Meanwhile, the expression of PTPN3 was found to be regulated by miR-199 in resistant ovarian cancer cells. These findings suggest that PTPN3 promotes tumorigenicity, stemness and drug resistance in ovarian cancer, and thus is a potential therapeutic target for the treatment of ovarian cancer.The current standard treatment for ovarian cancer is aggressive surgery followed by platinum-based combination chemotherapy. Recurrence and chemotherapeutic drug resistance are the two main factors that account for the high mortality of most ovarian cancers. Liposomal doxorubicin is primarily used for the treatment of ovarian cancer when the disease has progressed after platinum-based chemotherapy. However, relatively little is known about the genomic changes that contribute to both cisplatin and doxorubicin resistance in high-grade serous ovarian cancer (HGSC) under the selective pressure of chemotherapy. Here, we found that protein tyrosine phosphatase PTPN3 gene expression was substantially increased in both cisplatin and doxorubicin-resistant ovarian cancer cells. Silencing of PTPN3 restored sensitivity to cisplatin and doxorubicin in resistant ovarian cancer cells. Down-regulation of PTPN3 also inhibited cell cycle progression, migration, stemness  Ovarian cancer, the most common cause of gynaecologic cancer-associated death, is responsible for approximately 14,000 deaths in the United States annuallyA recent study on the whole-genome characterisation of chemoresistant ovarian cancer by The Australian Ovarian Cancer Study Group showed that CCNE1 amplification is common in primary resistant and refractory disease. Acquired chemotherapy resistance is mainly associated with the inactivation of tumour suppressors such as RB1, NF1, RAD51B and PTEN, germline BRCA1 or BRCA2 mutations, loss of BRCA1 promoter methylation, and overexpression of the drug efflux pump MDR1Here, we undertook a search for candidate genes that could confer both cisplatin and doxorubicin resistance in established resistant ovarian cancer cells. Using genomic analyses, we found that protein tyrosine phosphatase non-receptor type 3 (PTPN3) gene expression was substantially increased in both cisplatin and doxorubicin-resistant cells. Moreover, PTPN3 silencing restored the sensitivity of resistant ovarian cancer cells to cisplatin and doxorubicin. Silencing of PTPN3 also inhibited cell cycle progression, migration and stemness, and reduced the tumorigenicity of resistant ovarian cancer cells. These findings identify PTPN3 as a potential therapeutic target for ovarian cancer treatment and for overcoming cisplatin and doxorubicin resistance.To identify the critical factors associated with both cisplatin and doxorubicin resistance in ovarian cancer, we compared the gene expression differences in A2780, A2780CIS and A2780ADR cells obtained from the European Collection of Cell Cultures (ECACC) using ovarian cancer cell line panel gene expression dataCell cycle progression has been shown to play important roles in drug resistance. To investigate the molecular mechanism of PTPN3-mediated cisplatin and doxorubicin resistance in ovarian cancer cells, cell cycle analysis was used to examine the effect of silencing PTPN3 on cell signalling pathways. After transfection with PTPN3 esiRNAs (esiPTPN3), an endoribonuclease-prepared siRNA pool comprised of a heterogeneous mixture of siRNAs that all target the same mRNA sequence of PTPN3, a significant silencing effect of PTPN3 in both cisplatin and doxorubicin resistant ovarian cancer cells was confirmed by qRT-PCR . SubsequTo investigate the critical role of PTPN3 in ovarian cancer drug resistance and cell cycle progression, we examined the effects of silencing of PTPN3 on resistant ovarian cancer cell growth, migration and drug resistance. The cell growth curves indicated that cell growth was dramatically reduced by silencing PTPN3 in both cisplatin and doxorubicin resistant ovarian cancer cells . We nextin vitro. Soft agar colony formation assays showed that stable silencing of PTPN3 significantly inhibited the colony forming ability of both cisplatin and doxorubicin resistant ovarian cancer cells, suggesting that stable silencing of PTPN3 inhibits cellular anchorage-independent growth of resistant ovarian cancer cells in vitro  family, which is known to comprise signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitosis and oncogenic transformationPlatinum-based chemotherapy is currently the first-line treatment for advanced stage HGSC patients. However, resistance to platinum-based chemotherapy is the main obstacle in clinical practiceHere, we found that overexpression of PTPN3 promotes both cisplatin and doxorubicin resistance. Previous studies on the mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs , affecting 26% of colorectal cancers and a smaller fraction of lung, breast and gastric cancersProtein tyrosine phosphatases (PTPs) have been found to function as tumour suppressors or oncogenes, depending on the substrate involved and the cellular context. PTPN3 has been reported to be an oncogene in gastric2. They were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin mixture . Cisplatin and doxorubicin were obtained from Sigma-Aldrich and dissolved in DMSO. Cisplatin and doxorubicin resistant cells were developed from their parental cell line A2780 by gradual incremental doses of cisplatin or doxorubicin administration in cell culture medium in subsequent passages for about 6 months. Thereafter cisplatin resistant A2780CIS and doxorubicin resistant A2780ADR cells were maintained in presence of 1 μg/ml cisplatin or doxorubicin in cultured medium for 72 h in every alternate passage in accordance with the ECACC guidelines.The human epithelial serous ovarian cancer cell lines A2780 (cisplatin-sensitive) was purchased from the European Collection of Cell Cultures  and cultured in a humidified atmosphere at 37 °C, 5% CO6 cells per flank) of female BALB/c nude mice via subcutaneous injections. The study was approved by the Animal Ethics Committee of Yijishan Hospital Affiliated to Wannan Medical College, China. All animal related methods were performed in accordance with the relevant guidelines and regulations. Tumor volumes were determined each week by measuring their length (a) and width (b) using a vernier caliper. The tumor volume (V) was calculated according to the formula V = ab2/2. The statistical significance between tumor sizes in the shPTPN3 and shScramble control transfected groups was evaluated using the Student’s t test. The tumors were fixed in 10% neutral buffered formalin before being processed into paraffin blocks. The tissue sections were stained with PTPN3 primary antibody (Abcam) using standard IHC techniques.The validated shRNA stable transfected A2780CIS and A2780ADR cells were re-suspended in PBS and implanted into the right and left flanks . Samples were blocked with 10% goat serum before incubation with primary antibody. The samples were incubated overnight using following primary antibodies: rabbit anti-PTPN3 (1:100) or an isotype-matched IgG (Sigma) as a negative control in a humidified container at 4 °C. Immunohistochemical staining was performed with the Dako Envision plus System  according to the manufacturer’s instructions. The intensity of staining was evaluated by digital image analysis on the scale of 0 to 4 according to the percentage of positive tumors .The rest of materials and methods are provided in the online How to cite this article: Li, S. et al. Protein tyrosine phosphatase PTPN3 promotes drug resistance and stem cell-like characteristics in ovarian cancer. Sci. Rep. 6, 36873; doi: 10.1038/srep36873 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Rhizobium leguminosarum is of significant economic value because of its ability to provide fixed nitrogen to globally important leguminous food crops, such as peas and lentils. Discovery based research into the genetics and physiology of R. leguminosarum provides the foundational knowledge necessary for understanding the bacterium's complex lifestyle, necessary for augmenting its use in an agricultural setting. Transposon insertion sequencing (INSeq) facilitates high-throughput forward genetic screening at a genomic scale to identify individual genes required for growth in a specific environment. In this study we applied INSeq to screen the genome of R. leguminosarum bv. viciae strain 3841 (RLV3841) for genes required for growth on minimal mannitol containing medium. Results from this study were contrasted with a prior INSeq experiment screened on peptide rich media to identify a common set of functional genes necessary for basic physiology. Contrasting the two growth conditions indicated that approximately 10% of the chromosome was required for growth, under both growth conditions. Specific genes that were essential to singular growth conditions were also identified. Data from INSeq screening on mannitol as a sole carbon source were used to reconstruct a metabolic map summarizing growth impaired phenotypes observed in the Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, pentose phosphate pathway, and tricarboxylic acid cycle. This revealed the presence of mannitol dependent and independent metabolic pathways required for growth, along with identifying metabolic steps with isozymes or possible carbon flux by-passes. Additionally, genes were identified on plasmids pRL11 and pRL12 that are likely to encode functional activities important to the central physiology of RLV3841.The free-living legume symbiont  Rhizobium leguminosarum is a Gram-negative soil and rhizosphere colonizing bacterium that is also capable of engaging in endosymbiosis with specific leguminous plant genera. The host specificity of rhizobial infection is dependent upon the exchange of specific chemical signals between the infecting bacterium and host plant , lentils (Lens culinaris), and vetch (Vicia spp.). When in the endosymbiotic state, Rhizobium bacteroids reduce atmospheric nitrogen N2 to ammonia, which is then exported to the plant for assimilation. In return, the plant host provides fixed carbon and other micro-nutrients to the bacteroids to sustain the symbiosis   mutagenesis to study gene function at a genome scale  of RLV3841 and deconstruct central carbon metabolism for growth on mannitol, a preferred carbon source of rhizobia Vincent, . CompariR. leguminosarum bv. viciae 3841  or Vincent's minimal medium (VMM)  supplemented with 1.% (w/v) mannitol as a carbon source  and 50 μg/mL neomycin (Neo) for RLV3841, and 100 μg/mL ampicillin (Amp) and 50 μg/mL kanamycin (Kan) for E. coli.Vincent, . The donE. coli SM10λpir(pSAM_Rl) were grown in broth culture until late exponential phase. 1.0 ml of donor and 0.5 ml of recipient strains were mixed in a 1.5 mL microcentrifuge tube and pelleted at 12, 000 g for 3 min. The cell mixture was then washed twice with 1000 μL 1X phosphate buffer saline (PBS), and resuspended in a final volume of approximately 100 μL 1X PBS. Six independent conjugations were spotted onto pre-warmed VMM-mannitol plates and were incubated at 30°C overnight (~18 h). Following incubation each of the 6 conjugation spots were scraped and re-suspended in 1000 μL of 1X PBS and pooled into a total volume of 6 ml, representing the RLV3841 Tn mutant library.Mutant pools were generated as described in Perry and Yost  with min2 (Corning) Neo and Str containing VMM-mannitol agar plates. For each selection plate, 500 μL of the RLV3841 mutant pool was spread plated and allowed to dry. The agar plates were incubated at 30 C for 72 hr representing between 15 and 18 generations of RLV3841 growth on minimal media. Cells from each plate were harvested by scraping the thin film of cell growth and re-suspending in 5 mL of 1X PBS, vortexed thoroughly to homogenize the cells, and then a 1000 μL aliquot of each cell suspension was used for cell pelleting and DNA isolation  and appended to the dataset. Duplicate gene sequences were manually examined using reciprocal BLAST to the RLV3841 reference genome for all genes with <0.30 insertion density to avoid miss-classification as an essential gene due to a lack of mapped insertions as a result of the alignment penalty for multiple mapping locations. The final compiled and curated dataset is found in Supplementary File Outputs from the HMM for both the TY  were mutated to verify the INSeq growth phenotype data. Mutants were created using a single crossover mutagenesis approach with pJQ200SK  were calculated in early exponential phase (OD600 < 0.100) and late exponential phase (0.100 < OD600 < 0.200) by calculating the average time required to double the optical density of the cell cultures, within the defined growth phases.Growth curves of RLV3841, MA0920, and MA3335 were performed using a shaking head Synergy HT Microplate Reader (Biotek) with 250 μL of inoculated growth media per well in a 96-well NuncR Optical Bottom Plate (Thermo Scientific), and a 40 μL Anti-Evaporation Oil (Ibidi) overlay. Inoculated growth medium was prepared by scraping cells from freshly grown TYSm or SmGm plates, washing twice with 1XPBS, standardizing the cell suspension for an approximate initial ODThe RLV3841 genome contains 140,056 potential mariner insertion sites distributed across the chromosome and 6 mega-plasmids. Insertion densities within the genome ranged from 0.65 to 0.86 across the 7 replicons, with an average insertion density of 0.80 Table . The insComparison of the VMM-mannitol INSeq dataset with the TY INSeq dataset identified a set of genes that when mutated conferred a GI phenotype under both conditions; these 491 genes were assigned to the CFG Figure . Genes tOpen reading frames annotated as hypothetical proteins represent approximately 25.2% of the RLV3841 genome. Of these, 103 hypothetical proteins were observed to have a GI phenotype on VMM, TY, or both growth conditions , and MA3335 (INSeq predicted RL3335 VGI), in TY and VMM-Mannitol broth over 72 h are shown in Figure INSeq was used to identify a potential minimal central carbon metabolism pathway for growth on mannitol. Figure Mutation of genes encoded on RLV3841 megaplasmids that resulted in a growth impaired phenotype were assigned as plasmid growth impaired (PGI), while plasmid genes that when mutated impaired growth on VMM or TY uniquely were designated PVGI and PTGI respectively. Collectively, 48 genes distributed across the 6 megaplasmids were predicted to result in a PGI phenotype when mutated , cofactors and carriers 25 genes), nucleotides (20 genes), and metabolic intermediates (15 genes)  required for conversion of F6P into glucose-6P  were observed to be growth neutral, possibly due to functional redundancy between the two isozymes, which has been previously reported in Bradyrhizobium japonicum  were both VGI and TGI  were also observed to be growth impaired on both media  when mutated  predicted to be involved in cobalamin biosynthesis, that was severely PGI on VMM-mannitol, and moderately PGI on TY when mutated. Previous studies in R. etli identified similar growth phenotypes when a homologous cobalamin biosynthetic cluster on p42e was deleted  and pRL120210 (putative rpiB), which encode enzymes predicted to function in central carbon metabolism, were found to be PGI when grown on mannitol as a sole carbon source. Their chromosomal homologs RL2513 (tpiA) and RL2547 (rpiB) were both observed to be GN (Table R. leguminosarum bv. viciae strain VF39, the homologs of pRL120209 and pRL120210 are required for growth on erythritol as a sole carbon source (Yost et al., R. leguminosarum were unable to grow on minimal media (Hynes et al., Beyond identifying putative n pRL11065-32 predmariner transposon appears to outweigh its disadvantages. A sufficient saturation of neutral mariner insertion sites within the mutant community allows for confident identification of regions that lack insertions due to the negative selection resulting from a GI phenotype. In this and previous, work a sufficient level of neutral “TA” site saturation has been recovered to allow Bayesian methods of analysis, using a relatively modest amount of sequencing data when compared to other mariner INSeq studies. Considerations in inoculation density, the number of generations of growth during negative selection, and the ability to recover mutant populations needs to be carefully considered in order to ensure enough complexity is retained in the mutant pools post-selection to allow for statistical analysis.INSeq, like all high-throughput molecular techniques, is not without limitations. Genes with large regions of sequence duplication, or no mariner “TA” insertion sites, cannot be assayed using INSeq. These genes represent only 1.9% of the genome. However, the targeted library preparation method and robust statistical analysis afforded by the use of MmeI-adapted BP and CY conceived and designed the research; BP and MA conducted the experiments; BP conducted the data analysis; BP and CY prepared and finalized the manuscript.The presented research was conducted with support from a Discovery Grant awarded by the Natural Sciences and Engineering Council of Canada.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Isothermal amplification methods for detection of DNA and RNA targets have expanded significantly in recent years, promising a new wave of simple and rapid molecular diagnostics. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products. Here we report the amplification of discrete target fragments of several kilobases at 37 °C from both double- and single-stranded circular template DNA using specific primer pairs. In contrast to existing methods, this amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. In addition to the discrete amplicon products, this method also produces higher molecular weight products consisting of multiple repeated copies of the amplicon and template DNA. We demonstrate that two features of gp32 enable this amplification: a facilitation of primer strand invasion into double-stranded DNA, and a suppression of non-homologous primer annealing and nonspecific amplification. The ability presented here to produce long, discrete DNA products in an isothermal reaction extends the scope of isothermal amplification to enable more useful applications of these promising methods. Most methods involving DNA or RNA start with the molecules of interest at concentrations far below the necessary amount, and accordingly must be increased through amplification. DNA amplification can be performed using a variety of strategies, most commonly the polymerase chain reaction (PCR) which is enabled by cycles of thermal denaturation of double-stranded DNA (dsDNA). While routine and flexible, PCR is limited in application by its requirement for instruments capable of thermal control and cycling between high temperatures for dsDNA denaturation and lower temperatures for primer extension by the DNA polymerase. Additionally, the degree of amplification is intrinsically limited by the cycling of the reaction: each cycle can at most produce a doubling of the DNA. Recent years have seen growing adoption of methods that amplify DNA or RNA at a single temperature, accordingly referred to as “isothermal” amplification. These methods require simpler instrumentation, using only a single, moderate temperature. Additionally, isothermal methods can provide extremely rapid results, as fast as 10 minutes, and can produce a large amount of DNA, enabling simple, visual detection of amplificationBsu, Bst, and E. coli DNA Polymerase I, or phi29 DNA polymerase) separates dsDNA after initiation at a primer, with the initiation step proving the key limit to speed and efficiency of an isothermal reaction. Strategies to facilitate initiation provide the main variance among isothermal methods. For example, initiation approaches include: creation of nicks by a nicking enzyme as in strand displacement amplification (SDA)3 and nicking enzyme amplification reaction (NEAR)4; facilitated strand invasion as in strand invasion-based amplification (SIBA)5, recombinase polymerase amplification (RPA)6 and helicase-dependent amplification (HDA)7; or thermodynamic invasion and annealing as in the initiating first round of SDA, NEAR, loop-mediated isothermal amplification (LAMP)8, multiple displacement amplification (MDA) and rolling circle amplification (RCA)9. Many MDA and RCA applications alternatively use single-stranded DNA as a starting material. After initiation, primer design in methods like LAMP enable rapid exponential amplification as the primers form loop structures to create 3′ ends extendable by a DNA polymerase. Generally, all of these methods target very short amplicons, with length usually less than 150 base pairs (bp). LAMP and rolling-circle methods  produce much longer, complex products which are concatameric, highly branched, and difficult to use in any downstream application. A limitation of isothermal methods has been the creation of discrete (i.e. a single DNA species), long (>500 bp) dsDNA products. Here we present a novel isothermal amplification scheme capable of producing discrete, several kilobase (kb)-long dsDNA products using only two specific primers, a strand-displacing DNA polymerase, and the single-stranded DNA binding protein gp32 from bacteriophage T4.Each isothermal technique relies on enzymatic activities or primer design to bypass the need for thermal denaturation of dsDNA. Approaches vary, but generally strand displacement activity by a DNA polymerase  during phage DNA replication, recombination and repair processes. It binds ssDNA cooperatively as a monomer, and each gp32 protein covers ~7 bases of ssDNA with no observed sequence specificityBsu DNA Polymerase, Large Fragment) we were able to amplify discrete dsDNA amplicons of several kilobases from circular templates using a pair of target-specific primers, as well as higher molecular weight products consisting of multiple repeated copies of the amplicon and template. We also demonstrate a significant increase in specificity of amplification facilitated by gp32, with a suppression of nonspecific product in the presence of the single-stranded binding protein. Together these results show an unexpected role for gp32 in facilitating strand invasion and initiation of synthesis, with a novel isothermal amplification application uniquely capable of producing several-kilobase, discrete products from circular dsDNA.In the above applications the effect of gp32 was mostly attributed to its ability to bind single-stranded nucleic acid, such as the oligonucleotide primers or target DNA or RNA. In this report we demonstrate that gp32 alone is able to assist strand invasion of primers into dsDNA at moderate temperatures (30–45 °C) and facilitate formation of an initiation site for DNA amplification. Using a combination of gp32 and a mesophilic strand-displacing DNA polymerase  using circular double-stranded pUC19 plasmid template, DNA polymerase, dNTPs in a reaction buffer suitable for amplification Fig. . No therTo confirm the identity of the product species, the structures of the gp32-assisted amplification product were analyzed by restriction enzyme digestion and sequencing. Individual bands from an isothermal reaction producing a 700 bp amplicon of pUC19 were excised and purified following electrophoresis separation. The multimeric DNA products were digested with two restriction enzymes, each cutting pUC19 once, in single or double digests Fig. . The fraWe also tested gp32-mediated amplification using single-stranded circular DNA M13) as the template Fig. , the ssM3 as the We tested whether linear dsDNA could serve as a template. Using the same primers, we PCR-amplified the pUC19 fragment that supported gp32-based amplification  produced the same amplification patterns as Bsu and the maximum activity was observed at approximately 38 °C , and this result indicates Bst 2.0 retains enough polymerase activity at the lower, optimal temperature for gp32. Two strand displacing polymerases, phi29 DNA polymerase, commonly used in traditional rolling-circle amplification, and Vent (exo-) DNA polymerase were unable to support gp32-mediated amplification , and T5 exonuclease to remove plasmid that has nicked or gapped sites. None of these treatments affected the ability of plasmid to serve as a template for efficient amplification  on dsDNA could serve as efficient initiation sites for the strand-displacing DNA polymerase, we investigated the effect of gp32 with nicked circular dsDNA, and whether nicking can facilitate the gp32-assisted amplification  supported any amplification from ds pUC19 Fig. , lane 5,This enhanced specific binding by gp32 was further examined using single primers that either had the same sequence as the ssM13 template  or were complementary to it . In the absence of gp32, these two types of single primers produced a similar smear of high molecular weight product  DNA Polymerase and an incubation temperature of 60–65 °C. This method generates a ladder which was interpreted as simple multimers of linearized circle24. These reactions also included gp32 in the reaction, although at 5–10 fold lower concentrations than used in experiments detailed here. When this hyper-branched RCA was carried out using Bst DNA polymerase at similar temperature, no gp32 was required26. In contrast, the amplification described here works with both double- and single-stranded circular DNA templates, the reaction occurs at 37 °C and requires a high concentration of gp32 (~300 ng/μl). Analyses of the products indicate the smallest band is a discrete amplicon flanked by the opposing primer pair, and the rest of the ladder consists of concatamers of the linearized circle with a duplication of the single amplicon . The most common form of this technique, multiply-primed RCA, utilizes random hexamers annealing to denatured double stranded plasmid at multiple sites that enables amplification by phi29 DNA polymerase6 where two other T4 proteins, UvsX and UvsY, are also required. Both amplifications take advantage of the primer binding and dissociation cycles facilitated by UvsX to achieve exponential PCR-like amplification even with a linear template, although they adapt different strategies in primer designs. Both RPA and SIBA target relatively short amplicons. RPA prefers short amplicons (around 500 bp or shorter) for efficient amplification while SIBA only works with very short amplicons due to the requirement for a synthetic oligonucleotide for strand invasion. As we demonstrated, we were able to amplify amplicons of several kb in length in a simple system without the need for recombination enzymes, invasion oligo templates, or associated ATP regeneration systems. Products of this size and nature are much more easily utilized in downstream applications  and isothermal production of these products could enable clonal amplification of longer library inserts than is currently possible in next-generation sequencing platforms. Moreover, this gp32-mediated amplification also generated very long products (>25 kb), demonstrating a combination of benefits from synthesizing discrete amplicons generated by methods like SDA and RPA and high molecular weight products generated by LAMP and RCA.Similarly high concentrations of gp32 were required in RPA (~900 ng/μl) and SIBA (~250 ng/μl)In summary, we have demonstrated that with the assistance of a high concentration of gp32, it is possible to generate discrete dsDNA products at 37 °C using only single-stranded binding protein, DNA polymerase, and two target-specific primers. This study reveals that gp32 has some unexpected roles in promoting primer invasion on a dsDNA template, enhancing specific binding and preventing non-specific primer annealing. These properties may generally facilitate DNA amplification strategies at ambient and moderate temperatures. The ability to synthesize long, discrete dsDNA at low temperature expands the reach of DNA amplification methods and may have use in molecular diagnostics, clonal amplification for next-generation sequencing , and other biotechnology applications. Though the method requires relatively high input amount compared to sensitive diagnostic amplification techniques, the ability to produce long, discrete and concatameric products from a small and easily-prepared substrate would be useful in many of these methods. Although at this time it can only efficiently utilize circular DNA templates, future testing will determine the feasibility of extending the method to amplifying linear DNA as in PCR, but without the need for DNA denaturation by thermal cycling.Bsu DNA Polymerase, Large Fragment (New England Biolabs (NEB)) and 300–456 ng/μl (9.0–13.6 µM) T4 gene 32 protein (NEB). DNA oligos were from IDT and their sequences and target amplicons are listed in Supplemental Materials  Fragment; Sequenase Version 2.0 DNA polymerase (Affymetrix); phi29 DNA Polymerase; DNA Polymerase I (E. coli); T4 DNA Polymerase.Other polymerases tested were from NEB unless otherwise noted: Amplification reactions were stopped by addition of 1x SDS-containing loading buffer and analyzed by 1x TBE agarose (1%) gel electrophoresis containing ethidium bromide. DNA markers were 100 bp ladder or 1 kb ladder (NEB). For analyzing the structure of the amplified products, individual bands were excised from the gel and DNA was extracted using a Monarch® DNA Gel Extraction Kit (NEB). Purified DNA was then used for restriction analysis with either one or two restriction enzymes and for Sanger sequencing.No datasets were generated or analyzed under the current study.Supplemental Material


Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study. Telomere shortening has recently been identified as one of nine ‘hallmarks of aging’  and blooA central requirement of all methods of TL measurement is the extraction of a suitable quantity of high quality DNA. A considerable number of DNA extraction methods have been employed to date by researchers studying TL . In geneThe qPCR method measures RTL as the total amount of telomeric sequence relative to the amount of a non-variable copy reference gene sequence within the same DNA sample . StandarThe objective of the present study was to assess the effect of two different DNA extraction methods, and the use of different calibrators on RTL measurements. We compared RTL measurements of blood samples that were collected from a Holstein Friesian cattle population after extracting DNA using two silica membrane-based DNA extraction protocols and a salting out (non-membrane-based) method. To validate our results with samples from a different species we compared one of the two silica membrane-based methods with the salting out method using buffy coat samples from wild Soay sheep. We found high repeatability of RTL measurements, regardless of DNA extraction method, and no difference in mean RTL among extraction methods when a DNA extraction method specific (MS) calibrator was used.Whole blood samples were collected from Holstein Friesian cattle during 2009–2013 at the Crichton Royal Farm  as part of a long-term genetics study for which blood samples have been archived for many years . SamplesAdditionally, we used blood samples collected from a wild population of Soay sheep on the St Kilda archipelago in the Outer Hebrides (Scotland), which have been subject to individual-based monitoring and regular sampling since 1985 . Blood sBlood sampling from Holstein Friesian cattle and Soay sheep was approved by the Animal Experiments Committee .DNA from each cattle sample was extracted using the QIAGEN Gentra Puregene kit (PG) based on a non-membrane salting out method and two silica membrane-based protocols of the QIAGEN DNeasy Blood & Tissue kit: spin column (SC) and the 96-well plate (SP). DNA from each sheep sample was extracted using the PG and SC protocols.According to the PG protocol, DNA is first isolated by removing red blood cells and lysing white blood cells. RNA and proteins are removed by enzyme digestion and salt precipitation, respectively. DNA is recovered by alcohol precipitation and dissolved in DNA hydration solution. The SC and SP protocols rely on a silica-based extraction method during which cells are lysed and transferred onto silica membranes to which DNA binds specifically during a centrifugation step. DNA is washed and finally eluted using a DNA hydration buffer. When possible, we performed different DNA extraction methods simultaneously on each sample. We followed the manufacturer’s protocol with certain alterations to improve yield and quality of DNA samples. The most important alternation was that the silica protocols were started with a red blood cell lysis step that allowed us after centrifugation to transfer only the white blood cell pellet dissolved in PBS onto the silica membranes. This step removed impurities in the beginning of the protocol and improved purity measurements greatly. SC samples were also prepared in duplicates that were run through the same silica membrane to improve DNA yield. All alternations are detailed in We employed a strict quality control (QC) strategy during DNA extraction and qPCR to ensure that samples extracted by different methods were of similar quality, purity and integrity. Our aim was to minimize the risk of differences between DNA extraction measurements being due to sample quality rather than differences of methods themselves. Samples failing QC were excluded from our final analyses .We tested DNA yield and purity using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) with the software NanoDrop 2000. Samples with DNA concentrations less than 20 ng/μl were excluded from further investigation . The ave® 2.0 (Invitrogen) using a Qubit® dsDNA BR Assay kit (Invitrogen) according to the manufacturer’s manual. SP extracts were measured on a FLUOstar Galaxy microplate reader (BMG LABTECH) using a Quant-iT™ dsDNA Assay Kit (Invitrogen) following the manufacturer’s instructions. Both procedures are based on the detection of a fluorophore that becomes fluorescent when bound to double stranded DNA. Measurements are evaluated in relation to standards with known DNA concentrations. Because the signal is specific for double stranded DNA (dsDNA) fluorescence spectroscopy measurements are more accurate for DNA yield than NanoDrop measurements. Samples with average concentrations lower than 20 ng/μl calculated over two measurements on either fluorometer were excluded from further investigation. DNA integrity was assessed visually by running 200ng on a 0.5% agarose gel with ethidium bromide at a final concentration of 0.8 μg/ml. Gels were run at 100 mV and 200 mA for 45 minutes and then visualised with an AlphaImager TM 2200. Gels were visually scored for integrity on a scale of 1 to 5  gene (accession number: NM_001009284), which we selected as our reference gene. The selection of our reference gene was based on comparison of a panel of 12 candidate genes for sheep and 6 for cattle, supplied as part of the Primerdesign GeNorm kit . B2M showed completely stable qPCR results indicative of non-variable copy number, and is well conserved and located on chromosome 10 of the bovine genome and chromosome 7 of the ovine genome [CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT) and tel 2b (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT) primers were used [Leukocyte RTL was measured by qPCR  as the ae genome . For theere used . TelomerB2M primers were run in separate wells (monoplex qPCR) but on the same qPCR plate. Each qPCR plate was repeated four times over two days. Our calibrator sample came from a large volume of blood obtained from an individual cow or sheep, respectively. We extracted large quantities of DNA from each calibrator sample using different methods to match those applied to our experimental samples: PG, SC and SP for cattle, PG and SC for sheep. In the cattle experiment, each qPCR plate included three calibrator samples, one for each of the extraction methods used . In the sheep experiment, we only included the MS calibrator on each plate .The use of identical primers allowed us to use identical reaction conditions for both cattle and sheep qPCRs. We ran samples extracted by different methods and species on separate 384-well plates. Reactions for telomere and B2M primers were used at 300 nmol. Nuclease-free water was added to the master mix to have a final volume of 10 μl per well.Samples and calibrators were loaded at a dilution of 1 ng/μl onto a 96 well plate (sample plate) that also contained a four step 1:4 serial dilution of calibrator DNA starting with 10 ng/μl as standard and nuclease free water as non-template control. A Freedom EVO 2150 robot (by TECAN) was used to transfer all samples, standards, calibrators and negative controls in triplicate onto a 384 well qPCR plate. The robot mixed 1 μl of the contents of the sample plate with 9 μl of master mix in each qPCR plate well. The master mix for both reactions contained 5 μl of LightCycler 480 SYBR Green I Master (Roche) per well. Telomere primers were used at a concentration of 900 nmol, B2M primers rarely forming primer dimers in the negative controls. Telomere primers always form primer dimers due to the repetitive nature of their sequence. Evidence for primer dimer formation can be seen as melting peaks at slightly higher melting temperatures than the telomere qPCR product and also as amplification curves at very late cycles (average Cq for telomere negative controls: 38.1 (cattle) and 31.3 (sheep) compared to average Cq values of samples: 14.42  and 13.52 ).The qPCR was performed on a LightCycler 480 (Roche) using the following protocol: Enzyme activation: 15 min at 95°C; then 50 cycles of: 15 s at 95°C (denaturation), 30s at 58°C , 30 s at 72°C ; melting curve: 1 min at 95°C, 30s at 58°C, then continuous increase of temperature (0.11°C/s) to 95°C with continuous signal acquisition; Cool down: 10 s at 40°C. Melting curves showed a single peak with th percentiles, as our reaction efficiency for each amplicon group [B2M reaction, and 93.6%- 94.4% (cattle) and 92.5%-95.5% (sheep) for the telomere reaction. We set a constant fluorescence threshold within the window of linearity across all plates for the calculation of Cq values. The threshold was for B2M 0.221 in cattle and 0.1 in sheep and for the telomere amplification 0.256 and 0.1 in cattle and sheep, respectively.The software package LinRegPCR  was usedon group . The meaB2M and telomere reaction) for each qPCR plate using LinRegPCR. Samples were excluded from final analysis if at least one of their triplicate amplifications had a qPCR efficiency that was 5% higher or lower than the mean efficiency for the respective amplicon. Also, samples were excluded if their Cq values had a coefficient of variation (CV) > 5% across triplicates. Elimination of samples that failed quality control for qPCR efficiency or Cq values ensured high intra-plate repeatabilities and efficiencies, although less than 1% of our samples were excluded based on these criteria  a MS calibrator, (2) a calibrator extracted with a single method across all plates, arbitrarily choosing PG , and (3) a constant Cq value across all plates . We chose constants of 26 for the reference gene and 14 for telomeres, as these were the average sample Cqs for these amplicons in our cattle experiment. The use of a constant Cq in the above equations allowed us to examine how well the use of a plate-specific calibrator  accounted for plate-to-plate variation in RTL measures, whilst keeping RTL values on a similar scale as the RTLs calculated with MS and PG calibrators. In the subsequent sheep experiment, we only compared the MS calibrator with the no calibrator calculations (25.99 for reference gene and 13.71 for telomeres). We also examined variation in the raw Cq values for the telomere and Each sample was run on four identical qPCR plates per DNA extraction method. We calculated the Pearson’s correlation coefficient for the individual RTL measurements between all possible plate combinations. We took the average RTL for a sample across the four plates within each extraction method and calculated the Pearson’s correlation coefficient among methods. We calculated the CV–i.e. the standard deviation divided by the mean—across replicates of each sample both across all plates and within plate using the same extraction method. Pooled CVs across samples were calculated as the geometric mean CV.Linear mixed models were used to estimate the repeatability of RTL measurements and Cq values for a given sample, the degree of plate to plate variation, and the effect of DNA extraction method on mean RTL. The model of analysis included the random effects of sample, sample-by-extraction method interaction and plate, and the fixed effect of DNA extraction method. Variance components for the random effects were estimated using restricted maximum likelihood. The sum all variance components constituted the total phenotypic variance. The repeatability of sample RTL across plates and methods was calculated as the ratio of the sample variance to the total phenotypic variance. The ratio of the sample-by-extraction method interaction to total phenotypic variance provided an estimate of the proportion of variance attributable to differences in RTL among extraction methods within a sample, whereas the ratio of the plate effect to total phenotypic variance expressed the proportion of variance attributable to differences in the mean RTL among plates. We tested the significance of any differences in mean RTL associated with DNA extraction method by comparing models with and without extraction method as a fixed effect using a likelihood ratio test. We ran separate models for RTL calculated using MS calibrators (both species), PG calibrators (cattle only) and no calibrator (both species). We made the same comparisons for the reference gene and telomere Cq values in both species. All statistical analyses were performed in R Studio with R 3.1.2  with mixA total of 56 of our PG and SC cattle samples, 51 of our SP cattle samples, and 36 of our sheep samples passed all quality controls for all DNA extraction methods and were used for RTL measurement  and between methods for the two species are summarized in In both cattle and sheep, we found significantly (P<0.05) higher mean RTL in samples extracted using the non-membrane-based method (PG) compared to those extracted with the silica membrane-based methods (SC and SP), when using either the PG calibrator or no calibrator in calculations , Table 2In the present study, we addressed the effect of DNA extraction method on RTL measurements by comparing two silica membrane-based kits (SC and SP) with a kit that uses a non-membrane-based salting out method (PG). As expected , we founWe found that the rank order of RTL measurements among samples is largely unaffected by DNA extraction methods. Across extraction methods, our RTL measures showed reasonably high repeatabilities and inter-plate correlations and low inter-plate CVs that were close to those reported in the qPCR telomere literature ,14,16. TImportantly, our results show that it is possible to account for differences in mean RTL associated with DNA extraction method using a DNA extraction method-specific calibrator. Our reading of the literature suggests it is unusual for qPCR-based telomere studies in both epidemiology and ecology to provide much information about the source or preparation of the calibrator sample used. The five previous studies of DNA extraction method effects on RTL discussed above presumably used a calibrator sample extracted using only one extraction method, although most of them fail to explicitly state what kind of calibrator was used –13 and hIt is obviously preferable to use a completely consistent approach and extract DNA using the same method within a study. However, a major challenge in the study of telomere dynamics is to generate sufficiently detailed longitudinal data to determine whether variation in TL observed later in life is the result of differences set in early life or differences in attrition rates across life . AddressThis study adds to the emerging literature showing that DNA extraction methods may affect the mean RTL measurement produced by qPCR techniques. We present the first evidence for such effects in non-human vertebrates, documenting similar results in two ruminant species of considerable economic and agricultural importance in which TL variation has recently been examined with some exciting initial results –30. We aS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


The Ebola virus (EBOV) variant Makona (which emerged in 2013) was the causative agent of the largest outbreak of Ebola Virus Disease recorded. Differences in virus-host interactions between viral variants have potential consequences for transmission, disease severity and mortality. A detailed profile of the cellular changes induced by the Makona variant compared with other Ebola virus variants was lacking. In this study, A549 cells, a human cell line with a robust innate response, were infected with the Makona variant or with the Ecran variant originating from the 1976 outbreak in Central Africa. The abundance of viral and cellular mRNA transcripts was profiled using RNASeq and differential gene expression analysis performed. Differences in effects of each virus on the expression of interferon-stimulated genes were also investigated in A549 NPro cells where the type 1 interferon response had been attenuated. Cellular transcriptomic changes were compared with those induced by human respiratory syncytial virus (HRSV), a virus with a similar genome organisation and replication strategy to EBOV. Pathway and gene ontology analysis revealed differential expression of functionally important genes; including genes involved in the inflammatory response, cell proliferation, leukocyte extravasation and cholesterol biosynthesis. Whilst there was overlap with HRSV, there was unique commonality to the EBOV variants. The evolution of Ebolaviruses of varying pathogenicity complicates assessment of the public health significance of novel filoviruses. Ebolaviruses are a diverse genus within the Filoviridae family. Five species have been characterised; from those non-pathogenic in humans to those with high case-fatality rates (CFR) ranging from 25–90%13in vivo using Macaca fascicularis as an animal model identified a potential shift in the pathogenesis of infection with EBOV Makona compared to other variants. Results showed a reduced pace of disease progression correlating with viral titre1in vitro and in vivo has revealed changes in the strength of activation observed in molecular pathways during infection89Work A full appreciation of global cell biology changes induced by the recent Makona variant has not been investigated. The profiling of cellular changes elicited by virus infection has been used previously to identify fundamental differences in cellular interactions, which occur between high and low pathogenicity variants. For example, with pandemic influenza H1N1 and a lower pathology variant12A direct comparison of cellular effects induced by different variants of EBOV and comparison to Makona would provide useful indicators to the biological properties of this virus. Therefore, the aim of this study was to define gene expression signatures associated with the Makona variant of EBOV compared to the 1976 Ecran variant by identifying significantly changed patterns of gene expression using RNAseq.50 by the Reed & Muench method50 units/cell and cultured for 24 h, this was used as a comparator for pathway analysis. The HRSV strain used in this study is tissue culture adapted and was chosen as we and others have previously characterised this strain in A549 cells50/cell at Containment Level 4 and total cellular RNA purified at 24, 48 and 72 hours post-infection using the Qiagen RNeasy Mini Kit. Total RNA was purified from cells infected with HRSV at 24 h post-infection. After this time point, for this virus, cells undergo syncytia formation. Replicates of A549 and A549 NPro mock infected controls were generated (n = 3) and all 72 h infections for Makona and Ecran were performed in duplicate.A549 cells were obtained from the European Collection of Cell Cultures (ECACC) maintained by Public Health England. A549 NPro cells are a stably transfected cell line expressing the Bovine Viral Diarrhoea virus (BVDV) N protein and was kindly provided by Prof. Steve Goodbourn . The EBOV Makona variant was obtained from the European Mobile Laboratory as part of the response to the 2013–2016 West African outbreak, and is designated Ebola virus/H. sapiens-wt/GIN/2014/Makona-Gueckedou-C05. EBOV-Ecran was isolated during an outbreak in October 1976  designated Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Ecran. Both viruses used in this study were quantified by TCIDRNA yields were measured with a Nanodrop 1000 (Thermo Scientific) and were normalised to 60ng/μl before addition to qRT-PCR. Primers and minor-groove binder probes were using previously described sequencesWestern blots were performed on protein extracts collected in Laemlli Buffer (Sigma) supplemented with an additional 10% SDS to comply with local controls for the removal of samples from containment level 4. Samples were heated at 95 °C for 10–15 minutes prior to removal to containment level 2. Protein lysates were loaded on to Novex 4–12% gradient acrylamide gels (Life Technologies). The gels were run in a Novex X-2 vertical gel tank for 50 minutes at 200 V in MOPS buffer (Life Technologies). PVDF blots were performed using the IBlot semi-dry blotting system (Life Technologies) and blots blocked for minimum 2 h at room temperature with non-fat dried milk  in Tris-Buffered Saline–Tween20 . Blots were rinsed in TBST and primary antibody staining performed overnight with the IRF-3 and GAPDH specific rabbit polyclonal antibodies (Millipore). Secondary antibody staining was performed as per antibody instructions using goat anti-rabbit antibody conjugated to HRP. Luminescence assay was performed with ECL Prime reagent  after a 5 minute incubation and imaged in a GeneSys Gel Imaging System (GeneSys).The RNA samples were DNase treated and prepared for Illumina sequencing on a HiSeq 2500as described in previous workBriefly, base calling and de-multiplexing of indexed reads was performed by CASAVA version 1.8.2 (Illumina) to produce 30 samples from the 5 lanes of sequence data, in fastq format. The raw fastq files were trimmed to remove Illumina adapter sequences using Cutadapt version 1.2.1Viral mapped reads were further analysed using Cufflinks with the CuffDiff algorithm to calculate fragments per kilobase per million (FPKM) and viral mRNA levels determined for each virus at each time point. Mapped reads to the human genome were also analysed using EdgeR v. 3.3Two different EBOV variants were investigated in this study; the EBOV Makona isolated from the 2013–2016 West Africa outbreak and the progenitor virus, EBOV Ecran, isolated in 1976. The effect of the two EBOV variants on the host transcriptome of A549 cells was investigated. A549 cells were chosen for this study because they produce a robust innate response to virus infection and have been used to study the innate cell response to a number of different viruses1122232450 and cells infected with an equal MOI (0.5 TCID50/cell). The abundance of genome was compared by qRT-PCR analysis  distribution. Levels of mRNA were normalised to account for size factors and general linear models (GLM) were employedenerated  and a prenerated . This anTranscriptome changes in cells infected with the variants of EBOV may have reflected generic responses to viral infection, rather than being specific for EBOV. To investigate and control for this, A549 cells were infected with HRSV at an MOI of 0.5 PFU/cell and the total RNA purified at 24 h post-infection and analysed by RNASeq. The EBOV and HRSV data were then used to calculate pathway activation scores in IPA (Z-scores); reflecting the cumulative patterns of increased/decreased abundance of gene transcripts aligned with a pathway and thus the pathway activation state . This an31IL-10, TRAF6, STAT2, c-Jun and LTA all showed expression levels in Makona infected cells different from those of Ecran but similar to that of Ecran infected A549 NPro cells. LTA is a TNF-α family cytokine with potent immunomodulatory activityBAK and BCL-2 were increased in abundance in infected NPro cells compared to infected A549 cells, whilst transcripts encoding IFI35, IFIT3 and IFNAR were all decreased in abundance. Perturbation of interferon signalling should make cells more permissive to viral infection. To assess whether EBOV Ecran displayed accelerated replication in A549 NPro cells, qRT-PCR was performed to assess intracellular viral genome abundance . Related to this was the increased abundance of the Niemann-Pick disease, type C1 (NPC1) transcript in virus infected cells . NPC1 has been identified as a receptor for EBOV entryFor both Ecran and Makona, pathway analysis of the transcriptome changes in A549 cells indicated activation of the cholesterol biosynthesis pathways  have been observed in HRSV in cell culture15484925in vivo analysis of blood taken from patients with acute EVD from Guinea suggested that there was a robust interferon responseIFI35, OAS1, STAT1, TAP1 and IRF-7 gene transcripts were all increased in abundance in HRSV infected A549 cells compared with mock infected A549 cells, and decreased in abundance in EBOV Makona and Ecran infected A549 cells compared with mock infected A549 cells. BAK, BAX, FADD, IKKα and all showed decreases in abundance in Makona or Ecran infected A549 and NPro cells compared with mock infection. IL-6 expression was also induced in HRSV, Makona and Ecran infection of A549 cells and in Ecran infection of A549 NPro cells. IL-6 was evaluated in the Macaca fascicularis model system comparing the pathogenesis of EBOV Mayinga variant with Makona. This experiment indicated that IL-6 concentration in serum reached similar levels for both variants at pre-defined end points but rose more quickly in Mayinga infection47IL-10 and SOCS1, in Makona infected cells. More importantly, comparison of Ecran infected A549 cells with Ecran infected A549 NPro cells showed that disruption of interferon signalling in the A549 NPro cell line did not enhance the replication rates of Ecran. The implication is that interferon has little impact on viral growth due to the potent modulatory activity of EBOV virulence proteins.The interferon signalling pathways have been studied in EBOV infection in cell culture, leading to the discovery that the viral encoded proteins, VP35 and VP24 are able to disrupt interferon signalling810Activation of cholesterol pathways in infected cells was shown to be common in both EBOV variants but not found in HRSV. Cholesterol has been shown to be important for detachment of infected cells and this was induced by membrane anchored EBOV GPTRAF1, PLB1, CTU1, TNFAIP3, PTX3, ATF3 and CEBPB9EGR1 is differentially expressed as well as ATF3, both are regulatory factors governing cell activation in response to stress. Aligning genes to canonical pathways  variants of Ebola virus. Sci. Rep.7, 43144; doi: 10.1038/srep43144 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The mechanism of bile acid induced cholestatic liver injury remains controversial, thus hindering the development of new therapies for these diseases. In this research highlight, we briefly review the evolution of our understanding of the pathogenesis of bile acid induced liver injury, and summarize our recent findings on this topic. Our data suggests that under pathophysiological conditions bile acid induced liver injury is mediated by inflammatory responses that are initiated from stressed hepatocytes. We conclude by mentioning potential new therapeutic approaches for treating cholestatic liver injury based on these pathophysiologic concepts. In these studies, cultured human hepatocytes were exposed to submillimolar levels of toxic secondary bile acids. However, the concentration of these bile acid species in patient serum rarely reached these experimental levels . Subsequent studies proposed that the cause of cell death in cholestasis was due to bile acid induced hepatocyte apoptosis . This hypothesis was primarily based on observations in cultured rat hepatocytes treated with high levels (≥50 μM) of glycodeoxycholic acid or glycochenodeoxycholic acid (GCDCA) –8. However 1) GCDCA is not a major endogenous bile acid in rodents, and its concentrations in serum and liver are less than 10 μM even in bile duct ligated rats ; and 2) no apoptotic cells have been detected in cholestatic livers –12. In contrast, taurocholic acid (TCA), the major endogenous bile acid in rodents, did not cause apoptosis in these cells even at millimolar concentrations . Recently, Allen et al showed that TCA treatment of cultured mouse hepatocytes (200 μM for 6 h) stimulated chemokine expression . These authors proposed that bile acid induced liver injury was mediated via an acute inflammatory response. This hypothesis is also supported by finding neutrophil infiltrations in necrotic areas of liver following bile duct ligation in rodents . They also found that the bile acid induction of chemokines is FXR independent but partially Egr1-dependent . Since Egr1 is a transcription factor expressed in many cells and tissues, they speculated that bile acid activation of Egr1 was mediated through a membrane receptor although the nature of this signaling pathway remained elusive . In addition, the functional significance of bile acid induced chemokine production in liver injury remained to be explained.Chronic cholestasis, whatever the cause, results in liver injury where elevated hepatic levels of bile acid contribute to the exacerbation of the disease. However, the mechanism remains controversial. Earlier concepts held that high hepatic levels of bile acids killed hepatocytes through their direct cytolytic effects Mdr2 (Abcb4)−/− mouse , 16. In those studies, we sequentially assessed the development of liver injury over time in 10-day, 3-week, 6-week and 12-week old Mdr2−/− mice by examining liver histology, gene expression, neutrophil infiltration, and plasma levels of bile acids at these various time points compared to their littermate wild-type controls. Plasma levels of bile acids in these Mdr2−/− mice were significantly higher than in the wild-type controls at all time periods measured after birth and rosein parallel with the increased expression in the liver of proinflammatory cytokine mRNAs for Tnfα, Ccl2, Cxcl1, and Cxcl2. In 10-day old mice, the number of hepatic neutrophils were similar between Mdr2−/− and wild-type control mice. However, marked elevations in neutrophil infiltrations were first detected in the 3-week Mdr2−/− livers, while histological evidence of liver injury did not become apparent until these Mdr2−/− mice were 6-weeks of age. This chronological data clearly indicated that a hepatic inflammatory response precedes in time the development of liver injury, thus directly supporting the concept that it is the cytokine induced inflammatory response that first results in cholestatic liver injury.Our interest in the mechanism of bile acid induced liver injury came about by observations we obtained concerning the evolution and progression of cholestatic liver injury in the . First, we challenged Ccl2−/− mice and their wild-type controls with 1% cholic acid in their diet for 7 days. Significantly lower levels of plasma bile acids and alanine aminotransferase (ALT) were found in Ccl2−/− livers than in the wild-type controls, suggesting that deletion of the chemokine Ccl2 protects mice from cholestatic liver injury presumably by muting the inflammatory response. To confirm these results, we examined the effects of 7-day bile duct ligation in these Ccl2−/− mice. Assessment of liver histology revealed marked necrosis in the wild-type livers, that was nearly absent in Ccl2−/− livers. Plasma ALT in wild-type mice was 3 times the levels seen in Ccl2−/− mice, despite similar levels of plasma bile acids in both genotypes. FACS analysis and immunohistochemistry demonstrated significantly less infiltrates of neutrophils and T-cells but not macrophages in the livers from Ccl2−/− mice when compared to the wild-type control. Together, these findings confirmed that inflammatory chemokines play a key role in bile acid induced liver injury where neutrophils and T-cells may mediate these events.In our recent study, summarized below, we more directly assessed the role of proinflammatory chemokines in cholestatic liver injury To further investigate the mechanism of this bile acid induced inflammatory response in the liver, we first isolated liver parenchymal and non-parenchymal cells from the mice and treated them with the major species of endogenous bile acids for various times. Only hepatocytes were induced to stimulate the expression of proinflammatory chemokines  while bile acid concentrations of TCA (up to 200 μM) had no effect on the non-parenchymal cell fraction or on isolated cholangiocytes, whereas all of these cells had a cytokine response to LPS treatment. The induction of chemokines in hepatocytes was both dose- and time-dependent. Even at concentrations as low as 25 μM, albeit a pathophysiological relevant concentration, 24 h treatment of TCA significantly induced Ccl2 and Cxcl2 mRNA expression. Similar studies in human hepatocytes confirmed these observations. Further functional cell migration assays in transwell cultures, demonstrated that chemokines released into the culture medium from bile acid treated hepatocytes but not non-parenchymal cells were able to stimulate neutrophil chemotaxis. These observations confirmed that only hepatocytes but not other cells in the liver initiate an inflammatory response when exposed to pathophysiological relevant levels of bile acids.Ntcp(Slc10a1)−/− mice, where extremely high levels of bile acids were found in the plasma but both the patient and the Ntcp null mice were completely protected from cholestatic liver injury , 18. Because NTCP/Ntcp is a hepatocyte specific protein in humans and rodents, this explains why the other hepatic non-parenchymal cells are more resistant to bile acid induced tissue injury. These findings also argue against a role for a plasma membrane receptor in the hepatocyte in mediating this cascade of events .To better understand why bile acids stimulated the expression of chemokines in hepatocytes but not other liver cells, we examined the role of the bile acid uptake transporter, Ntcp/Slc10a1. We found that bile acid induction of the chemokine Cxcl2 was significantly diminished when Ntcp expression was knocked down by siRNAs or when bile acid uptake was blocked by a newly synthesized Ntcp inhibitor in these cells. These findings indicated that bile acids had to be transported into the hepatocyte in order to initiate the synthesis of these cytokines. This conclusion is also consistent with observations from a rare case of an NTCP-deficient patient and also in , 20. However, we did not detect ROS in these cells and the antioxidants N-Acetyl Cysteine or Mito-Q had little effect on repressing bile acid induction of Cxcl2. Alternatively, because bile acids caused mitochondrial damage, we speculated that toll-like receptor 9 (Tlr9) might be activated. As anticipated, bile acid induction of chemokine Cxcl2 was significantly diminished in Tlr9−/− mouse hepatocytes. Similar results were also obtained in hepatocytes from the MyD88/Trif double knock out mouse, two proteins that are down-stream signaling molecules of Tlr9. Conversely, bile acids and the Tlr9 agonist synergistically stimulated Cxcl2 expression in mouse hepatocytes. Most importantly, liver injury was also reduced in hepatic specific Tlr9-deficient mice although not eliminated. Therefore, we speculate that there are likely to be additional signaling pathways involved in bile acid induction of chemokine expression in hepatocytes and further mechanistic studies will be needed.To further investigate the intracellular events caused by bile acid accumulation in hepatocytes and also to better elucidate the signaling pathways involved in the induction of chemokines, we found that elevated hepatic levels of bile acid caused endoplasmic reticulum stress and mitochondria damage, evidenced by the organelle specific proteins leaking into cytosol as well as mitochondrial membrane potential changes after bile acid treatment, findings consistent with previous reports Finally, to verify whether our findings from mouse cells and cholestatic rodent models described above were representative of the pathogenesis of cholestasis in human patients, we treated primary human hepatocytes with bile acids and also examined liver histology from patients with various cholestatic disorders. We found that GCDCA, the major endogenous bile acid in humans significantly induced chemokine CCL2 CCL15, CCL20, CXCL1 and IL-8 mRNA expression after exposure to 50 μM, a pathophysiological relevant concentration. We also found that serum ALT levels in cholestatic patients, measured around the time of obtaining liver tissue, positively correlated with the degree of periportal neutrophil infiltration in these biopsies. These findings are consistent with our rodent studies and emphasize the importance of the bile acid induced inflammatory response in the pathogenesis of cholestatic liver disease in man.In summary, recent findings from our research and that of others indicate that a hepatocyte initiated inflammatory response plays a key role in bile acid induced cholestatic liver injury. Indeed, these studies point to several new targets for treatment of cholestasis since reducing the bile acid pool size, blocking bile acid uptake into hepatocytes, minimizing mitochondrial injury, and or blocking cytokine production or its inflammatory response are all distinct approaches that might reduce cholestatic liver injury. These findings emphasize that future therapeutic breakthroughs in this field may need to explore blocking multiple targets that are involved in the pathogenesis of this disorder as described in this report.


Because of the importance of their function, AMPAR dynamics, activity and subunit composition undergo a tight regulation which begins as early as prenatal development and continues through adulthood. Accumulating evidence suggests that the precise regulatory mechanisms involved in orchestrating AMPAR trafficking are challenged in the aging brain. In turn dysregulation of AMPARs can be linked to most neurological and neurodegenerative disorders. Understanding the mechanisms that govern AMPAR signaling during natural and pathological cognitive decline will guide the efforts to develop most effective ways to tackle neurodegenerative diseases which are one of the primary burdens afflicting an increasingly aging population. In this review, I provide a brief overview of the molecular mechanisms involved in AMPAR trafficking highlighting what is currently known about how these processes change with age and disease. As a particularly well-studied example of AMPAR dysfunction in pathological aging I focus in Alzheimer’s disease (AD) with special emphasis in how the production of neurofibrillary tangles (NFTs) and amyloid-β plaques may contribute to disruption in AMPAR function. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamate-gated channels that mediate most fast excitatory transmission in the central nervous system (CNS). Because of their crucial role in regulating brain function, AMPARs are under tight regulatory processes that control their biosynthesis, membrane trafficking, degradation and various post-translational modifications . In the U.S. alone, AD affects more than 5 million people with an annual cost of over 200 billionAt the molecular level, AD is characterized by the presence of two pathological metabolites: plaques formed by oligomeric clusters of amyloid-β (Aβ) and neurofibrillar tau tangles (NFTs). Aβ is a secreted proteolytic derivative of the amyloid precursor protein (APP) which has been linked to early deficits in AD pathogenesis  that triggers AMPAR mobilization. A prominent example of this form of plasticity is long-term potentiation (LTP). LTP is elicited in response to high frequency stimulation for brief periods of time (in the order of seconds) sufficient to activate NMDARs initiating a calcium-dependent mechanism that recruits synaptic AMPARs thereby increasing synaptic efficacy. Conversely, low frequency stimulation for several minutes leads to the removal of synaptic AMPARs to produce a long-sustained decrease in synaptic strength known as long-term depression (LTD). Both forms of NMDAR-dependent plasticity have been proposed to underlie important functions like information processing, learning and memory  with GluA1/GluA2  and GluA2/GluA3 heteromers being the predominant conformation  since blockade of the GluA2-NSF interaction results in a rapid run-down of AMPAR-mediated currents  by protein kinase A (PKA) also increases the peak conductance and open probability of the channel  serve as auxiliary subunits that regulate channel activity and synaptic stabilization (Bats et al., TARPs such as stargazin and γ5 are key regulators of AMPAR biophysical properties by reducing the sensitivity to polyamines of GluA2-lacking receptors (Soto et al., Before deepening into AD pathology, it is important to consider how the molecular processes that govern AMPAR-mediated transmission are affected during healthy aging. An older brain often exhibits mild defects in cognitive capabilities such as spatial and working memory with usually harmless consequences. This natural cognitive decline does not seem to be directly connected to significant neuronal loss which only seldom occurs in healthy elderly, but rather depends on the reorganization of synaptic structure (recently reviewed in Morrison and Baxter, To date, work focused in the events underlying healthy aging are limited to just a few studies tangentially addressing AMPAR trafficking by the means of analyzing synaptic plasticity. The primary findings of these studies conclude that in general LTP in older animals is less robust and requires stronger stimulation protocols to be elicited (Moore et al., A surge of instability at the postsynaptic density may have negative repercussions not only for synaptic plasticity but also for maintaining stable synaptic strength. In fact, overall AMPAR hypofunction in aged subjects is suspected however it is uncertain whether this is a consequence of a decrease in synaptic AMPARs or an increase of modified receptors with reduced stability at synapses and/or conductivity. Two recent studies in aged rats showed that the administration of positive allosteric modulators of AMPARs had a beneficial effect on restoring age-related memory and synaptic potentiation deficits (Bloss et al., The aforementioned scenario suggests two alternative hypotheses. One theory could postulate that aging alters presynaptic function thereby reducing glutamate release. A decrease in glutamate exocytosis would particularly affect AMPARs which bind to the endogenous agonist with much lower affinity than other glutamate receptors such as NMDARs (Edmonds et al., A second scenario could predict an increase of modified AMPARs which are either more labile at synapses or with reduced net conductivity. In this situation the administration of stimulants may overcome their loss of function, at least transiently. As discussed previously, increased instability and reduced conductivity can be triggered by altering AMPAR phosphorylation and/or by modifying their subunit composition. Currently, there are no reports assessing AMPAR phosphorylation in animal models of normal aging however a study by Hara et al.  suggesteIn the pathology of AD, neuronal function is compromised in two fronts: extracellularly by the accumulation of secreted Aβ oligomers and intracellularly by deposits of aberrant tau protein. However it is possible that neurons may not have to face these challenges simultaneously. Cellular and animal models indicate that Aβ accumulation precedes NFTs formation (Hardy and Selkoe, Aβ induces synaptic aberrations by altering the morphology and composition of synapses that lead to significant dendritic spines loss (Lacor et al., In accordance with synaptic and cognition decline, LTP is impaired in animal models of AD and in response to acute administration of Aβ (Walsh et al., Intriguingly, the presence of Aβ plaques do not necessarily lead to neurodegeneration (Mintun et al., Interestingly, Aβ oligomers have been shown to preferentially target different AMPAR subunits (Zhao et al., By inducing the selective endocytosis of GluA2/3s, Aβ could trigger synaptic depression and reduce synapse stability. Thus it seems reasonable to speculate that synapses which reduced levels of GluA2/3-containing AMPARs may be protected from Aβ toxicity. In fact, a recent screening for gene-expression profiles associated with mild cognitive impairment found GluA3 among the genes that showed a strong negative correlation with neurodegeneration (Berchtold et al., in vitro and in vivo models. To complicate things further, in recent years there has been a growing appreciation that NFTs may be less efficient in disrupting neuronal function than soluble tau (de Calignon et al., in vivo microscopy in an animal model of taupathy (rTg4510; Santacruz et al., Although the role of tau in disrupting AMPAR trafficking has been less studied than the effect of oligomeric Aβ, there is substantial evidence of synaptic abnormalities associated with tau-mediated signaling with numerous studies exposing that AMPARs appear particularly vulnerable to tau pathology. These body of work described a significant loss of surface receptors in cultured neurons treated with mutated tau proteins (Hoover et al., As aforementioned, there is ample evidence indicating that synaptic strength is altered in models of tau abnormalities (D’Amelio et al., But how a microtubule-associated protein, mostly located at presynaptic compartments may affect postsynaptic organization and dynamics? A potential explanation may lay in recent findings showing that tau can be localized not only in axonal microtubules but also at postsynaptic densities, albeit at much lower levels (Ittner et al., Although phosphorylated tau is usually considered potentially neurotoxic, recent evidence suggests a critical role of phosphorylated tau in maintaining normal synaptic transmission and plasticity. Thereby, using biochemical and electrophysiological assays, Regan et al. , have shIn addition to phosphorylation, tau acetylation is emerging as an interesting target of tau regulation during pathological aging as abnormal acetylation of tau lysines K274 and K281 have been associated with dementia in AD (Tracy et al., Aggregates of Aβ oligomers and NFTs are hallmarks of AD, however it remains unknown how extracellular Aβ and intracellular NFTs trigger synaptic dysfunction. Do they employ different mechanisms or share common pathways to accelerate synaptic decline? There are also questions regarding the time-course of AD progression. Since pathological effects associated with plaques usually appear earlier (Hardy and Selkoe, Despite the answer to these questions remains largely unknown, there is ample evidence of a causal association between oligomeric Aβ and tau (Rapoport et al., Regulation of tau by Aβ-mediated NMDARs activation entails that common signaling pathways linked to these receptors may be implicated in facilitating the pathological effects of both molecules. In agreement, GSK3β regulates both tau phosphorylation and Aβ production and its inhibition prevents Aβ-induced impairment of LTP and improve AD pathology (Shipton et al., The dynamic regulation of AMPARs is a crucial factor in supporting baseline synaptic transmission and plasticity. Thus AMPAR dysregulation underlies synaptic decay during natural and pathological aging. Over the last years, great progress has been made in understanding how AMPAR signaling is affected during neurodegenerative diseases such as AD in which both NFTs and Aβ disrupt glutamate receptors and their downstream pathways. Interestingly, several findings indicate that selective dysregulation of GluA2/3-containing AMPARs may be an important target during both natural and pathological aging. Removal of GluA2 has profound consequences in trafficking and AMPAR calcium permeability resulting in an increase of calcium influx which may contribute to neuronal excitotoxicity. Selective targeting of GluA3 and GluA2-containing AMPARs during pathological conditions suggests the intriguing possibility that the molecular mechanisms involved in their regulation may be the foundation of novel therapies for neurodegenerative diseases. In this regard, personalized medicine catered to various genetic backgrounds may be helpful to identify the potential underlying causes of synaptic malfunction in a case-by-case basis. In order to accomplish this, a better understanding of the molecular underpinnings involved in AMPAR trafficking in healthy synapses will be crucial.SJ prepared the figures, wrote and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.