homoplasy. Our results also disagree with the conclusions derived from short mtDNA sequences such as partial cytb reported by Kuehn and colleaguesCervus-Megaloceros clade and attributed the palmate antlers in Megaloceros and Dama to homoplasy. Instead, our results support the hypothesis of a Megaloceros-Dama clade suggested by previous studies based on morphological features and phylogenetic analyses from cytb239We obtained nearly complete mtDNA sequences from two ancient cervid bones from the Swabian Alb dated to 12,175 ± 50 uncal ya  and 12,370 ± 30 uncal ya , respectively. Both sequences are distinct from 44 mtDNAs of extant cervids. The phylogenetic analyses suggest that the reconstructed mtDNAs are maternally closely related to fallow deer  mitochondrial DNABone samples were exposed to UV-light overnight to remove surface contamination. A sample of 50mg was removed from the inside of the longbone of each bone using a dentistry drill. DNA extraction was carried out using a guanidinium-silica based methodCapreolus capreolus (NC_020684.1). To achieve a higher resolution in the topology, in a second round sequence reads were mapped to a full mitochondrial genome of the fallow deer Dama dama (JN632629.1), and in a third round to the consensus sequence of our ancient putative Megaloceros giganteus sample HF/65/100, which was generated by mapping to the Dama dama mitochondrial reference sequence as described.Library pools were sequenced on the Illumina Hiseq 2500 platform using two index reads (2*100 + 7 + 7 cycles) following the manufacturer’s protocol. De-indexing was performed by sorting all sequences corresponding to their p7 and p5 combinations using the CASAVA software version 1.8. Forward and reverse reads were merged into single sequences if they overlapped by at least 11 bpC to T and G to A substitution patterns were obtained from the sequences using a custom software developed as an extension package to handle the output format of the mapping iterative assemblerMegaloceros samples using ClustalW . Alignments and phylogeny constructions were conducted in MEGA 6.0.6. The mitochondrial D-Loop region was excluded using the BioEdit sequence alignment editor ) and phylogenies were constructed from a total of 14,147 positions. Both maximum-likelihood and maximum-parsimony topologies were generated for all positions for which coverage was at least three-fold in each of the ancient sequences. Alignment columns with gaps or missing data were eliminated. Bootstrap support values were obtained over 1000 replicate data sets, using alpine musk deer as an outgroup . The phylogenetic trees were edited in FigTree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree).A multiple sequence alignment was generated from 44 full mitochondrial genome Genbankcytb) coordinates within our reconstructed mitochondrial sequences, these were aligned to previously published cytb sequences of Megaloceros giganteus , fallow deer (AJ000022.1), and red deer (AB924664.1) using MEGA 6.0.6 and sequences outside of the aligned regions were discarded. Nucleotide sequence differences were then calculated by pairwise alignment between each of our ancient cytb sequences and each of the previously published cytb sequences using BLAST.To identify cytochrome b (13C) and nitrogen (15N) were measured. The results were combined with stable isotope data obtained from ancient reindeer (Rangifer tarandus) and red deer (Cervus elaphus) remains of the Swabian, French, and Swiss Jura dating 13,000 to 12,000 uncal year ago, which corresponds roughly to the GI-1e interstadial1819Megaloceros giganteus) dating to the pre-LGM from southwestern France (SW France) and Belgium18To study the habitat pattern revealed by stable isotopes, collagen was extracted from both bone fragments and carbon  was conducted at the Department of Geosciences of Tübingen University using a Thermo Quest Delta+XL mass spectrometer coupled to a NC2500 CHN-elemental analyzer, which provides elemental analysis . The international standards used include marine carbonate (V-PDB) for δ13C and atmospheric nitrogen (AIR) for δ15N. Analytical error, based on within-run replicate measurement of laboratory standards , was ± 0.1‰ for δ13C values and ± 0.2‰ for δ15N values. Reliability of carbon and nitrogen isotopic values was established by measuring the chemical composition, with C/Ncoll atomic ratio within the range of 2.9 to 3.6Isotopic analysis .


Mapping millions of DNA reads from such samples to reference protein databases requires long run-times, and short read lengths can result in spurious hits to unrelated proteins (loss of specificity). We developed ShortBRED  to address these challenges, facilitating fast, accurate functional profiling of metagenomic samples. ShortBRED consists of two components: (i) a method that reduces reference proteins of interest to short, highly representative amino acid sequences (“markers”) and (ii) a search step that maps reads to these markers to quantify the relative abundance of their associated proteins. After evaluating ShortBRED on synthetic data, we applied it to profile antibiotic resistance protein families in the gut microbiomes of individuals from the United States, China, Malawi, and Venezuela. Our results support antibiotic resistance as a core function in the human gut microbiome, with tetracycline-resistant ribosomal protection proteins and Class A beta-lactamases being the most widely distributed resistance mechanisms worldwide. ShortBRED markers are applicable to other homology-based search tasks, which we demonstrate here by identifying phylogenetic signatures of antibiotic resistance across more than 3,000 microbial isolate genomes. ShortBRED can be applied to profile a wide variety of protein families of interest; the software, source code, and documentation are available for download at  High throughput DNA sequencing of the human microbiome presents a tremendous resource for researchers interested in studying microbial community functions such as antibiotic resistance. However, assigning DNA reads to protein families remains a challenging problem, as reads derived from a given protein-coding gene may map spuriously to homologous regions from unrelated proteins, which results in false positives. We addressed this problem with our method ShortBRED, which first identifies short peptide sequences (“markers”) that are highly representative for specific protein families, and then searches for these markers in metagenomic sequencing data to accurately detect and quantify protein families. In this work, we applied ShortBRED to profile antibiotic resistance in the healthy human microbiome of individuals worldwide and across bacterial genomes. ShortBRED can be similarly applied to profile many other protein families of interest. Quantifying proteins of interest from metagenomic sequencing data in a fast and accurate manner is a central challenge in microbial community analysis. Whole metagenome shotgun (WMS) sequencing provides millions of short nucleotide sequences (often 100–250 bases long) from the DNA of organisms in a sample; we refer to these short DNA sequences as “reads.” A common approach to profiling protein families from these data involves (i) mapping reads to a database of reference protein sequences followed by (ii) interpreting the mapping results to estimate protein family relative abundance. This process is complicated by regions of local similarity in otherwise unrelated protein families: reads drawn from such regions will map non-specifically, which can result in false positive identifications (reduced specificity). Reducing the time spent on mapping reads is also an important task, as typical metagenomic sequencing depths and reference database sizes continue to grow rapidly.de novo and then annotating them via comparison with reference databases , but it can be tuned to be more conservative.S1 FigValues reflect area under the ROC curve (AUC) as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing ARDB sequences.(PDF)Click here for additional data file.S2 FigValues reflect area under the ROC curve (AUC) as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing VFDB sequences.(PDF)Click here for additional data file.S3 FigValues reflect specificity as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing ARDB sequences.(PDF)Click here for additional data file.S4 FigValues reflect specificity as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing VFDB sequences.(PDF)Click here for additional data file.S5 FigValues reflect sensitivity as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing ARDB sequences.(PDF)Click here for additional data file.S6 FigValues reflect sensitivity as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing VF sequences.(PDF)Click here for additional data file.S7 FigValues reflect Spearman correlation between estimated abundances and true abundances as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing ARDB sequences.(PDF)Click here for additional data file.S8 FigValues reflect Spearman correlation between estimated abundances and true abundances as minimum marker length and initial clustering ID are varied. This analysis was based on the 5%-spiked synthetic metagenomes containing VFDB sequences.(PDF)Click here for additional data file.S9 FigA) and (B) report the sensitivity and specificity of the two methods for mapping reads to their correct families on six synthetic 454 metagenomes, spiked with 5%, 10%, and 25% of their material from the ARDB (panel A) and VFDB (panel B). (C) and (D) display scatterplots of protein family “predicted by mapping”, the abundance values calculated by ShortBRED and the centroids, vs. “expected from gold standard”, the abundance values of the protein families in the 10% synthetic metagenome. This figure is an analog of ((PDF)Click here for additional data file.S10 FigPhylogenetic tree of bacterial genomes from IMG  overlaid(PNG)Click here for additional data file.S1 TableThis table displays measures of the performance of ShortBRED and centroids to profile synthetic metagenomes. The results for ShortBRED using its default settings on the six synthetic metagenomes are in bold. Illumina_05,Illumina_10, and Illumina_25 represent synthetic Illumina metagenomes with reads from input dataset sequences comprising 5%, 10%, and 25% of the metagenome. Illumina-mutated-3pct and Illumina-mutated-5pct metagenomes had 3% and 5% of their amino acid content mutated before being incorporated into the metagenome. Centroids were also obtained by clustering at 85% identity. Any result mapping to centroid with length ≥30 and ID ≥95% was considered a “match”.(XLSX)Click here for additional data file.S2 TableThese are yeast centroids which had false positive matches to reads from a synthetic bacterial metagenome at high identity (≥95%) and long length ≥30 amino acids. Source: Kyoto Encyclopedia of Genes and Genomes (KEGG) (XLSX)Click here for additional data file.S3 TableEach row of this table represents a family of antibiotic resistance (AR) proteins and each column corresponds to a sample in our dataset. The abundance of the AR family in the sample is given in RPKM.(XLSX)Click here for additional data file.S4 TableEach row represents a family of antibiotic resistance (AR) proteins. Column B lists the corresponding AR class. (The classes are groups of AR families.) The columns provide summary statistics of the ShortBRED RPKM counts for each family. Sample statistics for columns C through I are based on the entire sample of all three datasets. Summary statistics are also provided for the individual three datasets. Please note that the Yatsunenko et al dataset results were based on running ShortBRED using the centroids, as opposed to the markers from ShortBRED-Identify. This is because this particular dataset has shallower coverage than the Illumina datasets (HMP and T2D).(XLSX)Click here for additional data file.S5 TableEach row in this table represents summary statistics for the abundance of a particular AR class in a genus of bacteria. We used 3,305 bacterial genomes obtained from the Integrated Microbial Genomes database. In "annotated genome" mode, ShortBRED takes a fasta file of open reading frames from the genome, calls USEARCH to build a database, and then checks the markers against the database for hits.(XLSX)Click here for additional data file.S6 TableThis table presents the differences between counting antibiotic resistance (AR) abundance in microbiome samples using ShortBRED, and inferring AR abundance in the same samples based on the abundance of bacteria in the microbiome multiplied by the median count of each AR class found in available bacterial genomes. Each row is a class, each column is sample from the HMP and the value in each cell equals [ShortBRED Count in RPKM—SumClick here for additional data file.


Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these ’Proteotypic peptides for Quantification by SRM’ (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile  Epitope tags are widely used in cell biology and biochemical analysisSRM experiments are typically performed on triple quadrupole instruments where the first (Q1) and the last (Q3) quadrupoles are used in a static manner to filter specific predefined precursor and fragment ions respectively, while in the second quadrupole (Q2) collision-induced dissociation of the precursor ion takes placeIn targeted proteomics, samples are first processed by the use of a robust protocol, followed by complete proteolytic digestion with trypsin or other proteases. Depending on sample complexity and target concentration, separation schemes can vary, with complex orthogonal fractionation strategies for sensitive detection of low abundant proteins1215Despite the clear use for targeted proteomics, especially as a verification step in biomarker studies to reduce the number of candidates coming from discovery screensBy using cleavable reporter peptides, equimolar amounts of a protein reference peptide and a universal peptide can be introduced in the sample, facilitating precise analysis21Co-immunoprecipitation (Co-IP) is a classical approach to show an interaction between two proteins. Transient expression of both tagged bait and prey proteins is typically used for these experiments, although the application of specific primary antibodies for both proteins is becoming increasingly popular and often required to ascertain an interaction on the endogenous level. In Co-IP, protein complexes are typically eluted from the purification beads and analyzed directly by Western blot which results in a semi-quantitative read-out.Pyrococcus furiosus. After several rounds of selection, two peptides were retained and evaluated for direct sensitive detection and quantification in complex lysates. These PQS peptides were also introduced as peptide tags in different target proteins, providing a universal quantitative read-out system for the tagged proteins. In addition, we performed binary protein-protein interaction (PPI) assays for different pairs of tagged proteins expressed from vectors in human cells, and for a binary interaction between two endogenous proteins after introduction of the PQS peptide sequences in the genome sequence of the target proteins.In this report, we describe the selection of Proteotypic peptides for Quantification by SRM (PQS peptides) from the unique proteome of the hyperthermophile To enable sensitive SRM-based quantification using a protein tagging strategy, we first needed to define optimal peptides that could be developed into a universal SRM assay. We rationalized that such peptides should adhere to the following criteria. 1) The peptides need to show good LC-MS(/MS) properties, including ionization and fragmentation, to maximize assay sensitivity. Ideally, such peptides can be detected in complex samples such as digested whole cell lysates without the need for fractionation prior to detection. 2) The SRM peptides are unique or proteotypic for a wide range of commonly used model organisms in life sciences research. 3) The peptides allow for quantification in complex samples. 4) The peptides should be generated very efficiently by trypsin, minimizing potential quantification deviations when performing Stable Isotope Dilution (SID) experiments. 5) Finally, heavy labeled counterparts should be straightforward to produce and easy to handle (e.g. not prone to precipitation).P. furiosus, a well-characterized hyperthermophile archaeon which thrives at an optimum growth temperature of 100 °CP. furiosus are encoded in a genome that is drastically different from most other species which leads to an overlap of merely seven tryptic peptides upon comparison with human protein tryptic peptideset al. showed that peptides from both proteomes show a very similar behavior in LC-MS systemsP. furiosus extract) was digested, fractionated and analyzed by LC-MS/MS. This standard is predicted to contain the majority of the approximately 2,000 known proteins that P. furiosus encodes for and led here to the identification of 1,581 unique peptide sequences, originating from 582 unique proteins, that were retained for further selection and validation. An overview of the complete peptide triage workflow is provided in To maximize the chance of retrieving unique peptides with all of these properties, we reasoned that a proteomic analysis of P. furiosus discovery experiments were further processed to create a shortlist of peptides that show optimal LC-MS characteristics. To this end, average matched intensities were calculated for each peptide that uniquely matched a single protein. Differential scores were subsequently calculated for all peptides based on the Huber median and mean absolute deviation (MAD) values as defined in experimental procedures. These differential scores were then averaged for each peptide across the three discovery experiments. A second calculation was done for the fragmentation efficiency, represented by the fragment ion coverage. For each identified peptide, the number of annotated ions for every spectrum were summed and averaged over the three discovery experiments.To narrow down the number of suitable peptides, the data obtained from the Eukaryota, and for all strains of E. coli. None of these peptide sequences were found in these protein databases, which include the vast majority of model organisms.Following calculations, the peptides were filtered in an elimination process where peptides with at least one of the following criteria were discarded: 1) peptides that contain methionines or cysteines (oxidation effects), 2) peptides that show missed cleavages, 3) peptides that start with glutamine (non-quantitative pyro-glutamic acid formation), 4) peptides that show consecutive prolines (elution peak broadening) and 5) peptides that are shorter than 9 amino acids. From the remaining list, a first set of peptides was selected by considering only peptides having a differential score >6 in all three replicates which resulted in a set of 7 peptides  and GLGASPGIGAGR (PQS2), we spiked a dilution series of these peptides in a tryptic digest of HCT116 cells. Here, peptide levels varied over a dilution range from 6.4 fmol to 0.1 fmol detectable peptide in 0.5 μg of HCT116 proteome digest  algorithm (LTLIFR-PQS1-FAYLYD and VAEAYR-PQS2-FLETEN)In addition to Mal-MyD88, we next tested the PQS peptides as readout for the LCP2-GRAP2 interaction involved in T-cell signaling3132TP53+/PQS1-FLAG). The tryptic context was also introduced to ensure optimal release upon tryptic digestion. We also generated a cell line containing the 3xHA-PQS2 tag fused to the C-terminus of MDM2 (HCT116 MDM2+/PQS2-HA) (We further explored the use of the PQS peptides by their introduction at the C-terminus of endogenous proteins. We used recombinant adeno-associated virus (rAAV) in combination with Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 to generate a cell line that expresses an endogenous p53 protein containing a C-terminal combinatorial 3xFLAG-PQS1 tag (HCT116 PQS2-HA) . Cell liPQS2-HA) . Next, wPQS2-HA) .TP53+/PQS1-FLAGMDM2+/PQS2-HA cells  normalization approaches incorporated in the heavy labeled counterparts19In this work we present small peptides flanked by optimal tryptic contexts that are characterized by optimal MS properties and thus allow SRM-based detection and quantification of proteins upon incorporation of the peptide sequence as a tag to the protein. The selected peptides are universally applicable being proteotypic for all Clearly, a possible disadvantage of our approach is the need to fuse the PQS peptide(s) to the protein(s) of interest. This can cause artifacts as the PQS tag may perturb protein folding, or have unexpected effects on transcription or translation. We focused all genome engineering efforts on the C-terminus of the native protein, as we rationalized the risk of interference with regulatory elements is lower at this end of the protein36et al. clearly demonstrates a need for lysis optimization or even complementary lysis conditions to obtain a comprehensive view on a protein complex44Classical Co-IP approaches typically rely on semi-quantitative Western analysis as a read-outWhile we rely on the combinatorial use of well-defined affinity sequences to enrich for the protein(s) of interest which causes our tag sequences to grow considerable in length, it is theoretically possible to only depend on the PQS peptide for both enrichment and detection by generating  antibodies against the sequence4614P. furiosus proteins using a classical shotgun approach. The exact experimental details can be found in the For initial selection of the PQS peptides, we first analyzed the Agilent complex proteomics standard as a source for P. furiosus proteome was retrieved from ms_limshuber’ function of the R ‘MASS’ package. This function applies iterative winsorization to reduce the effect of outliers on the distribution of valuesk parameter that determines the outlier range was set to 1.2. The Huber median and MAD values were then used to compute a differential score for each peptide using the formula Ipep is the average matched peptide intensity for peptide i of protein j, mj is the huber median of the average matched peptide intensities for protein j, and sj is the huber MAD of the average matched peptide intensities for protein j. An additional calculation was done for the fragmentation efficiency, indicated by the fragment ion coverage. For each identified peptide, the number of annotated ions for every spectrum were summed and averaged over the three discovery experiments.Identification data from the P. furiosus peptides was then verified by mapping the peptide sequences back, irrespective of any cleavage rules, to the corresponding UniProt protein databases of all Eukaryota , and all Escherichia coli strains  with DBToolkitUniqueness of the final selection of in silico predicted PQS peptides were synthesized in-house using standard solid-phase Fmoc chemistry on a Syrol peptide synthesizer (Biotage) and purified by RP-HPLC. For the initial evaluation of in silico predicted peptides, 100 fmol of each peptide was analyzed on the TSQ Vantage LC-SRM system operated as described in the The 15 best P. furiosus discovery experiments were uploaded using Panoramahttps://panoramaweb.org/labkey/PQSpeptides.All Skyline files used for obtaining data presented in this work, both targeted and The two most intense PQS peptides, designated PQS1 and PQS2, were spiked in a tryptic digest of HCT116 cells (0.5 μg injected) in separate dilution series  for each peptide. To each sample, the appropriate heavy labeled counterpart was added to a final injection amount of 5 fmol as reference for quantification, instrument stability and peak identification facilitation. The final LOD values correspond to the lowest detectable amount of light peptide (i.e. S/N > 3) in the sample series for each peptide. Standard deviations were calculated based on the extracted mean standard error values from Skyline.in silico evaluated for their tryptic propensity when inserted into the human proteome. We therefore first determined all tryptic peptides from the human proteome with the aid of DBToolkitThe two selected PQS peptides were http://horfdb.dfci.harvard.edu/)cDNA coding for proteins of interest was obtained from the human ORFeome collection  was inserted for selection purposes. Homology regions were picked up by genomic PCR on HCT116 DNA. Separate inserts were simultaneously ligated using In-Fusion cloning kits (Clontech). All primer sequences and DNA constructed by DNA synthesis can be found in +/PQS2-HA cells, the PQS2 peptide was inserted and the 3xFLAG sequence was exchanged for a 3xHA-tag. 24 h after transfection, the medium was refreshed and after 48 h of additional incubation viral stocks were prepared using the AAV purification ViraKit (Virapur) according to the protocol provided by the manufacturer. Engineering of p53 was performed by a combination of rAAV and CRISPR/Cas9 whereby rAAV infection was done one day after Cas9 transfection using FugeneHD (Promega). CRISPR guide RNAs (gRNAs) (in silico activity prediction using the online Zhang Lab CRISPR design tool (http://crispr.mit.edu/) and constructed in Streptococcus pyogenes Cas9 in the pSpCas9(BB)-2A-Puro vector  according to the protocol published by Ran et al.® mutation detection kit (Integrated DNA Technologies) -based genome editing was performed on HCT116 cells cultured in McCoy’s 5A medium in presence of 10% fetal bovine serum (Gibco). Virus production was performed in AAV-293 cells (Agilent) by Polyethylenimine (PEI) co-transfection of a modified pAAV-MCS plasmid (Agilent), hereafter referred to as targeting vector and the pDG vector (Plasmidfactory). For generation of HCT116  (gRNAs)  were useologies) . Only th2 petri dish engineered clonal HCT116 populations were lysed in 100 μl 20 mM ammonium acetate by freeze-thawing after detaching the cells with cell dissociation buffer (Gibco). Digestion occurred overnight by addition of trypsin equal to 1% of the total protein concentration of the sample as determined by Bradford assay. Prior to transferring the sample to a MS vial, Trifluoroacetic Acid (TFA) was added to a final concentration of 0.1% and the sample was centrifuged at 16,000 g for 10 min to remove any debris.For direct detection of the SRM peptides in cell lysates without prior enrichment, subconfluent 145 cmProtocols regarding endogenous affinity purification experiments are described in How to cite this article: Vandemoortele, G. et al. An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics. Sci. Rep.6, 27220; doi: 10.1038/srep27220 (2016).


Anopheles midgut is a critical step for malaria transmission; the parasite numbers drop drastically and practically reach a minimum during the parasite's whole life cycle. At this stage, the parasite as well as the vector undergoes immense oxidative stress. Thereafter, the vector undergoes oxidative stress at different time points as the parasite invades its tissues during the parasite development. The present study was undertaken to reconstruct the network of differentially expressed genes involved in oxidative stress in Anopheles stephensi during Plasmodium development and maturation in the midgut. Using high throughput next generation sequencing methods, we generated the transcriptome of the An. stephensi midgut during Plasmodium vinckei petteri oocyst invasion of the midgut epithelium. Further, we utilized large datasets available on public domain on Anopheles during Plasmodium ookinete invasion and Drosophila datasets and arrived upon clusters of genes that may play a role in oxidative stress. Finally, we used support vector machines for the functional prediction of the un-annotated genes of An. stephensi. Integrating the results from all the different data analyses, we identified a total of 516 genes that were involved in oxidative stress in An. stephensi during Plasmodium development. The significantly regulated genes were further extracted from this gene cluster and used to infer an oxidative stress network of An. stephensi. Using system biology approaches, we have been able to ascertain the role of several putative genes in An. stephensi with respect to oxidative stress. Further experimental validations of these genes are underway.Ookinete invasion of  Maintenance of redox homeostasis is critical for proper functioning of cellular processes and disruption of this prooxidant-antioxidant balance in a cell results in oxidative stress. Oxidative stress may be caused by the normal functioning of the cell  or as an immune response to pathogens Several studies have established that generation of ROS can be endogenous due to the leakage of activated oxygen from mitochondria during oxidative phosphorylation, peroxisomes, and activated inflammatory cells Plasmodium, the causative agent of malaria, leads a complex life cycle, alternating between two hosts, vertebrate and invertebrate, with diverse environmental and physiological regimens. Further, within these two hosts, the parasite also exists as intra- and extracellular forms thereby being exposed to extreme surroundings. Several studies have revealed that Plasmodium undergoes immense oxidative stress during their erythrocyte cycle, considering that they live in a pro-oxidant environment in the red blood cells that contains oxygen and iron Plasmodium overcomes this obstacle by using its defense mechanisms to protect against oxidative damage Plasmodium, its vector, Anopheles also undergoes tremendous oxidative stress due to the high proliferative rate of the parasite and invasion of several of its organs by the parasite. The zygote transforms into motile ookinetes within 24 hours of ingestion of an infected blood meal and invades the mosquito midgut epithelium. Once inside, the ookinete develops into the oocyst between the basal lamina and the midgut epithelium. Upon maturity, the oocyst produces thousands of sporozoites that are released from the midgut into the hemocoel and finally reach the salivary glands. Here, they invade the salivary glands and mature to form the salivary gland derived sporozoites that are ready to infect the host during the next mosquito bite. During each of the invasion process and subsequent increase in parasite numbers, the mosquito undergoes extreme oxidative stress and several of the signaling pathways and innate immunity pathways are activated to protect the mosquito Just as in the case of In the post-omics era, it is becoming clear that integration of genome-scale technologies provide better tools for understanding biological function An. stephensi during successive stages of the development and maturation of Plasmodium vinckei petteri. Using next generation sequencing platforms, we obtained the transcriptome of the midgut of An. stephensi during P. vinckei petteri oocyst stage. We identified those transcripts that were differentially expressed and evaluated the dynamics of the An. stephensi redox system during oocyst development. Using Support Vector Machines (SVM) we classified unannotated genes of the transcriptome dataset into oxidative stress pathways. Additionally, we identified microarray datasets from public databases that studied An. gambiae during Plasmodium development, and arrived upon the set of An. gambiae genes involved specifically in oxidative stress during Plasmodium midgut invasion. Using all the above information, we inferred an almost complete network of the oxidative stress of An. stephensi during Plasmodium invasion.We have undertaken the present study to elaborate perturbations in the redox system of Animal experiments were performed in accordance with National animal ethics guidelines of the Government of India after approval by Institutional Animals Ethics Committees of International Centre for Genetic Engineering & Biotechnology, New Delhi (Permit number: ICGEB/AH/2011/01/IR-8).Anopheles stephensi were reared at 28–30°C and humidity maintained at 70–75%. Mosquitoes were maintained by feeding with raisin soaked in 2% sterile glucose solution and water. 4–5 days old female mosquitoes were fed on P. vinckei petteri 279 BY (gametocytemia  = ∼0.05%) infected mice. Midguts from the infected mosquitoes were dissected on 5th day post infection and checked for the presence of oocysts for the confirmation of infection.Anopheles stephensi midgut samples were collected from three different stages, namely, sugar fed (PVpSF), blood fed (PVpBF5D) and blood fed 5 days post P. vinckei petteri infection (PVpiBF5d). In case of infection and blood feed, around 150–200 An. stephensi mosquitoes were fed on P. vinckei petteri 279 BY (gametocytemia  = ∼0.05%) infected mice. Fully fed mosquitoes were separated from unfed and partially fed mosquitoes and reared in cages until day 5 post feeding. Midguts were dissected and stored in trizol in −80°C. The feeding experiments were performed for a minimum of three times for both blood and infected blood feeding and a total of 200–300 midguts were collected over a period of time for each sample. Total RNA was isolated separately from each lot and finally pooled during RNA seq library preparation. The libraries were made following manufacturer's instruction. RNA sequencing was performed using Illumina platform. The Total RNA quality was verified using RNA 6000 Nano Kit  on 2100 Bioanalyzer , with a minimum RNA Integrity number (RIN) value of 7.An. stephensi were generated, one each from total RNA extracted from sugar fed mosquitoes (PVpSF), mosquitoes 5 days post blood feed, (PVpBF5D) and mosquitoes 5 days post infected blood feed (PVpiBF5D). The RNA-Seq library construction and sequencing was performed by commercial service providers . Total RNA was used to enrich mRNA using Oligotex mRNA midi prep kit . 2 µg of total RNA using oligo(dT) magnetic beads and fragmented into 200–500 bp using divalent cations at 94°C for 5 min. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase  and random primers. Fragments were A-tailed and end repaired after second strand cDNA synthesis. The cDNA libraries were constructed for the samples using the TruSeq RNA Sample Preparation Kit  with alternate fragmentation method for generating 200–500 bp fragments, according to manufacturer's instructions. The Paired-End RNA-Seq libraries were diluted and sequenced using TruSeq SBS Kit V3 on HiSeq2000  for generating 2×100 bp sequencing reads.Three Paired-End RNA-seq libraries of An. stephensi genome was used as the reference genome which was downloaded from VectorBase A simplified workflow of transcriptome assembly and analysis performed in this study is shown in An. stephensi consisted of three steps . In order to build the predictive model we used SVM, a powerful supervised machine learning algorithm for prediction, that classifies an object into one or more classes based on the set of input feature vectors. In our study, PSSM generated by PSI-BLAST was used as the input feature vector for SVM. PSSM based classifiers has been reported as the most suited among the SVM classifiers Evolutionary information can be extracted using Position Specific Substitution Matrix (PSSM), hence, for the functional characterization of the m number of SVM models for m pathways. The training data for each of the ith model consisted of protein sequences of ith pathway of interest as positive set and protein sequences of the m-i pathways as negative set  curve and Area under Curve (AUC). ROC is a plot of false positive rate (1-specificity) on x-axis and true positive rate (sensitivity) on y-axis. The plot depicts the trade-off between specificity and sensitivity. The mathematical representations of the expressions can be represented as:Sensitivity =  (TP/TP+FN)×100Specificity =  (TN/TN+FN)×100Accuracy =  (TP+TN/TP+FN+TN+FP)×100MCC = (TP×TN) – (FP×FN)/sq.root [(TP+FN)×(TN+FP)×(TP+FP)×(TN+FN)]Where TP means True positive, TN means True Negative, FN means False Negative and FP means False Positive.The differential expression analyses of the libraries were performed with Bioconductor package edgeR. edgeR uses TMM  approach for the normalization of read counts. We identified differentially expressed genes by comparing all the three libraries with each other i.e. PVpSF vs PVpBF5D, PVpSF vs PVpiBF5D and PVpBF5D vs PVpiBF5D. edgeR analysis were performed by taking disperson value of 0.1 and p value < = 0.05.An. stephensi corresponding to differentially expressed genes were downloaded from VectorBase and were analyzed using KOBAS web server. Hypergeometric test was selected as statistical method and FDR correction was performed using Benjamin and Hochberg method (1995). Pathways with p< = 0.05 were considered as significant pathways.Differentially expressed genes from each comparison were taken separately and subjected to pathway enrichment analysis. For the identification of significantly enriched pathways the protein sequences of Plasmodium”, “Drosophila”, “Oxidative stress” and “Infection” as key elements and wherever required, the related terms and alternative terms were also used. Online library and databases namely, PubMed, ArrayExpress and Gene Expression Omnibus (GEO) were searched for the data using the key words and the relevant data were downloaded  or an invasion-deficient, CTRP (Circumsporozoite- and TRAP-related protein) knockout (ko) strain (2) E-MEXP-1859: Transcription profiling of Drosophila transformed with two Plasmodium cell surface antigens, circumsporozoite protein (CSP) and Thrombospondin-related adhesive protein (TRAP) and (3) GSE11012: An analysis of the impact of infection by Buchnera aphidicola APS on gene expression of Drosophila S2 cells. All the three datasets were background corrected and normalized using LOESS and Aquantile normalization. The normalized data were further analyzed using Bioconductor packages LIMMA For the purpose of maximizing the information on redox dynamics of datasets  from pubAn. stephensi gene co-expression network was generated using ExpressionCorrelation plugin of Cytoscape (http://apps.cytoscape.org/apps/expressioncorrelation) An. stephensi fed on human blood) was used to generate the gene co-expression network. Common genes among all the libraries, with their expression values were used as an input for generation of the gene co-expression network using ExpressionCorrelation plugin of Cytoscape. The identified and predicted genes related to oxidative stress were mapped onto the An. stephensi gene interaction network and a sub-network consisting of only these mapped genes was drawn out from the network.An. stephensi during P. vinckei petteri development by integrating datasets originating from Illumina RNA-seq technology and gene expression microarrays.Integration of different types of genomic data provided new insights into the interactions that exist between genes that are otherwise not distinguishable while studying single data sets P. vinckei petteri oocyst development in the midgut, we obtained the transcriptome of the midgut during the time point of mature oocyst development in the midgut. Physiologically, at this stage, the oocysts are mature and were ready to invade the midgut with oocyst derived sporozoites which is ideal for our study purpose of studying the oxidative stress in Anopheles during Plasmodium invasion. Analysis of such a cellular state is likely to reveal metabolic homeostasis facilitating parasite maturation and most importantly minimal metabolic penalty on vector. We dissected the midguts at day 5 post infection and processed those midguts that had maximum oocysts 8 reads , which includes 4.21×107 reads from PVpSF library, 4.24×107 reads from PVpBF5D library and 4.37×107 reads of PVpiBF5D library were further processed  values of these pathways supported the accuracy of the pathway models. The AUC value for the Glycolysis ∼0.9801, Citric acid cycle with AUC value of 0.8571, PPP with highest value 1, oxidative phosphorylation ∼0.9704, TGF ∼0.9978, WNT ∼0.944, for MAPK it is 0.9821 and for JAK is predicted to be 0.9762.Comparative genomics can be used for the functional annotation of genomes that are not annotated completely. We annotated assembled transcriptome of the M scores . The robM scores . These gAn. stephensi during P. vinckei petteri development and to understand the interactions of these participating genes during Plasmodium invasion of the midgut. For this purpose, it was important first to generate a broad interaction network of An. stephensi before segregating the redox related sub-set. For this purpose, we utilized the annotated genes of our transcriptome dataset to arrive upon a reference network on to which the oxidative stress genes identified and predicted in our study were mapped to infer the oxidative stress sub-network network of An. stephensi upon P. vinckei petteri infection with 516 nodes and 2904 edges  involving bacterial infection was selected as a control Previous studies have highlighted the impact of bacterial infection on the redox status in mosquitoes Anopheles during Plasmodium development, detailed computational analysis was performed on the selected microarray datasets  analysis of data E-MEXP-378 and E-MEXP-1859 revealed six pathways each having significant P-values and OXPHOS pathway was found to be common among them. GSEA results showed cytoskeleton organization and biogenesis, Wnt signaling pathway, JAK-STAT, p53 signaling pathway, pentose phosphate pathway and OXPHOS as significant pathways for E-MEXP-378 and likewise oxidative phosphorylation, response to oxidative stress, Toll pathway, response to ROS, melanization defense response and hydrogen peroxide catabolic response as significant pathways for E-MEXP-1859 dataset. The 13 common differentially expressed significant genes in dataset E-MEXP-1859 and GSE11012 were removed from the further analysis so as to reduce the false positive results. Detailed analysis of these datasets revealed 40 genes of  gambiae  and 145 Anopheles, a three prong approach was used. Our aim was to integrate different types of data sets and extract information for most of the genes that are playing role in oxidative stress in Anopheles including unannotated genes, information of genes in other insects, extract information available in public domain and our own experimental data. Previous studies performed by integrating such information have yielded much information in predicting gene function For the purpose of inferring the redox system of Anopheles Click here for additional data file.Table S1Table showing the data used in the study.(DOCX)Click here for additional data file.Table S2Table representing the GSEA predicted Anopheles genes (microarray data) which may play role in oxidative stress.(DOCX)Click here for additional data file.Table S3The excel sheet shows the SVM summary and the predicted SVM scores of unannotated transcripts.(XLS)Click here for additional data file.


Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels  and improved intestinal barrier function at multiple levels . Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota  High fat high cholesterol containing Western diet-dependent changes in the genetic makeup of human gut microbiome are thought to underlie this epidemic of chronic diseases such as obesity, insulin resistance and atherosclerosis. Although diet-induced changes in gut bacteria Curcuma longa, with demonstrated anti-inflammatory as well as anti-carcinogenic properties. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it preferentially targets intestinal epithelial cells We examined whether “selective decontamination” of the gut by the use of non-absorbable antibiotics that are active in the intestinal lumen could attenuate the total release of bacterial LPS into circulation and thereby reduce chronic inflammation linked diseases namely Western-diet induced atherosclerosis and glucose intolerance. The combination of antibiotics used for standard selective decontamination (neomycin and polymyxin B) eliminates aerobic Gram-negative bacteria from the mucosal surfaces of the digestive tract, while the majority of the anaerobic flora persists and support colonization resistance Here we demonstrate that Western diet-induced increase in intestinal permeability leading to translocation of bacterial endotoxin LPS is reduced by selective decontamination of the gut via administration of non-absorbable antibiotics as well as by oral supplementation with Curcumin resulting in attenuation of inflammation-linked diseases namely atherosclerosis and glucose intolerance. This study establishes that targeted improvement of intestinal barrier function is a viable therapeutic strategy to attenuate Western diet-induced effects on intestinal mucosa, the resulting endotoxemia and development of associated diseases.All chemicals were obtained from Sigma-Aldrich  unless indicated otherwise.All animal procedures were approved by the Institutional Animal Care and Use Committees of Virginia Commonwealth University. LDL receptor knockout mice (LDLR−/−) originally obtained from Jackson Laboratory and maintained in the laboratory were used for all studies. Where indicated, at 10 weeks of age mice (littermates) were fed a high fat, high cholesterol Western type diet  which contained 21% fat, 0.15% cholesterol, and 19.5% casein by weight with no sodium cholate for 16 weeks. In one set of studies, non-absorbable antibiotics (100 mg/L Neomycin and 10 mg/L Polymyxin B) were administered in drinking water; untreated or control mice received normal drinking water. In second set of studies, Curcumin (100 mg/kg in 0.5% carboxy methyl cellulose made fresh and administered within 15 minutes) was given as daily oral gavage; untreated or control mice were gavaged with vehicle alone. Administration of antibiotics or curcumin did not affect the food intake or WD-induced weight gain .Mice were fasted overnight and at the time of necropsy, blood was collected by cardiac puncture and plasma was frozen at −80°C until analyzed. Plasma LPS levels were determined by Endpoint Chromogenic LAL (Lamilus Amebocyte Lysate) assay  according to the manufacturer's instructions, with the following modifications: samples were diluted 5–10-fold to avoid interference with background color and preheated to 70°C for 10 minutes prior to analyses.Intestinal alkaline phosphatase activity was measured as described Mice were orally gavaged with FITC-dextran 4 kDa (600 µg/kg body weight) dissolved in deionized water and plasma was collected after 4 h. Using three aliquots of plasma in duplicate, FITC fluorescence (excitation 488 nm and emission 518 nm) was measured using Perkin Elmer Victor 3 plate reader and concentration of FITC in plasma determined using a standard curve and expressed as ng/µl of plasma.10-week-old LDLR−/− mice were fed a Western diet for 16 weeks. After an overnight fast, a single bolus of glucose (2 mg/g body weight) was given intraperitoneally. Blood glucose levels were determined by commercially available glucometer using tail vein blood at 0, 15, 30, 60, 120 min.The aorta was dissected from the heart to the iliac bifurcation, cleaned of any surrounding tissue, opened longitudinally, pinned on black wax and fixed for 24 h in 10% buffered formalin. The fixed aortas were imaged on a black background using a Canon Digital Camera fitted with a 60 mm, f/2.8 Macro Lens. Total area and the area occupied by the lesions in the aortic arch and total aorta was determined using AxioVision Image Analysis software as described earlier Thioglycollate elicited peritoneal macrophages were plated in 24-well plates. Medium was replaced after 4 h to remove non-adherent cells and cells were transduced with AdNF-κB-Luc (5 MOI) . After 24 h, cells were treated with 50 µM oleic acid Thioglycollate elicited peritoneal macrophages were plated in 24-well plates. Medium was replaced after 4 h to remove non-adherent cells and cells were treated with LPS (50 pg/ml) ±50 µM oleic acid for 24 h. Conditioned medium was used to determine the levels of secreted IL-6 and MCP-1 levels by ELISA .5 cells/cm2 and grown in DMEM  containing fetal calf serum (10% v/v), penicillin (100 U/ml) and streptomycin (100 mg/ml) for 21 days at 37°C in 5% CO2 in air. Prior to the start of the experiment the trans-epithelial electrical resistance (TEER) of the Caco-2 cultures was measured with an EVOM  to confirm their fully differentiated state. TEER is a measure of tight junction integrity and indicator of differentiation. Cultures displaying a TEER greater than 700 Ω cm2 were considered suitable for the study. Cells were pre-treated with 5 µM curcumin (in DMSO) for 48 h before stimulating with LPS (1 µg/ml) in serum free medium for 24 h.Caco-2 cells, obtained from the American Type Culture Collection , were plated on polycarbonate Transwells at 1×1014C] Mannitol   in HBSS was added to the apical side. Medium  was collected from the basolateral side at 0.5, 1, 2 and 4 hours to measure paracellular permeability.After 24 hours, 1 ml -mannitol or FITC dextran 4 KDa from the apical to the basal side. Time dependent increase in the translocation of [3H]-mannitol  with or without pre-treatment with curcumin and permeability of the monolayers determined by monitoring the translocation of [mannitol  and FITCmannitol  was indu3H]-mannitol and FITC in LPS-treated Caco-2 cells as well as attenuation of LPS release in vivo, these data provide strong evidence that curcumin can beneficially affect intestinal barrier function.Intestinal epithelium maintains its selective barrier function through the formation of network of tight junction proteins such as claudins and ZO-1. These proteins interact intracellularly with adaptor proteins that link to the actin cytoskeleton The findings reported in this study not only demonstrate Western diet-induced changes in intestinal barrier function (reduction in IAP and tight junction proteins resulting in increased permeability) leading to increased endotoxemia, macrophage activation and subsequent development of glucose intolerance and atherosclerosis but also establish that improvement of this barrier function can significantly attenuate this pathological sequelae . While sIntestinal barrier is a multi-layer defense system Low grade chronic inflammation is central to the development of obesity and Western diet-induced metabolic diseases and trace, yet pathological, amounts of intestine-derived LPS released into the circulation accompanies many of these conditions Curcumin, a polyphenolic natural compound and a principal ingredient of Asian spice Turmeric is reported to possess anti-inflammatory and anti-oxidant properties. However, the mechanisms underlying its observed effects have not been completely understood. Dietary curcumin is poorly absorbed and undergoes glucuronidation and excretion limiting its release into the circulation. Consequently, significant efforts are currently being directed to the development of novel methods for systemic or targeted delivery of curcumin In conclusion, the data presented here provide direct evidence for the role of Western diet-induced disruption of intestinal barrier function in the development of metabolic diseases such as diabetes and atherosclerosis. Furthermore, these studies also identify curcumin as an agent effective in restoring the intestinal barrier function by modulating multiple components of this barrier including IAP and paracellular permeability. Future studies will examine the effects of curcumin on other components of the intestinal barrier to further advance our current understanding and to develop curcumin as a dietary supplement to attenuate multiple inflammation-linked diseases.


In control steatotic hepatocytes, we found higher generation of ROS, increased lipoperoxidation, an altered redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates, as compared to intact lean hepatocytes. Fatty hepatocytes exposed to tBHP exert more severe damage, lower reduced glutathione to total glutathione ratio, and higher formation of ROS and production of malondialdehyde and are more susceptible to tBHP-induced decrease in mitochondrial membrane potential. Respiratory control ratio of complex I was significantly reduced by tBHP in both lean and steatotic hepatocytes, but reduction in NADH-dependent state 3 respiration was more severe in fatty cells. In summary, our results collectively indicate that steatotic rat hepatocytes occur under conditions of enhanced oxidative stress and are more sensitive to the exogenous source of oxidative injury. This confirms the hypothesis of steatosis being the first hit sensitizing hepatocytes to further damage.Oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of nonalcoholic fatty liver disease and toxic liver injury. The present study was designed to evaluate the effect of exogenous inducer of oxidative stress ( Then fluorescence intensity was measured for 40 min . Results are expressed in percent where control nonsteatotic hepatocytes are 100% of fluorescence intensity difference (at 40′ minus at 0′).The production of ROS was evaluated using 5- and 6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate . After incubation, the cells were washed in William's E medium and loaded by 1 Secondary end product of lipoperoxidation MDA in culture medium was determined by the assessment of thiobarbituric acid reactive substances .Functional capacity of cultured hepatocytes was evaluated by the amount of albumin secreted into the culture medium using Rat Albumin ELISA Quantification Kit .λem 525 nm). At higher potentials, JC-1 forms red-fluorescent “J-aggregates” (λem 590 nm). Hepatocytes were incubated with 10 μM JC-1 (dissolved in William's E medium) in humidified atmosphere containing 95% air and 5% CO2 at 37°C for 30 min, then the cells were washed twice with fresh media. MMP was visualized using fluorescence microscope Olympus IX51  equipped with the digital camera Olympus E600 . Results are expressed as percentage of cells containing mitochondria with high membrane potential.MMP was depicted using hepatocyte uptake of JC-1 , a cationic carbocyanine dye that accumulates in mitochondria according to its membrane potential. At low membrane potential, JC-1 exerts a green fluorescence (μg/mL) hepatocytes  were incubated in 2 mL of K+-medium [2 consumption rate in state 3 to state 4) was calculated. Oxygen uptake at state 3 and state 4 is expressed as pmoles oxygen per second per million cells. For evaluation of oxygen uptake OROBOROS software  was used.Oxygen consumption was measured using a High Resolution Oxygraph 2K . Digitonin-permeabilized . When significance was detected, Tukey-Kramer's post hoc test was used for comparisons between the different groups. Steatotic hepatocytes, compared to nonfatty cells, exert significantly lower activity of cellular dehydrogenases . RCR of complex I and oxygen consumption at state 3 were reduced by tBHP in both lean and steatotic cells; NADH-dependent respiration at state 3 was significantly lower in fatty hepatocytes than in lean controls .μM TFP for a period of 60 min (30′ + 30′) did not lead to significant changes in LDH activity in culture medium  only when cells were firstly preincubated with TFP prior to tBHP exposure. Therefore we only present results with preincubation followed by coincubation.We compared two protocols of cell incubation with 5 TFP was able to partially prevent tBHP-induced elevation in LDH activity in culture medium of steatotic hepatocytes , increasIn this experiment, we studied oxidative stress-induced changes in hepatocytes isolated from nonfatty and steatotic rat liver. For evaluation of peroxidative damage, hepatocytes were exposed to tBHP, a prooxidant compound frequently used for assessment of mechanisms involving in oxidative stress in biological systems. Oxidative stress plays commonly a key role in the pathogenesis of both xenobiotic/drug-induced hepatotoxicity  and NAFL in vivo in patients [ in vitro in mouse hepatocyte line (AML12 cells) treated with free fatty acids [tBHP is known to cause peroxidation of membrane lipids  and deplpatients  and in epatients  and in vty acids  or in raty acids . In contty acids . Similarty acids  observedty acids ; thus outy acids . We show2 cells. Nevertheless, TFP was not significantly effective in prevention of reduced activity of cellular dehydrogenases, decreased albumin synthesis, enhanced lipoperoxidation, and surprisingly altered mitochondrial complex I activity (data not shown).Mitochondrial functions are often altered in the liver affected by NAFLD . Thus inIn steatotic liver, there is a higher offer of fatty acids to be peroxidized which together with an insufficient antioxidant capacity of the liver  leads toMitochondria, as the main energy provision system, is a crucial site of action of many hepatotoxic substances . In the In summary, we demonstrated that there are higher production of ROS, increased lipid peroxidation, lower redox state of glutathione, and decreased ADP-stimulated respiration using NADH-linked substrates in control fatty hepatocytes, as compared to control lean hepatocytes. We provided evidence that steatotic rat hepatocytes isolated from fatty liver are more susceptible to oxidative injury caused by tBHP in primary culture. According to the partial effect of TFP in the prevention of tBHP induced injury, MPT seems to participate in the toxicity of tBHP in lean and steatotic hepatocytes and the onset of MPT appears to be caused by lower concentration of tBHP in fatty cells. In addition, the present study confirmed the significance of inhibition of complex I activity induced by tBHP in both lean and steatotic cells. Our results collectively indicate that steatotic rat hepatocytes in primary culture are under conditions of enhanced oxidative stress. Moreover, these fatty hepatocytes are more sensitive to the exogenous source of oxidative injury. Free radicals are not only a cause but also a consequence of human pathologies, such as NAFLD. Mitochondria play an essential role in the generation of ROS and at the same time mitochondria are an important target for toxic action of free radicals . Our res