The polymorphisms involved in drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1 CRF_BC, the most prevalent HIV-1 strain in China, have been poorly characterized.pol region of HIV-1 CRF_BC from 631 treatment-naïve and 363 treatment-experienced patients using the selection pressure-based method. We calculated an individual Ka/Ks value for each specific amino acid mutation. Result showed that eight polymorphic mutations  in RT for treatment-experienced patients were identified, while they, except for R135L, were completely absent in those from treatment-naïve patients. The I132L and T139K/R mutants exhibited high-level resistance to DLV and NVP and moderate resistance to TMC-125 and EFV, while the K101Q and H221Y mutants exhibited an increased resistance to all four NNRTIs tested. The W88C, R135L, and L228R may be RTI-induced adaptive mutations. Y181C+K101Q mutant showed a 2.5-, 4.4-, and 4.7-fold higher resistance to TMC-125, NVP and EFV, respectively, than Y181C alone mutant, while Y181C+H221Y or K103N+H221Y mutants had significantly higher resistance to all four NNRTIs than Y181C or K103N mutants. K103N+T139K and G190A+T139K mutant induce higher resistance  to all four NNRTIs than K103N or G190A alone mutation.To reveal the drug resistance mutations, we compared the gene sequences of I132L and T139K/R are rare but critical mutations associated with NNRTI-resistance for some NNRTIs. K101Q, H221Y and T139K can enhance K103N/Y181C/G190A-assocated NNRTI-resistance. Monitoring these mutations will provide useful information for rational design of the NNRTI-based antiretroviral regimen for HIV-1 CRF_BC-infected patients. Human immunodeficiency virus type 1 (HIV-1) has been categorized into nine genetically distinct subtypes within the M group, including subtypes A, B, C, D, F, G, H, J, and K. Recombination between genomes of two viruses of different subtypes results in generation of a circulating recombinant form (CRF) pol region have been poorly characterized. Particularly, the mutation sites associated with NNRTI-resistance in RT of HIV-1 CRF_BC viruses have not been reported Rapid evolution and high mutation rate of HIV allow the virus to gain the ability of drug resistance. It is possible that HIV-1 genetic diversity may influence the type of resistance mutations that might eventually emerge upon drug exposure as well as the rate of emergence of resistance pol region of HIV-1 CRF_BC isolated from treatment–naïve and experienced patients, and then conducted the selection pressure analysis to identify rare but critical sites of mutations potentially associated with NNRTI-resistance. The association was further confirmed by using infectious clones with or without the newly identified mutations.In the present study, we compared the gene sequence of pol sequences of the viruses obtained from plasma samples of the HIV-1-infected patients using PCR technique. The ART-experienced patients were receiving highly active antiretroviral therapy, including 2 NRTIs and 1 NNRTI. The NRTIs are lamivudine(3TC) plus zidovudine(AZT) or stavudine(d4T), while the NNRTI is either nevirapine(NVP) or efavirenz(EFV). Specifically, 13.5% of the patients had been treated with 3TC/AZT/EFV, 6.1% with 3TC/d4T/EFV, 58.7% with 3TC/AZT/NVP, 15.7% with 3TC/d4T/NVP, and 6.1% with unknown regimen. The mean treatment time was 18 months, including 28.0% for 0–6 months, 11.0% for 7–12 months, 23.1% for 13–18 months, 13.5% for 19–24 months, 17.9% for >24 months and 6.1% for unknown time.This study involved 994 HIV-1-positive patients, including 631 treatment-naïve patients  and 363 ART-treated patients . All the patients were identified to be infected by HIV-1 CRF_BC as determined by Neighbor-joining genetic analysis of P = 0.0012, fisher exact test).We used the selection pressure-based method, an important way to explore the rare but critical sites of drug resistance P<0.05), and H221Y mutant had a moderate increase (approximately 2-fold) of resistance to these four NNRTIs (P<0.05), while the W88C, R135L and L228R mutations had no significant effect on the viral resistance to RTIs , Rescriptor (DLV), Viramune (NVP), and Sustiva (EFV), were determined. CRF_BC strains with K103N and Y181C in RT were included as controls. The K101Q, I132L and T139K/R mutants exhibited significant (2∼28-fold) increases in resistance to all the four NNRTIs tested  is greater than 1 and LOD is greater than 2, the influence of X to Y (X→Y) was considered significant. Then, software Cytoscape was used to construct the relationship among these predicted drug resistance mutations as reported The network represents the comprehensive relationship among the predicted drug-resistance mutations, and the arrows from the source node to the target node indicate the influence of one to another. In the network, the size of the node represents the mutation frequency of that site from one amino acid to another, while the width of line represents the influence strength between two mutations. As shown in The mutation T139K may be induced by other mutations, including K103N, Y181C, M184V and G190A, and the selection pressure ratio from G190A to T139K reach to 7. Notably, T139K mutation had a significant influence on G190A, indicating a correlation between the two mutations. The mutual influence between T139K and G190A hints that these two mutations may form as a mutation pattern to function synergistically. Interestingly, H221Y was associated with Y181C and/or K103N mutations. For example, K103N, Y181C and H221Y are three mutations formed by pairwise interactions. Y181C and H221Y, in particular, have strong mutual influence , suggesting that H221Y and Y181C may form combinatorial mutation patterns to synergistically resist the drug treatment.pol with mutations Y181C, G190A or K103N was constructed through site-directed mutagenesis with or without the newly identified mutations in this study. We tested the phenotypic resistance of these combinations Y181C, G190A or K103N with different mutation sites in RT of HIV-1 CRF_BC to NNRTIs using an in vitro phenotypic assay. As shown in P<0.05). Y181C+H221Y mutations resulted in significantly higher resistance to all four NNRTIs than Y181C alone mutation, ranging in 3.00∼4.24 FC (P<0.05). K103N+T139K mutant induce higher resistance to all four NNRTIs, with FC ranging from 2.00 to 14.15. K103N+H221Y mutations exhibited an increased (1.69- to 2.96-fold) resistance to the four NNRTIs tested (P<0.05), while K103N+K101Q mutants did not displayed a higher NNRTI-resistance than K103N alone mutant. G190A+T139K also showed a higher increased (1.48- to 7.21-fold) resistance to all four NNRTIs than G190A alone mutation.We next examined the effect of the single mutation sites listed above in combination with Y181C or K103N on viral resistance to NNRTIs. The pNL4-3 clone containing HIV-1CRF_BC Most of the current anti-HIV drugs have not been tested in the clinical trials in China, drawing attention to the effectiveness of these drugs against the HIV-1 strains circulation in China. We recently have shown that Fuzeon and Maraviroc, the only two HIV entry inhibitors approved for clinical use by the US FDA, are much less effective against the HIV-1 subtypes circulating in China than the B subtype predominating in the United States and Europe At present, the antiretroviral drugs have been used not only for treatment, but also for prevention of HIV infection/AIDS in vitro and in vivo observations suggest that the various subtypes may respond differently to NNRTIs Some pol region of HIV-1 strains isolated from 631 treatment-naïve patients and 363 ART-treated patients who were verified to be infected by HIV-1 CRF_BC. We found that the frequencies of 15 polymorphism sites in RT of CRF_BC strains isolated from the treatment-experienced patients were significantly different from those isolated from the treatment-naïve patients. Especially, seven mutations at six positions  were completely absent in the RT of CRF_BC strains isolated from drug-naïve patients. In contrast, their frequencies in strains isolated from ART-treated patients were significantly increased, suggesting their specific association with ART treatment. Since the ART regimen of these patients contained two NRTIs and one NNRTI, in vitro experiments were tested for susceptibility to 3TC, d4T, AZT, TFV. The results demonstrated that these mutations were not associated with the resistance to NRTIs  of RT, which is involved in the formation of the base of the NNRTI-binding pocket The increasing evidences suggest that in addition to those currently known mutations, more and more unidentified mutations may also be involved in the development of NNRTI resistance, which contribute to NNRTI therapy failure In summary, our data suggest that I132L and T139K/R mutations that exhibited high-level resistance to NNRTIs are the rare but critical mutants associated with NNRTI-resistance in RT of CRF_BC strains that are predominantly circulating in China, while K101Q and H221Y mutations are associated with the increased resistance to all the four NNRTIs tested, although at codons 101 and 221 were reported relating to NNRTI resistance. The co-presence of H221Y, T139K or K101Q with the well-known RTI-resistance mutations K103N, G190A or Y181C may strengthen the drug-resistance effect. Further study is needed to determine how these mutations and combined mutations affect the binding kinetics of NNRTIs. We suggest that these newly identified mutations should be considered for the improvement of algorithms that predict clinical responses to antiretroviral drugs and for assessing the efficacies of next-generation drugs. This information will aid in designing initial treatment strategies for persons infected with CRF_BC viruses and interpreting genetic resistance among the CRF_BC-infected patients whose antiretroviral therapy has failed.http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html). Furthermore, to confirm these sequences, we conducted a Neighbor-joining genetic analysis of pol sequences obtained from plasma samples of all HIV-1-infected patients using the PCR technique as previously described The study population included pre-selected HIV-1-positive patients with treatment-naïve and experienced antiretroviral therapies, who participated in a multicenter AIDS Cohort Study including China Global Fund AIDS Program, and “Eleven Five” major projects in Xinjiang and Sichuan provinces of China during 2007–2011. The individuals who newly HIV-infection screened and confirmed were investigated without experiencing ART were chosen as the treatment- naïve patients in Xinjiang and Sichuan province of China during that time. The HIV/AIDS patients who received ART with 2 NRTIs and 1 NNRTIs regimen in the two provinces were investigated to detect viral load and CD4 count periodically. When the patients encountered virological failure during ART according to WHO ARV therapy failure criteria  pol sequence was carried out by an in-house polymerase chain reaction protocol as previously described CTGTATTTCAGCTATCAAGTCTTTTG ATGGG). A nested PCR was then employed using the generated cDNA as template. The nested PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen Inc) and sequenced with the ABI 3100 DNA Sequencer.HIV YaXa is the number of samples with the same amino acid mutation both at site Y and X; and NYsXa is the number of samples with a synonymous mutation at codon Y and an amino acid mutation at codon X. NYaXo and NYsXo are the number of samples with the amino acid mutation and a synonymous mutation at codon Y in the absence of any mutation at X respectively.The Ka/Ks values for specific amino acid substitutions were determined as described by Chen et al YaXa+NYsXa and q as defined above.The LOD score by which we evaluated the significance of apparent amino acid pairs was calculated using the following formula:If LOD>2, the positive selection is significant.GGTCACCAAATGAAAGATTGTACTGAGAGGGAA and ACCGGTTCTTTTAG AATCTCCCTGTTTTCTGCCTGT) were used for second-round PCR, which underlined sequences mark the relevant restriction sites. The nested PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen Inc), digested with BstE II and AgeI (NEB) and then ligated to BstE II - and AgeI-digested pNL4-3. The mutations were introduced into CBJB257 RT regions inserted in T-vector by using site-directed mutagenesis with DNA polymerase  and site mutation primers. DNA sequencing was performed in both directions across the entire RT-coding region to verify the absence of spurious mutations and the presence of the desired mutation. It should be noted that the cloned fragment of CRF_BC RT encompass just about 300 aminos of N-terminus. Although the mutations of the other region in RT may enhance resistance to HIV drugs, such as some mutations in the connection domain, such situation should be ruled out because pNL4-3 was wild type reference strain without such mutations.The infectious molecular clone was constructed by incorporating amplified PR and RT regions of CRF_BC into pNL4-3 using BstE II and Age I restriction sites after BstE II at position 2049 (RT region) of pNL4-3 was created by replacing A with T. HIV-1 CRF_BC (CBJB257), which was isolated from treatment-naïve intravenous drug user in Xinjiang, China 50) and the antiviral activity of NNRTIs were determined using TZM-b1 cells as previously described HIV-1 (HIV-1WT) and HIV-1 with the mutations (HIV-1MT) were generated by transfection of the plasmids into 293T/17 cells by using Fugene 6 Transfection Reagent (Roche Applied Science) according to the manufacturer's instructions. The 50% tissue culture infectious dose Click here for additional data file.Table S2Sensitivity and resistance of different mutation sites in HIV-1 CRF_BC RT region to NRTIs.(DOC)Click here for additional data file.


The clonal efficiency of MADM is intrinsically low because it relies on Cre/loxP-mediated mitotic recombination between two homologous chromosomes rather than within the same chromosome, as in the case of conditional knockout (CKO). Although sparse labeling enhances in vivo resolution, the original MADM labels too few or even no cells when a low-expressing Cre transgene is used or a small population of cells is studied. Recently, we described the usage of a new system, MADM-ML, which contains three mutually exclusive, self-recognizing loxP variant sites as opposed to a single loxP site present in the original MADM system (referred to as MADM-SL in this paper). Here we carefully compared the recombination efficiency between MADM-SL and MADM-ML using the same Cre transgene, and found that the new system labels significantly more cells than the original system does. When we established mouse medulloblastoma models with both the original and the new MADM systems, we found that, while the MADM-SL model suffered from varied tumor progression and incomplete penetrance, the MADM-ML model had consistent tumor progression and full penetrance of tumor formation. Therefore MADM-ML, with its higher recombination efficiency, will broaden the applicability of MADM for studying many biological questions including normal development and disease modeling at cellular resolution in vivo.Mosaic Analysis with Double Markers (MADM) is a mouse genetic system that allows simultaneous gene knockout and fluorescent labeling of sparse, clonally-related cells within an otherwise normal mouse, thereby circumventing embryonic lethality problems and providing single-cell resolution for phenotypic analysis  By design, MADM generates a pair of GFP and RFP labeled daughter cells from a colorless progenitor cell via Cre/loxP-mediated mitotic recombination between two homologous chromosomes followed by G2-X type chromatid segregation  nor change the protein concentration of Cre recombinase in a given transgenic line, the only way to enhance the reaction rate relies on introducing more loxP sites into each chromosome. A string of wildtype loxP sites would not achieve this goal since they could recombine with each other hipp11 locus, MGI:5461148  transgene . We chos For quantification analysis, we collected cerebella at two ages, P5  when the majority of GNPs are actively proliferating in the EGL, and P14 when most GNPs migrate into the IGL and differentiate into granule neurons. All mice analyzed were heterozygous for the Math1-Cre transgene so any differences in recombination efficiency would be attributed to the number of loxP sites rather than the varied level of Cre expression. Based on the developmental process, we counted all labeled cells within the appropriate cerebellar region according to the age of samples, EGL for P5 and IGL for P14. To establish a reliable counting scheme, we carefully examined many brain sections at these ages to look for regions of consistent labeling, and chose the EGL at the base of the sulcus between the fifth and sixth folia in P5 brains , suggest The increased recombination efficiency in MADM-ML could be explained by two hypotheses. The first hypothesis is that the presence of multiple loxP sites increases the local Cre concentration at the steady state, thereby enhancing recombination efficiency . The sec To test these hypotheses, we compared the recombination efficiency in three scenarios: two alleles of single loxP (SL/SL), one allele of multiple loxP and one allele of single loxP mice (ML/SL), and two alleles of multiple loxP (ML/ML). Because the data clearly showed that there is no significant difference in labeling efficiency between SL/SL and ML/SL  have greatly increased incidences of medulloblastoma before age 5 , the, the1], in vivo, we envision that in the future, each of the mutant alleles generated by KOMP could be analyzed using the MADM system. With increased chromosomal coverage, optimization of recombination efficiency and the capacity for both gene knockout and overexpression, MADM should have ever-increasing impact on many fields of biology. Recent efforts in genome sequencing and human genetics have identified a number of mutations as candidate contributing factors to a variety of human diseases ,26. To fAll mice were housed and used in strict compliance with the approved protocol 11-09 by the Institutional Animal Care And Use Committee at the University of Oregon. The multiple-lox MADM11 mice will be available from The Jackson Laboratory:  GT11ML (JAX stock number 022976) and TG11ML (JAX stock number 022977).  The Jackson Laboratory also has Math1-Cre transgenic (JAX stock number 011104), Ptc-null (JAX stock number 003081), p53-null (JAX stock number 002101) and both single-lox GT11 and TG11 (JAX stock number 013749 and 013751).GATCTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATAACTTCGTATA andWe had two important technical considerations in designing the MADM-ML targeting cassette from the MADM-SL cassette. First, there is a risk of illegitimate recombination between WT loxP and lox variants. Although Lee and Saito (1998) showed that such events rarely occur, an illegitimate recombination would create a new loxP variant that can’t recombine with any lox sites. Considering the risk, we decided to flank the added lox variant sites with FRT so we could remove them with Flippase transgene if that would become a problem. FRT sites can’t be recognized by Cre so won’t interfere with our experiments. The final configuration of multi-lox string in the MADM-ML system is FRT-lox5171-lox2272-FRT-loxP. Secondly it was difficult to synthesize two long oligonucleotide primers that encompass FRT and lox variants. Therefore, we designed two pairs of shorter primers that can be annealed and ligated together into the original MADM-SL cassette cut with Bgl II to make the MADM-ML cassette. All these primers are phosphorylated at the 5’ end for the ligation reaction to occur. The sequences of first pair of primers are ATGTGTACTATACGAAGTTATGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGA  The sequences of the second pair of primers are GTACACATTATACGAAGTTATATAACTTCGTATAGGATACTTTATACGAAGTTATGandGATCCATAACTTCGTATAAAGTATCCTATACGAAGTTATATAACTTCGTATAFRT, lox5171, lox2272, FRT, loxP) AGATCTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCThe resulting sequence is Just for comparison, the equivalent site in the original MADM-SL is , multiple loxP (ML/ML), and mixed (ML/SL). For the SL/SL group, we generated TG11/GT11; Math1-Cre mice from the crosses of TG11/TG11; Math1-Cre/Math1-Cre with GT11/GT11 mice. For the ML/ML group, we generated TG11ML/GT11ML; Math1-Cre mice from the crosses of TG11ML/TG11ML with GT11ML/GT11ML; Math1-Cre/Math1-Cre mice. For the ML/SL group, we generated TG11/GT11ML; Math1-Cre mice from the crosses of TG11/TG11; Math1-Cre/Math1-Cre with GT11ML/GT11ML mice. P14 analysis was performed on for the SL and ML groups.p53KO/TG11; Math1-Cre/Math1-Cre with GT11/GT11; Math1-Cre/Math1-Cre; Ptc-KO/WT for the MADM-SL model; and TG11ML,p53KO/TG11ML; Math1-Cre/Math1-Cre with GT11ML/GT11ML; Math1-Cre/Math1-Cre; Ptc-KO/WT for the MADM-ML model. Mice used for tumor latency and penetrance analysis were sacrificed for time course sampling, because they showed signs of illness, or for other experimental reasons. Tumor size data reported in  For analysis of latency and penetrance of medulloblastoma model, we used mice derived from the cross of TG11,Ptc-KO/WT; p53flox/p53flox; Math1-Cre; ROSA26-floxed stop-tdTomato from the cross of Ptc-KO/WT; p53flox/p53flox; ROSA26-floxed stop-tdTomato with p53flox/p53flox; Math1-Cre. We also used a CKO model to compare with the MADM-based medulloblastoma model, with the genotype of P5 brains were collected and directly fixed in ice-cold 4% paraformaldehyde for 48hrs. After three 10 min washes with 1xPBS, brains were incubated in 30% Sucrose at 4°C for 24 hrs and then embedded in Tissue Tek OCT embedding medium and stored at -80°C. P14 brains were collected after perfusion with 4% paraformaldehyde, and then processed the same as P5 brains, except for 24hr post-fix. Embedded brains were cryosectioned sagittally at 30 μm thickness, sequentially collected, 5-7 slices per slide, and five slides per brain. Sectioned slices were stained with chicken anti-GFP  and goat anti-myc [RFP/tdTomato is myc-tagged]  in PBT (PBS with 0.3% Triton-100) with 5% donkey normal serum. After multiple washing with PBT, slices were stained with secondary antibodies, including donkey anti-chicken-Cy2  and donkey anti-goat-Alexa 555 . Brains for tumor analysis were collected and processed with the same procedure as P14 brains. In addition to the anti-GFP and anti-myc antibodies, some tumors were stained with rabbit anti-Ki67  and mouse anti-NeuN . After multiple washing with PBT, slices were stained with secondary antibodies used above, plus either donkey anti-rabbit-Alexa647or donkey anti-mouse-Alexa647  to detect Ki67 or NeuN staining, respectively.Immuno-stained sections were examined and photographed using an Olympus FV-1000 upright laser confocal microscope. Images were taken using the 20x objective, line sequential and kalman at 512x512 pixels. P5 brains were analyzed in a defined square, (10x10cm as viewed on a computer screen) between the fifth and sixth folia at the base of the sulcus, and only cells in the EGL were counted. P14 brains were analyzed in a defined square (5x5cm as viewed on a computer screen) at the base of the third folia, and only cells in the IGL were counted. To ensure systematic sampling, slices at three distinct medial to lateral planes from each brain were used for cell counting. For gross tumor imaging, half brains cut sagittally were imaged with an Olympus MVX-10 macroscope for tumor size assessment.Statistical analysis was performed using MatLab software, GraphPad Prism and Microsoft Excel. Cell counts from all three slices of each brain were summed for each data point. P5 data were analyzed using one-way ANOVA, and Multiple Comparisons using the Tukey HSD test . The average total labeled cells  counted for SL/SL=86.29, ML/ML=831.22, and ML/SL=48.94. P14 data were analyzed using an independent samples t-test with unequal variance (n=5 for SL/SL and n=7 for ML/ML groups). The average total labeled cells counted for SL/SL=317.6 and ML/ML=880.62.Figure S1Potential uses for MADM. (A) Illustration of the application of MADM for studying cell-autonomous gene functions in cellular morphogenesis. Illustrated is a hypothetical example of how MADM could reveal the subcellular details of dendritic morphology to demonstrate the role of a given gene in dendritic branch formation. (B) Illustration of how MADM can be used to trace lineages and study the fate of both green and red cell lineages. Hypothetically, in a tissue consisting of two cell types of unknown lineage relationship, MADM can determine if the two cell types are generated directly from a multipotent stem cell or indirectly from unipotent intermediate progenitor cells. If the pattern shown were observed, one would conclude that the latter is true.(TIF)Click here for additional data file.Figure S2Math1-Cre labels a small fraction of GNPs with MADM. At P5, Math1-Cre labels almost all GNPs with a floxed-stop tdTomato Cre reporter . However, the labeling of MADM with Math1-Cre is very sparse (right panel). Scale bar: 20um.(TIF)Click here for additional data file.


There are three classes of HIV-1 based on diversity of the viral envelope: M (major), O (outlying) and N (new). The M group is subclassified into nine major subtypes including A—D, F—H, J and K, as well as several recombinant forms. There is a growing need to evaluate antiretroviral (ARV) treatment in these diverse subtypes.env gene for this post hoc analysis.M06-802 was an open-label, global, 48-week phase III trial. 599 ARV-experienced, HIV-1-infected subjects were randomized 1:1 to receive LPV/r 800/200mg QD or 400/100mg BID with ≥2 investigator-selected nucleoside/nucleotide reverse transcriptase inhibitors. Classification of HIV-1 viruses by subtype was determined by analyzing sequences of the P=0.465) and non-B subtype subjects   or non-B  subjects; however, subtype B subjects were more likely to have previously been treated with PIs.Of the 595 subjects with available sequence data, 386 (65%) were infected with subtype B, 121 (20%) were infected with subtype C, 88 (15%) were infected with another subtype. Subjects infected with subtype B were more likely to be male (75%), white (56%), and living in North America (N.A.) (52%), while subjects infected with subtype C were more likely to be female (56%), black (83%), and living outside of N.A. or Western Europe (W.E.) (99%). Subjects infected with other subtypes were more likely to be male (56%), white (88%) and living outside of N.A. or W.E. (82%). The proportion of subjects with subtype B responding to treatment at week 48 as analyzed using the FDA-TLOVR method (53%) was similar to that of subtype C subjects (57%, LPV/r demonstrated similar efficacy and safety among subjects infected with different HIV-1 subtypes.


The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H2-utilizing Methanosarcina acetivorans from H2-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of Hc that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c and MP in the energy-conserving electron transport pathway of non-H2-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.The results support roles for ferredoxin, cytochrome  The CO-dependent reduction of ferredoxin with CdhAE was not used to avoid binding of CO to high spin heme in cytochrome c and potentially inhibiting membrane-bound electron transport. The NADPH:CoM-S-S-CoB oxidoreductase activity was monitored by detecting the sulfhydryl groups of HS-CoM and HS-CoB  cleaves the C-C and C-S bonds of acetyl-CoA releasing a methyl group and CO that is oxidized to CO2 with electrons transferred to ferredoxin. The CdhAE component of M. acetivorans was isolated independently from the other subunits and both copies encoded in the genome were represented. Although it was not possible to determine which CdhAE component reduced ferredoxin, the high percent identities  suggests it is the electron acceptor for either or both copies. In both pathways, ferredoxin is the electron donor to a membrane-bound electron transport chain that terminates with MP donating electrons to the heterodisulfide reductase HdrDE that catalyzes the reduction of CoB-S-S-CoM. Proteomic and genetic evidence  ferredoxins, the one purified from M. acetivorans is most closely related to that isolated from acetate-grown M. thermophila [Methanosarcina species.Of the nine putative 2 × [4Fe-4S] ferredoxins annotated for the genome of rmophila , a resulMethanosaeta thermophila [2 suggesting still other alternative electron transport pathways coupled to generation of ion gradients driving ATP synthesis in acetate-utilizing methanogens. The physiological significance of these diverse electron transport pathways is yet to be determined; however, it has been suggested that avoiding H2 is advantageous to the marine isolate M. acetivorans since sulfate reducing species that dominate this environment outcompete methanogens for H2 potentially disrupting electron transport [M. acetivorans is incapable of growth with H2/CO2 it synthesizes all of the enzymes necessary for reduction of CO2 to methane and is capable of robust growth via the CO2-reduction pathway albeit with electrons derived from the oxidation of CO [Interestingly, genes encoding subunits of Ma-Rnf or Ech hydrogenase are absent in the genome of the acetate-utilizing isolate rmophila  that is ransport . It is ion of CO -36.M. thermophila is an acetotrophic Methanosarcina species incapable of metabolizing H2 [c of M. acetivorans with deduced sequence identities ranging from 86 to 98% . All other chemicals were purchased from Sigma-Aldrich or VWR International . All chromatography columns, resins and pre-packed columns were purchased from GE Healthcare .M. acetivorans [2-free and manipulations were performed anaerobically in an anaerobic chamber  containing 95% N2 and 5% H2. Frozen cells were thawed, re-suspended (1 g wet weight/ml buffer) in 50 mM MOPS buffer (pH 6.8) containing 10% (v/v) ethylene glycol and passed twice through a French pressure cell at 6.9 × 103 kPa. The lysate was centrifuged at 7,200 × g for 15 min to pellet cell debris and unbroken cells. Membranes were purified from the cell extract using a discontinuous sucrose gradient comprised of 2 ml 70% sucrose, 4 ml 30% sucrose and 1.5 ml 20% sucrose contained in 50 mM MOPS buffer (pH 6.8). A 2 ml volume of cell extract was overlaid on the gradient and centrifuged at 200,000 × g for 2 h in a Beckman type 50 Ti rotor. The brown band containing membranes at the 30% and 70% sucrose interface was collected and stored at -80°C until use.tivorans  was culttivorans  and the M. acetivorans was centrifuged at 200,000 × g for 2 h to pellet the membrane fraction. The supernatant solution (200 mg of protein in 10-ml) containing the soluble fraction was loaded onto a Q-Sepharose FF column (50 ml bed volume) equilibrated with 50 mM MOPS (pH 6.8). The column was developed with 500 ml of a 0-1.0 M NaCl linear gradient. Each 10 ml fraction was assayed for CO dehydrogenase activity by monitoring the CO-dependent reduction of methyl viologen as previously described [4)2SO4 contained in 50 mM MOPS (pH 6.8) was added to the concentrated protein solution to final concentration of 900 mM and loaded onto a Phenyl-Sepharose FF (low sub) column (20-ml bed volume) equilibrated with 50 mM MOPS (pH 6.8) containing 1.0 M (NH4)2SO4. The column was developed with 100 ml of a 1.0-0.0 M (NH4)2SO4 decreasing linear gradient. Fractions from the peak of CO dehydrogenase activity were pooled and concentrated followed by addition of a volume of 50 mM MOPS (pH 6.8) to lower the (NH4)2SO4 concentration to below 100 mM and then loaded on a HiTrap Q-Sepharose HP column (5 ml bed volume) equilibrated with 50 mM MOPS buffer (pH 6.8). The column was developed with 50 ml of a 0-1.0 M NaCl linear gradient. The peak containing CO dehydrogenase activity that eluted at approximately 0.3 M NaCl was collected and stored at -80°C until use.All purification steps and biochemical assays were performed anaerobically in the anaerobic chamber. Crude cell extract of acetate-grown escribed . The pooA320 (ε320 = 9300 M-1 cm-1) similar to that described previously [All purification steps and biochemical assays were performed anaerobically in the anaerobic chamber. Ferredoxin was assayed by the ability to couple CO oxidation by CdhAE to the reduction of metronidazole followed by the decrease in eviously . One uniM. acetivorans was loaded onto a Q-sepharose FF column (20 ml bed volume) equilibrated with 50 mM MOPS (pH 6.8) containing 10% (v/v) ethylene glycol. The column was developed with 200 ml of a 0-1.0 M linear NaCl gradient. The fraction with the highest activity was then diluted 10-fold with 50 mM MOPS (pH 6.8) containing 10% (v/v) ethylene glycol. The solution was loaded on a Mono Q column (1.7 ml bed volume) to which 10 ml of a 0-1.0 M NaCl linear gradient was applied. The fraction containing ferredoxin that eluted at 600 mM NaCl was loaded on a Sephadex G-75 gel filtration column (100 ml bed volume) and developed with 50 mM MOPS (pH 6.8) containing 10% (v/v) ethylene glycol and 150 mM NaCl. The peak containing the purified ferredoxin was concentrated to A402 > 0.2 with a Vivacell 70 protein concentrator equipped with a 5-kDa cutoff membrane and stored at -80°C until use. The protein concentration was estimated by the ratio of absorbance at 230 and 260 nm as described [The soluble fraction of cell extract from acetate-grown escribed .All protein concentrations except for ferredoxin were determined by the bicinchoninic acid assay using the reagent from Thermo Scientific, Inc.. Detection of the free sulfhydryl groups of CoM-SH and CoB-SH was performed as previously described . The bufM. thermophila have been deposited in the GenBank database under accession number JN173061, JN173062, JN173063, JN173064, JN173065, JN173066, JN173067, JN173068, JN173069, JN173070, JN173071, JN173072, JN173073, JN173074, JN173075  .The sequences of DNA encoding Rnf and Mrp of The authors declare that they have no competing interests.MW carried out the biochemical studies, participated in sequence analysis and drafted the manuscript. J-F T carried out the genomic sequencing and sequence alignments. JGF conceived of the study, participated in its design and coordination, and finalized the manuscript. All authors read and approved the final manuscript.Figure S1. UV-visible absorption spectra of purified ferredoxin. As-purified (--), dithionite reduced (...). The protein concentration was 20 μM.Click here for fileFigure S2. Phylogenetic analysis and sequence alignment of ferredoxins. The M. mazei and M. acetivorans sequences, labeled with the prefix MA, were derived from the CMR database [M. thermophila (M.t.) sequence is published [Clostridium pasteurianum is published [Spinacia oleracea ferredoxin was obtained from the NCBI database (accession number O04683). Panel A, Phylogenetic analysis of ferredoxins. The tree was constructed by the neighbor-joining method with the MEGA4 program [Methanosarcina species. Motifs predicted to ligate two 4Fe-4S clusters are highlighted. The alignment was performed with ClustalX2 [database . The M. ublished . The sequblished  and the  program . BootstrClick here for filernf genes between Methanosarcina thermophila and Methanosarcina acetivoransFigure S3. Comparison of . Panel A. Organization of rnf genes in Methanosarcina thermophila versus Methanosarcina acetivorans. Numbers next to the arrows indicate deduced sequence identity. Panel B. Alignment of the deduced sequences of rnf genes between Methanosarcina thermophila (Mt) and Methanosarcina acetivorans (Ma). Highlighted are: conserved heme binding sites (CXXCH and CXXXCH) in Cyt c, the flavin binding motif (SGAT) in RnfG, and cysteine motifs binding iron-sulfur clusters in RnfC and RnfB.Click here for filemrp gene clusters between Methanosarcina thermophila and Methanosarcina acetivoransFigure S4. Alignment of . Numbers next to the arrows indicate deduced sequence identity.Click here for file


Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight  are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the  Genetically-encoded fluorescence protein (FP) voltage probes convert voltage changes across biological membranes into optical signals . In prinCiona intestinalis voltage-sensing domain with super ecliptic pHluorin A227D, exhibited significantly improved response amplitudes (-30 to -40% ΔF/F in response to a 100mV depolarization) in mammalian cells [in vivo in the mouse olfactory bulb , mouse barrel cortex , individual Drosophila neurons [C. elegans . The recently published FP voltage probe, ArcLight, based on fusion of the an cells . Linker  neurons  and C. eCiona-based ArcLight is relatively slow compared with small molecule voltage sensitive dyes [Danio rerio) voltage-sensing domain, has fast kinetics, although its signal to noise characteristics are likely too small for use in vivo [Ciona voltage-sensing domain and Kv3.1 also exhibit fast kinetics [In some experimental situations, a voltage sensing probe with fast response kinetics would be useful. Unfortunately, the response kinetics of the ive dyes  or some ive dyes . Baker e in vivo . Two othkinetics ,18, but Ciona voltage-sensing domain in ArcLight with voltage-sensing domain orthologs from five species: chicken, frog, zebrafish, mouse and human. We found that probes utilizing the chicken or zebrafish voltage-sensing domain exhibited fast responses, and a larger response amplitude than Zahra. We demonstrated that chicken ArcLight can resolve individual action potentials in cultured neurons as well as trains of high frequency voltage steps in the HEK293 cells. The increase in response amplitude afforded by the ArcLight’s super ecliptic pHluorin A227D can be transferred to probes utilizing other voltage sensors and is capable of capturing rapid movements of the voltage sensing domain. These new probes should be useful for detecting fast and high frequency voltage changes in excitable cells.In this study, we sought to combine the large signal size afforded by super ecliptic pHluorin A227D with the speed improvements seen with voltage sensing domains from other phosphatase orthologs. We replaced the Ciona voltage-sensing domain used in the previously published ArcLight probe with voltage-sensing domain orthologs from five species: chicken , frog , zebrafish , mouse  and human . The fusion sites between the voltage-sensing domain and super ecliptic pHluorin A227D in these probes were: Q174 for the frog, Q175 for the chicken and zebrafish, Q331 for mouse and Q193 for human. The fusion sites were chosen to correspond to the fusion site of the Ciona-based ArcLight-Q239 probe , we could detect evoked action potentials in single trials in neurons expressing this probe . It is unclear why the voltage sensing domain of different species would result in reporters with different kinetics. It appears that the coupling between the voltage sensor and the fluorescent protein favors faster kinetics in some species. We previously reported that voltage sensing domain orthologs from different species have faster response kinetics . The curThe chicken and zebrafish ArcLight have similar response kinetics as Zahra, but the response amplitude is larger (-9% for chicken and -8% for zebrafish ArcLight compared with -2% for Zahra). While the mechanism of the superior signal size of the ArcLight voltage probe is unknown, the current study demonstrates that the relatively large signal resulting from the use of super-ecliptic pHluorin A227D is transferrable to other voltage probes containing voltage sensing domain orthologs from different species. Probes containing super-ecliptic pHluorin A227D produce larger signals when compared to similar probes using other FPs . In addition it demonstrates that this enhanced modulatory behavior can respond rapidly. These finding indicate the portability of this fluorescent protein and its mechanism to other probe designs. Ciona-based voltage sensitive probes have a point mutation in the S4 transmembrane region that changes arginine to glutamine (R217Q) and causes a shift of the probe’s voltage dependent fluorescence change to a more physiological range [X. laevis, R309Q for the mouse and R171Q for the human) with a similar effect. Although we cannot be sure whether this effect is due to a change in sensing per se or in the coupling between the voltage-sensing domain and the fluorescent protein [All previously published al range ,19. The  protein , a similX. laevis [The frog ArcLight used for this study is based on Xl-VSP1, one of the two voltage sensitive phosphatase isoforms found in . laevis . This prThe human ArcLight used for this study is based on Hs-TPTE, one of the two voltage sensitive phosphatase isoforms found in humans ,23. Hs-TA single FP, super ecliptic pHluorin A227D, is used in ArcLight and in the newly developed chicken ArcLight. Although FRET based FP sensors enable ratiometric processing of signals and cancelation of bleaching or movement artifacts, there are also advantages for using a single FP as the reporter. One advantage is that it reduces the size of constructs needed for the viral vector delivery . -4 and 9x10-4, respectively [Arch (Addgene Plasmid 22217) and Arch D95N (Addgene Plasmid 34616) are microbial rhodopsin based fluorescence voltage probes and feature fast response to membrane voltage changes and slow bleaching rates . Howeverectively . As a reIn the future, it may be necessary to exploit the voltage-sensing domain from more species in designing improved FP voltage sensors. It may also be useful to introduce mutations to the voltage sensitive domain, especially to the transmembrane domains in screening for probes with fast kinetics. Ciona voltage-sensing domain (R217Q) in ArcLight with voltage sensitive domains from chicken, X, laevis, zebrafish, mouse or human. The coding sequence of the voltage sensitive domains were amplified with the polymerase chain reaction (PCR) using the pfu DNA polymerase , digested with restriction enzymes  and inserted between the HindIII and BamHI sites of the ArcLight construct. An extra codon GAG was introduced immediately after the starting ATG to create an optimal Kozak consensus sequence. Primers used for amplification of chicken voltage-sensing domain were:ArcLight derivatives based on the voltage sensing domains from voltage sensitive phosphatases from three species were generated by replacing the GCGAAGCTTCGCCACCATGGAGACGACTGTGAGGTATGAACAG-3 and 5: CCGGGATCCCCAGAGACCATTCTTCTGGTTAC-3. Primers used for amplification of X. laevis voltage-sensing domain were: 5-GCCGCTAGCGCCACCATGGCCTCAAGTGTAACAGACGAGCAG-3 and 5-CCGGGATCCCCAGAGACCAGCCTCCTTGTTAC-3. Primers used for amplification of zebrafish voltage-sensing domain were: 5-GCCGCTAGCGCCACCATGGCCACGTCTGTGCATTTTAACCCTG-3 and 5-CCGAGATCTCCTGAAACCATTCTCCTGGTGAC-3. Primers used for amplification of mouse voltage-sensing domain were: 5-GCGAAGCTTCGCCACCATGTATGGAGAAAAGAAGAGCCATTTGTATCTC-3 and 5-CGCGGATCCCCTTGATGAGCCAGTTGCAGAATCC-3. Primers used for amplification of human voltage-sensing domain were: 5-GCGAAGCTTCGCCACCATGGAGAATGAAAGTCCTGATCC-3 and 5-CGCGGATCCCCATGAAACAGATGAAAAATTCTTAACAG-3. An arginine to glutamine mutation was introduced to the S4 of the voltage-sensing domains , corresponding to the location of R217Q in ArcLight. Point mutation and modification to the linker sequences between the chicken voltage-sensing domain and super ecliptic pHluorin were introduced by using the QuickChange II XL site-directed mutagenesis kit . All DNA constructs were verified by sequencing using the dye-termination method 5- HEK 293 cells  were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 8% fetal bovine serum . Cortical neurons were isolated from E18 mouse embryos and maintained in Neurobasal medium with 0.5 mM Glutamax-I and 1 ml of B-27 supplement  per 50 ml of cultured medium. Cells were plated on coverslips coated with poly-D-lysine hydrobromide  and kept in an incubator at 37°C with 5% CO2. Transient transfection was accomplished by using half of the manufacturer’s recommended amount of DNA (2 μg per 35 mm dish or 0.4 μg per 12 mm coverslip in 24-well dish) and Lipofectamine2000 .Microelectrode recordings were performed in a perfused chamber with the bath temperature kept at 33°C by a temperature controller and bath solution containing: 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose, and 5 mM HEPES, pH7.4. We used a 3-5 MΩ glass patch pipettes  that were pulled on a P-97 Flaming/Brown type micropipette puller . The pipette solution contained 120 mM K-aspartate, 4 mM NaCl, 4 mM MgCl2, 1mM CaCl2, 10 mM EGTA, 3 mM Na2ATP and 5 mM HEPES, pH 7.2. Voltage-clamp recordings in the whole-cell configuration were performed using a Patch Clamp PC-505B amplifier  with a holding potential of -70 mV. Cultured cortical neurons were recorded in current clamp mode. Action potentials were evoked by current injections. The pipette solution for neuron recordings contained: 120 mM K-gluconate, 3 mMKCl, 7 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 20 mM HEPES and 14 mM Trisphosphocreatin, pH adjusted with KOH to pH 7.3. The senWhole-cell patch clamped cells were imaged with a Nikon Eclipse E6000FN upright microscope with a water immersion objective, Nikon Fluor 60×/1.00 N.A. A MLL-FN-473 nm 50 mW  was used as the excitation light source. The laser light was transmitted into the microscope by a multi-mode fiber coupler , a quartz light guide and an Achromatic EPI-Fluorescence Condenser . The filter cube contains a dichroic mirror 505DCXR and an emission filter HQ510LP . The fluorescence image was demagnified by an Optem® zoom system, A45699  and projected onto the 80×80 pixel chip of a NeuroCCD-SM camera controlled by NeuroPlex software . The images were recorded at a frame rate of 1000 fps. Confocal images were obtained with a Zeiss 780 LSM  confocal laser scanning microscope using a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. A 488 nm wavelength Argon laser was used for chromophore excitation. The dichroic beam splitter was a MBS 488. The emission filter was 493 - 598nm. Zeiss Zen 2009 software was used for image acquisition and processing.NeuroPlex software  was used to view the image sequences and output optical and electrophysiological recordings. The % ΔF/F was calculated by first subtracting the dark image from all frames, then the average of a region of interest in each frame (F) is subtracted from the average of the region taken from one hundred frames prior to the event of interest (F0) and this value is then divided by F0, i.e. % ΔF/F = ((F-F0) / F0)*100. The data were further processed and statistically analyzed with Origin8.1  and Excel .The probe dynamics are fitted with either a single exponential equation: or a double exponential equation:vs V plot was analyzed with the Boltzmann equation:The ΔF/F 0.5 is the membrane potential in mV at half maximal ΔF/F, and dx is the slope.where xvs V plot was calculated from the Boltzmann fit: The normalized ΔF/F vs V plot of a probe based on the chicken voltage-sensing domain and the Mermaid FRET pair was calculated with the area version of the Gaussian function:The ΔF/F Figure S1Protein sequence alignment of N-terminal regions of the voltage sensitive phosphatase from six species. Conserved residues are shaded. Transmembrane domains are indicated with orange and the S#. The arginine to glutamine point mutation that shifts the F-V curve of the probes to more physiological range is marked in red. The fusion site corresponding to that of ArcLight-Q239 is highlighted with yellow.(TIF)Click here for additional data file.Figure S2Expression of human or mouse ArcLight in transfected HEK 293 cells. Representative confocal images of human ArcLight-Q193 or mouse ArcLight Q331 expression in the HEK 293 cells. The probes carry either an arginine to glutamine mutation or not in the S4 domain.(TIF)Click here for additional data file.


E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR.cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT).The deletion of the cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion caused transcription inhibition of the bet operon in E. coli K-12 (JM109) but did not affect this operon transcription in E. coli B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the E. coli B (BL21) strain more resistant to environmental changes.The transcriptional changes caused by the deletion of  E. coli [Acetate accumulation is one of the main concerns during high cell density growth of E. coli ,2. It waE. coli -5. AcetaE. coli  and is aE. coli ,7. MethoE. coli -10.E. coli B (BL21) is different from that of E. coli K-12 (JM109) especially when the bacteria grow to high densities at high glucose concentrations [E. coli K-12 (JM109) accumulates acetate up to 11 g/L and its growth rate slows down; E. coli B (BL21) on the other hand, accumulates acetate to about 3 g/L and its growth rate is not affected. Careful evaluation of these two strains revealed that E. coli B (BL21) has active glyoxylate shunt, gluconeogenesis, anaplerotic pathway, and TCA cycle compared with E. coli K-12 (JM 109) [E. coli B (BL21), the central carbon metabolism pathways associated with glucose consumption are operating at the same rate regardless of the glucose concentration. Based on the above finding, it was suggested that FruR is responsible for the difference in the glucose metabolism of these two E. coli strains.The acetic acid production pattern of trations . E. coli(JM 109) . It seemfruR gene modulates the direction of carbon flow in E. coli by transcriptional activation of genes that encode enzymes associated with oxidative and gluconeogenic carbon flow and by repression of genes that are associated with fermentative carbon flow [ppsA, fbp, pckA, aceA, which are vital for acetate uptake. Sugar catabolites tend to bind to Cra and displace it from the operator sites in target operons [ppsA, pckA, and aceBA), are generally subject to catabolite repression, while genes that are repressed by Cra , are subjected to catabolite activation [E.coli have used E.coli K-12 such as DH5α, BW25113, and MG1655 at low cell densities. The inactivation of Cra in plasmid-bearing E. coli DH5α altered metabolic gene expression, improved growth rate [cra gene had no distinct effect on the phenotype of E. coli BW25113 [cra deletion in E. coli MG1655 contributed to accumulation of pyruvate by suppressing gluconeogenesis due to the decreased expression of ppsA and pckA [FruR, also known as, Cra (Catabolic repressor/activator), is a global transcription regulatory protein in enteric bacteria that regulates gene expression by binding to a specific DNA sequence . It was bon flow ,16. Cra wth rate , and impwth rate , while t BW25113 . cra deland pckA .E. coli K-12 (JM109) and E. coli B (BL21) in regard to glucose metabolism, it was suggested that in E. coli B (BL21) cra is constitutively expressed, which explains why acetate is not accumulated. In the present work, we have investigated the effect of cra deletion on the central carbon metabolism in E. coli B (BL21) and E. coli K-12 (JM109) to verify the above assumption and to understand further the function of Cra in high cell density at high glucose, by following growth kinetics and gene expression.Considering what is currently known about Cra regulation, and the difference between E. coli B (BL21) and E. coli K-12 (JM 109), without modification and with the cra gene deletion (cra‾), are shown in Figure cra deletion in E. coli B (BL21) minimally affected growth, final cell yield, glucose consumption and maximal acetate and pyruvate accumulation. Unlike E. coli B (BL21), cra deletion in E. coli K-12 (JM109) affected the cell growth pattern. The final OD600 was 30 in E. coli K-12 (JM109) and 18 in E. coli K-12 (JM109) cra‾ , maximal acetate and pyruvate concentrations in E. coli K-12 (JM109) were 9 g/L and 14 g/L respectively, and in E. coli K-12 (JM109) cra‾ acetate and pyruvate concentration reached 6.6 g/L and 13 g/L respectively.Growth kinetics, glucose consumption and media conductivity profile of oli B BL2 and E. cE. coli B (BL21) and from 10 mS/cm to 13 mS/cm in E. coli B (BL21) cra‾. In E. coli K-12 (JM 109), the conductivity reached 20 mS/cm and in E. coli K-12 (JM109) cra‾, reached 21 mS/cm.Media conductivity values are shown in Figure E. coli B (BL21) with and without cra, and E. coli K-12 (JM 109) with and without cra, were grown in regular LB media containing 5 g/L (conductivity 10 mS/cm) and in LB media containing 15 g/L NaCl (conductivity 22 mS/cm).To evaluate the effect of the media conductivity  on the bacterial growth, E. coli B (BL21) strains were not affected by the high conductivity media both strain grew to an OD600 of 44 and the media conductivity reached 26-27 mS/cm. However, there was a difference between the two E. coli K-12 strains; E.coli K-12 (JM109) grew to final OD600 of 30 in regular LB media and to 27 OD600 in the high conductivity media, while E. coli K-12 (JM109) cra‾ grew to final OD600 of 18 in the regular media and to 11 OD600 in the high conductivity media. During the growth of both E. coli K-12 strains, the media conductivity reached 40 mS/cm.The growth of the different strains and media conductivity are shown in Figure cra - negative strains and parental (cra - positive) stains, are shown in Figure cra -negative strains and negative ratio is an indication that the gene transcription is lower in the cra negative strain. The transcription comparison was done on the following pathways of the central carbon metabolism: the TCA cycle, the glyoxylate shunt, the gluconeogenesis, and the glycolysis. No significance transcription differences were identified in the glycolysis and the TCA cycle pathways (Data not shown) but differences were identified in the expressions of the glyoxylate shunt pathway genes (aceA and aceB), the acetate producing gene (poxB), and the gluconeogenesis gene (ppsA); all these genes were down regulated in both cra‾ strains while acetate uptake gene (acs) was down regulated significantly only in E.coli B (BL21) cra‾.Results of microarray analysis, expressed as log 2 ratios of samples taken during the log phase growth from bet operon genes (betABT): the results are shown in Figure bet operon genes (betABT) in E. coli K-12 (JM109) cra‾ are low compared with their expression in the cra - positive stain. This information was confirmed by Real Time PCR  and E. coli B (BL21) as a result of its effect on the central carbon metabolism [E. coli B (BL21) but not in E. coli K-12 (JM109) [cra deleted mutants of these two strains and by analyzing their growth, metabolism and gene expression, we anticipated to have more information on the role of the Cra regulating the central carbon metabolism in E. coli, and perhaps be able to create a strain tolerant to high glucose concentrations. The results showed that cra deletion in E.coli B (BL21) minimally affected the growth and the maximal acetate accumulation, while in E.coli K-12 (JM109), cra deletion showed considerable effect. E. coli K-12 (JM109) grew to an OD of 30 and accumulated 9.0 g/l acetate, while E. coli K-12 (JM109) cra‾ stopped growing as soon as acetate concentration reached 6.6 g/L at an OD of 18; at this point, the media conductivity reached 21 mS/cm, indicating a possible effect on the ability of the mutant cell to keep its osmolarity.The purpose of this work was to find out if the global regulator Cra is responsible for the difference in glucose consumption and acetate production between coli B BL as a restabolism , we conc (JM109) . By creacra deletion affected the transcription of genes associated with acetate metabolism. ppsA (gluconeogenesis gene), aceA , aceB (the glyoxylate shunt pathway genes) and poxB (the acetate producing gene) were all down-regulated in both knockout strains, while acs (acetate uptake gene) was down regulated only in E.coli B (BL21) cra¯. These results are similar to previous reports that Cra represses transcription of genes that encode glycolytic (sugar-catabolizing) enzymes such as key enzymes in the Embden-Meyerhof and Enter-Doudoroff pathways, but activates transcription of genes that encode biosynthetic and oxidative enzymes such as key enzymes in the Krebs cycle, the glyoxylate shunt, the gluconeogenic pathway, and electron transfer [cra deletion on acetate and pyruvate production. These observations are different from previous reports that Cra deletion in E. coli MG1655 contributed to accumulation of pyruvate by suppressing gluconeogenesis due to the down regulation of ppsA and pckA [Our microarray results showed that transfer . Concernand pckA . It is pand pckA .acnA/B (aconitase), icdA (isocitrate dehydrogenase), eda (2-keto-3-deoxy-6-phosphogluconate adolase), edd (6-phosphogluconate dehydrase), pfkA (phosphofructokinase), and pykF (pyruvate kinase isoenzymes), but the transcription patterns of these genes in this work were not significantly different between the two strains (results not shown). Another global regulator that was reported to affect the glyoxylate shunt operon (aceBAK), is ArcA/B; this two-component system is considered to respond to oxygen limitation under microaerobic conditions [E.coli: acetyl-CoA synthetase is under the control of the sigma factors RpoS and RpoD and requires cAMP receptor protein, and the oxygen regulator (Fnr) for full expression [cra deletion does not affect the transcription patterns of cyaA, crp, and rpoD in E. coli B (BL21) and E. coli K-12 (JM109)  cra‾ but not in E. coli K-12 (JM109) cra‾. The down regulation of rpoS was reported to result in the high transcription of poxB and the low transcription of acs in E. coli B (BL21) [Cra has been known to affect positively and negatively the transcription of several other genes such as nditions . In addipression . Pyruvatoli B BL2 and E. cB (BL21) .bet operon, which has a role in protecting E. coli against high osmotic pressure [E. coli K -12 (JM109) cra¯ strain. This phenomenon can provide a possible explanation to the growth inhibition of this strain when the media conductivity was above 18 mS/cm. The bet operon consists of three genes: betT (choline transporter), betA (choline dehydrogenase), and betB  [bet genes are induced by choline, oxygen, and osmotic stress [E. coli K-12 (JM109) cra ¯ is likely the result of the limited transcription of the bet operon  and not in E. coli B (BL21); it is possible that Cra affects the bet operon indirectly. This is an additional difference between these two strains that may point to less regulation in E. coli B. The insensitivity of E. coli B (BL21) to the cra deletion, together with its insensitivity to high glucose concentration, enables this strain to be less affected by environmental stress such as glucose concentration and ionic strength. This may be the results of lacking an internal control mechanism that may be responsible for E. coli K-12 inability to cope with environmental stress.An unexpected outcome of our work is the discovery that the pressure -26, was ogenase) . The Choogenase) . The betc stress . The inhE. coli K and E. coli B, the cra gene was deleted and the mutant strains were compared to the parental strains in their gene expression, growth and metabolic behaviour. The transcriptional changes caused by the deletion of cra gene support the assumption that Cra is a major regulatory molecule that has an effect on the expression of ppsA, aceBAK and acs in E.coli B (BL21), but these transcriptional changes did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone, rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion in E. coli K-12 (JM109) caused transcription inhibition of the bet operon which is responsible for maintaining the salt concentration in the cells. In comparison, the bet operon expression was not affected in E. coli B (BL21). This property, together with this strain insensitivity to high glucose concentrations, makes this strain more resistant to environmental changes and may be one of the reasons for the growth properties of this strain.To evaluate the role of the global regulator Cra in the central carbon metabolism of E. coli B (BL21) (λDE3) , dcm, gal, Cmr) and E. coli K-12 (JM109) (λDE3) , relA1, supE44, Δλ-, Δ(lac-proAB), F', traD36, proAB, lacIqZΔM15) were used. Both strains were obtained from Promega Corp. .cra gene knock-out was carried out by recombination using plasmid pKD46 as previously described [cra gene using two primers escribed . A kanam5'-GTGAAACTGGATGAAATCGCTCGGCTGGCGGGAGTGTCGCGGACCACTGCTACGGTCTGCGTTGTCGGGAAGATGCG and 5'-CTTAGCTACGGCTGAGCACGCCGCGGCGATAGAGATTACGTTTAATGCGCGTACAAAGCCGCCGTCCCGTCAAGTCA.cra¯strain was confirmed by PCR with primers which can hybridize upstream and downstream of deleted cra genes. The PCR products were also electroporated into MG1655 harboring pKD46 to make MG1655 cra¯strain. P1 lysates of MG1655 cra¯were used to transduce cra mutation carrying a kanamycin marker into BL21(DE3) strain to make BL21(DE3) cra¯ which were confirmed as described above. PCR products carrying kanamycin marker and homologous region (50 bp) were electroporated into JM109 (DE3) harboring pKD46 where lambda recombinase was fully expressed by L-arabinose during culture at 30°C, and subsequently cells were spread on LB agar containing kanamycin. Candidate colonies were grown at 42°C to cure plasmid pKD46 carrying the temperature sensitive origin of replication. Specific gene knock-out in JM109(DE3) 2HPO4. After sterilization, 10 mM MgSO4, 1 ml/L trace metal solution [4OH, and dissolved oxygen was kept at 30% air saturation. For high-conductivity media, the NaCl was adjusted to 15 g/L, the other compositions were the same. Samples for acetic acid, pyruvate, glucose concentration, and conductivity analysis were collected at regular intervals, centrifuged at 14,000 × g for 5 min and the supernatant was analyzed with HPLC. Samples for total RNA purification were collected and centrifuged at 14,000 × g for 1 min at 4°C, the supernatant was removed, and the cell pellets were quickly frozen in dry ice and stored at -80°C.The four strains were grown at 37°C in modified LB medium containing 10 g/L tryptone, 5 g/L yeast extract (15 g/L for JM109), 5 g/L NaCl, and 5 g/L Ksolution , and 40 The concentrations of acetate and pyruvate were analyzed at 35°C (for acetate) and 65°C (for pyruvate) on an HP 1100 series  using an Aminex HPX-87H column . The mobile phase was 0.008 N sulfuric acid and flow rate was 0.6 mL/min. After injection of a 20-μL sample, absorbance was monitored at a wavelength of 210 nm using a UV detector. Glucose in the culture supernatant was determined using an YSI glucose analyzer . Conductivity was analyzed at 30°C using CDM210 conductivity meter .260) using a NanoDrop 1000 . Three biological replicates of RNA samples from E. coli B (BL21) and E. coli K-12 (JM109) cra knockout strain and wildtype were hybridized to Affymetrix E. coli Genome 2.0 Array. DNA-chip Analyzer (dChip version 2006) was used to process the raw data generated at the NIDDK Genomics Laboratory.Total RNA was isolated using a MasterPure RNA Purification Kit  and RNeasy Mini Kit  according to the manufacturers' protocols (MCR 85102 & 74104). RNA concentration and purity were determined by measuring absorbance at 260 nm (AReverse transcription (RT) was carried out with the High Capacity RNA-to-cDNA Kit using the manufacturer's protocol (Applied Biosystems). A 20-ul RT reaction mixture included 2 ug of total RNA, 10 ul of 2X RT buffer, 1 ul of 20X RT enzyme mix, and DEPC-treated water. Real-time PCRs were carried out on a 7900 HT Fast Real-Time PCR System (Applied Biosystems) with the Power SYBR Green PCR Master Mix (Applied Biosystems). The PCR mixtures were incubated at 95°C for 10 min to activate the AmpliTaq DNA polymerase, followed by 40 cycles of amplification . A final extension step was performed at 60°C for 10 min. Real-time PCR results were also analyzed using the 7900 HT SDS software (Applied Biosystems).The authors declare that they have no competing interests.YS performed experiments and analysis, literature search and writing the manuscript, JP hypothesised the idea, survey the literature, analyze results and writing the manuscript, LT performed fermentation experiments, SL genetic work, JS conceived the study participated in the designed and coordination and the writing of the manuscript. All authors read and approved the final manuscript.Supplemental Figure S1 and S2. Microarray data  and real time PCR data .Click here for file


Puberty is a period of increased susceptibility to factors that cause increasedbreast cancer risk in adulthood. Mammary end buds (EBs) that develop duringpuberty are believed to be the targets of breast cancer initiation. Whereas therole of estrogen (E) has been extensively studied in pubertal mammary glanddevelopment, the role of progesterone (P) during puberty is less defined.Pubertal and prepubertal ovariectomized mice were treated with vehicle control(C), E, P, or E+P. Mammary glands from these mice were analyzed for changes inmorphology, proliferation, and expression of the downstream targets amphiregulin(AREG) and receptor activator of NF-κB ligand (RANKL).P, acting specifically through the progesterone receptor, induced increases inmammary gland proliferation and EB formation that were associated with increasedAREG expression in ducts and EBs. E, acting specifically through the estrogenreceptor, produced similar responses also mediated by AREG. Blocking AREG actionby treatment with an EGFR inhibitor completely abrogated the effect of P on EBformation and proliferation and significantly reduced proliferation within ducts.P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc,an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesserextent in EB, but did not cause EB regression.These results demonstrate a novel P-specific effect through AREG to cause EBformation and proliferation in the developing mammary gland both before and duringpuberty. Thus, hormones and/or factors in addition to E that upregulate AREG canpromote mammary gland development and have the potential to affect breast cancerrisk associated with pubertal mammary gland development. The mouse mammary gland is used as a model for development of the human breast. Beforepuberty, both the mouse and human mammary glands grow and develop at an isometric rate,at the same rate as the body. At puberty, rapid changes in the hormonal milieu driveincreased proliferation and expanded ductal development to fill the fat pad (reviewed in ). DuringThere is increasing interest in factors influencing pubertal mammary gland developmentand breast cancer risk later in life . EB stru-/- mice with IGF-I plus P iscapable of stimulating mammary gland development in the absence of E, suggesting that Phas E-independent effects in the pubertal mammary gland [The ovarian hormone estrogen (E) is the primary hormone required for ductal developmentduring puberty , whereasry gland . A role ry gland -15. Consry gland . Thus, PIn this study, we sought to elucidate further the role of P in pubertal ductaldevelopment. We found that P acting through PR caused pubertal EB formation, which wasdependent on an increase in amphiregulin (AREG) expression. P-induced AREG wasassociated with proliferation in both the EBs and ducts. P also increased expression ofreceptor activator of NF-κB ligand (RANKL), a known paracrine mediator of P, whichprimarily affected proliferation in ducts. Further showing the importance of AREGdownstream of P, we found that the prepubertal mammary gland was highly sensitive to lowdoses of P that also increased AREG expression and led to EB formation. These studiesfurther emphasize the importance of AREG for EB formation in the pubertal gland anddemonstrate that hormones and/or factors, in addition to E, may be important for theearly regulation of AREG.BALB/c and C57BL/6 mice were purchased from Harlan  andJackson Laboratory , respectively. Two time frames of mammarygland development were examined. In the first case, pubertal 4-week-old BALB/c andC57BL/6 mice that initiated estrus cycling and exhibited EBs were ovariectomized(OVX). Three weeks post-OVX recovery was allowed for complete EB regression beforehormone treatments . OVX micIn the second case, prepubertal 3-week-old BALB/c mice that had not started estruscycles or EB formation were OVX. Two weeks post-OVX mice received a single injectionwith saline control (C), or low-dose E2 (0.1 μg), P (0.1 mg), or E2+P (0.1μg E + 0.1 mg P) with or without 5 mg ICI 182,780 (ICI) (Tocris Bioscience), andthen killed 48 hours later. Hormone dosages were based on the minimal concentrationsof E2 capable of EB formation in the pubertal gland . To blocAll mice were injected with 5-bromo-2'-deoxyuridine (BrdU) (70 μg/g body weight)2 hours before being killed. Mammary glands were fixed and processed as whole mounts , or paraPR was detected by using mouse monoclonal anti-PR . Estrogen receptor α (ERα), AREG, RANKL, and BrdU were detected byusing mouse monoclonal anti-ERα , goat polyclonal anti-amphiregulin , goat polyclonal anti-RANKL , or mousemonoclonal anti-BrdU primary antibodies followed by appropriate secondary antibodies conjugated to Alexa488  . Double Whole mammary gland total RNA was extracted by using TRIzol  following the manufacturer's suggested protocol. cDNA was produced byreverse transcription with random hexamer primers and reversetranscriptase-polymerase chain reaction (RT-PCR), and quantitation of murineamphiregulin (Mm00437583_m1)  and 18S RNAs(Hs99999901_s1) was performed, as previously described .P< 0.05 with the Student t test or ANOVA, as appropriate.BrdU and PR were quantitated for the number of positive luminal epithelial cellnuclei from captured images by using MetaMorph software, as previously described . A minimBefore puberty and the onset of estrus cycles, mammary ductal outgrowth is limitedand hormone independent. Increasing hormone levels during puberty produce localizedmammary ductal proliferation at sites called end buds (EBs) that drive ductalelongation (reviewed in ). To exa+) luminal epithelial cellscompared with vehicle controls, whereas treatment with E2 or E2+P did notsignificantly change the percentage of PR+ cells. Hormonal regulation ofPR expression was similar in both BALB/c and C57BL/6 mice  cells was significantly increasedthroughout the mammary gland in both ducts and EBs by E2, P, and E2+P treatment cells incorporated BrdU in the pubertal mammary gland afterhormone treatment. Treatment with E2, P, or E2+P produced similar BrdU uptake inPR+ cells and represented about one third of the total BrdU+cells in EBs , E2 (24.0 fold), and E2+P (39.6 fold)compared with vehicle controls , whereas the percentage of BrdU+ cells in ductsin response to P was reduced 66% by Iressa (P < 0.05 versus P).To examine further the role of AREG in pubertal P-induced EB formation, theEGFR-inhibitor Iressa was used to block AREG action through the EGFR. Inhibition ofEGFR by Iressa completely inhibited both E2- and P-induced pubertal EB formationFigure  and EB pReceptor activator of NF-κB ligand (RANKL) is another downstream effector of Pin the mammary gland ,29. FiveP = 0.2)  and by 46% in EBs (P <0.05 versus P) . Treatment with RANK-Fc had a small effect to reduce P-induced EB formationthat did not achieve statistical significance  Figure . In cont) Figure .To examine the sensitivity and response of the mammary gland to P before the onset ofpuberty, 3-week-old BALB/c mice were OVX and treated with a low dose of E2 or P . Notably+ and PR negative (PR-)) within EBs was significantlyhigher than in ducts after the single low-dose treatment and was similar among E2, P,and E2+P treatments  or E2 (22.95 fold) compared with vehiclecontrols -specific effect to cause endbud (EB) formation and proliferation in the developing mammary gland both before andduring puberty. This effect was primarily mediated through P-induced amphiregulin (AREG)via a progesterone receptor (PR)-mediated mechanism, and AREG expression was increasedby P in both ducts and EBs. Blocking AREG action by treatment with an epidermal growthfactor receptor (EGFR) inhibitor completely abrogated the effects of P on EB formationand proliferation, and significantly reduced proliferation within ducts. Thus, AREG isan essential mediator of P-induced EB formation and proliferation. To our knowledge,this is the first report to demonstrate the sensitivity of the prepubertal mammary glandto P.Four EGF-family receptors are expressed in the mammary gland at puberty: ErbB1 (EGFreceptor/HERl), ErbB2 (c-neu/HER2), ErbB3 (HER3), and ErbB4 (HER4) . MembersOur current studies extend the importance of AREG in pubertal mammary gland developmentto also include P-induced AREG as a mediator of EB formation and proliferation. Theability of P acting through PR to upregulate AREG expression is consistent with previousstudies in the adult mouse mammary gland  and mous+ cells in EBs in the pubertal mammary gland after 5days of treatment. A similar pattern of proliferation of PR+ cells was alsodetected after E2 treatment. A subset of luminal epithelial cells is hormone receptorpositive, expressing both ERα and PR [Proliferation of the epithelium in response to AREG may be through direct stimulation byAREG or through growth factors produced in the stroma . P exertα and PR . These rα and PR . AREG haα and PR , and a sα and PR . It is cPuberty, with increased hormone production leading to EB stimulation and ductaldevelopment, has been shown to be a critical period for mammary cancer susceptibility -8. E is Further supporting the importance of AREG in the mammary gland, we found similarproliferation and AREG expression in both pubertal BALB/c and C57BL/6 mice. Mice ofthese two different genetic backgrounds respond differently to P in the adult mammarygland , yet res+ cells that is dependent on cyclin D1, followed by a more robustproliferation of PR- cells through RANKL [-cells within the epithelium [Another downstream effector of P, receptor activator of NF-κB ligand (RANKL), wasalso increased by P treatment in BALB/c mice, and RANKL expression was detectedprimarily in ducts in the pubertal mammary gland. Inhibition of RANKL in the BALB/cmammary gland significantly reduced P-induced proliferation in ducts and to a lesserextent in EBs, but did not cause EB regression. Proliferation in the adult mammary glandis mediated by P via two distinct mechanisms; an early, direct mitogenic effect onPRgh RANKL . Consistithelium ,44-46. Iithelium . OverallIn conclusion, these studies demonstrate that the prepubertal and pubertal glands aresensitive to P, and lend further support for the ability of P to influence mammaryductal development, even in the absence of E. Given that puberty may be an importantperiod of cancer susceptibility, an understanding and elucidation of possible endocrinedisruption by factors that influence P responses and/or AREG regulation in theprepubertal and pubertal mammary gland must be considered, in addition to factors thatinfluence E responses. We propose that factors that influence P responsiveness and otherpathways that influence AREG production, in addition to E, warrant further investigationin the prepubertal and pubertal mammary gland.E: estrogen; E2: 17-β-estradiol; EB: end bud; EGFR: epidermal growth factorreceptor; ER: estrogen receptor; P: progesterone; PR: progesterone receptor; RANK:receptor activator of NF-κB; RANKL: receptor activator of NF-κB ligand.The authors declare that they have no competing interests.MDA designed and carried out all aspects of the mouse experiments, designed andparticipated in all immunofluorescence staining, analyzed and interpreted the collectiveresults of all the experiments, and wrote the manuscript. JRL carried out theimmunofluorescence experiments. JB participated in the execution of the mouse studies.RCS participated in the design of the inhibitor studies and reviewed the manuscript. SZHconceived of the study, participated in its design and data analysis, and reviewed themanuscript for important intellectual content. All authors read and approved the finalmanuscript.Figure S1. Both 17-β-estradiol and progesterone induce morphologicresponses in the pubertal C57BL/6 mammary gland. Pubertal 4-week-oldC57BL/6 mice were OVX, allowed to recover for 3 weeks, and then treated for 5days with vehicle control (C), E2, P, or E2+P, as described in the Materialsand Methods section. (A) Morphologic response to 5-day C, E2, P, orE2+P. Scale bar, 1 mm. (B) Immunofluorescent detection of PR expression.The values represent the mean ± SEM PR-positive luminal cells . The percentage of PR-positive cells in 5-dayP-treated BALB/c mice was less than control (*P < 0.05). Thepercentage of PR-positive cells in 5-day P-treated C57BL/6 mice was less thancontrol (#P < 0.05). Figure S2. Both 17-β-estradiol andprogesterone induce similar proliferative responses in the C57BL/6 pubertalmammary gland. Pubertal 4-week-old C57BL/6 mice were OVX, allowed torecover for 3 weeks, and then treated for 5 days with vehicle control (C), E2,P, or E2+P, as described in the Materials and Methods section. Proliferationanalysis by dual immunofluorescence was detection of BrdU and PR. Totalpercentage of BrdU-positive cells and percentage of BrdU-positive cellsco-expressing PR in ducts and end buds are presented. The percentages ofBrdU-positive luminal epithelial cells in ducts in response to E2, P, or E2+Ptreatment are greater than control (*P < 0.05). Figure S3. Both17-β-estradiol and progesterone regulate amphiregulin, butco-stimulation with both 17-β-estradiol and progesterone is required toregulate RANKL in the C57BL/6 pubertal mammary gland. Pubertal4-week-old C57BL/6 mice were OVX, allowed to recover for 3 weeks, and thentreated for 5 days with vehicle control (C), E2, P, or E2+P, as described inthe Materials and Methods section. (A) Immunofluorescent detection ofAREG (green) after E2, P, and E2+P treatment. (B) Immunofluorescentdetection in the mammary gland with antibody against RANKL (green) inE2+P-treated C57BL/6 mammary glands. Nuclei  were counterstained withDAPI (blue). Scale bar, 25 μm. Figure S4. Progesterone fails to induceRANKL in the prepubertal mammary gland. Prepubertal 3-week-old BALB/cmice were OVX, allowed to recover for 2 weeks, and then treated once with P, asdescribed in the Materials and Methods section. No RANKL (green) expression wasdetected by immunofluorescence. Nuclei were counterstained with DAPI (blue).Scale bar, 25 μm.Hormone responses in the pubertal and prepubertal C57BL/6 mammary gland.Click here for file


GB virus C (GBV-C) is prevalent globally and particularly among individuals at risk of parental exposures. Based on genetic diversity, this virus is now classified into six genotypes and many subtypes with distinct geographical distribution. In this study, 120 Injecting Drug Users (IDUs) were recruited from Yunnan province, China. Among them, 43 (35.8%) were positive for GBV-C RNA, 70 (58.3%) and 103 (85.8%) sero-positive for HIV-1 and HCV respectively. This revealed 18.3% of IDUs having GBV-C/HIV/HCV triple infection, which is significantly higher than 7.5% of GBV-C/HIV-1 and 10% of GBV-C/HCV dual infection rates (P<0.05). Based on 5′UTR sequences, the identified 43 viral isolates can be classified into three phylogenetic groups: one (2.3%) and two (4.7%) belonged to genotype 3 and 4, respectively, and the remaining 40 (93%) formed a new group with 97% of bootstrap support. This new GBV-C group was further confirmed by characterizing the E2 region and full-length genome sequences. Analysis of 187 nt 5′UTR sequence showed three previous reported isolates from Southeast Asia were re-classified into this new group. It implies they have the same origin with strains from Yunnan. Although we provisionally assigned this new group as GBV-C genotype 7, a simpler five groups of GBV-C nomenclature is recommended. Genotype 4, 6 and the newly designated genotype 7 could be reclassified as one group, which may represent a single GBV-C genotype. The classification of the other four groups was corresponding to that of previous reported genotype 1, 2, 3 and 5. Furthermore, the diversity of amino acid sequence in the E2 region was analyzed. The inhibitory effect of GBV-C genotype 7 on HIV-1 cell entry could be deduced. Since GBV-C may have a beneficial effect on AIDS disease progression and interact with HCV during co-infection, this finding may raise interests in future studies on this virus that was previously thought to be a “non-pathogenic virus”. Flaviviridae family. GBV-C has a single-stranded positive RNA genome of about 9.3 kb and contains a single open reading frame (ORF) encoding two structural (E1 and E2) and five non-structural  proteins Discovered by two independent groups in the mid-1990s, GB Virus C (GBV-C)/Hepatitis G Virus (HGV) has now been classified as a member of the GBV-C infection has been found worldwide. High prevalence is observed among subjects with the risk of parenteral exposures. The subjects include those with exposure to blood and blood products, those on maintaining hemodialysis, and those with intravenous drug using Comparing with HCV, the genetic diversity of GBV-C is lower with 11–14% of nucleotide difference in the polyprotein coding region between genotypes Although GBV-C infection has not been found in association with any particular disease, its co-infection with HIV-1 may produce some favorable outcomes, with a lower mortality rate, slower disease progression, and longer survival term The Yunnan province is situated in southwest China. It borders Southeast Asia countries of Laos, Vietnam and Myanmar to the south. As a hub on the trans-continental drug trafficking road, Yunnan has played a critical role in spreading many blood-borne infections in China Some studies have revealed that GBV-C infection occurs among IDUs in Southern China P<0.05).A total of 120 IDUs were recruited from six drug detoxification centers in five prefectures of Yunnan province, China. Among these prefectures three  border Myanmar and Vietnam, while two  are located at the central part of the province . Of the tro was included as the out-group. In this rooted phylogenetic tree (The amplification of GBV-C 5′UTR was successful for 43 IDUs and the expected amplicons corresponded to nucleotides -414 to -37 of the prototype GBV-C genome (U36380). Based on the 5′UTR sequences, a phylogenetic tree was reconstructed. The tree showed that the obtained 5′UTR sequences were divided into three phylogenetic groups. A sequence (2.3%) from Kunming (KM07) was classified into genotype 3, the reported predominant genotype in China. Two sequences (4.7%) from Dehong (DH019 and DH021) were classified into genotype 4, which was common in Southeast Asia. Unexpectedly, the remaining 40 sequences (93%) formed a solid cluster with a 97% of bootstrap support. This cluster was distinct from the known six GBV-C genotypes, suggesting the existence of a new genotype . For a btic tree , the thrThe three full-length GBV-C sequences, KY117, DL185, and DH028, were further characterized. They all had a 389 nt of 5′UTR, followed by a single ORF of 8529 nt and a 316 nt of 3′UTR. They shared common sizes in seven protein-encoding regions: E1(608 nt/212 aa), E2(1161 nt/387 aa), NS2(843 nt/281 aa), NS3(2031 nt/677 aa), NS4(945 nt/315 aa), NS5A(1245 nt/415 aa), and NS5B(1692 nt/564 aa). Against six reference sequences representing six known GBV-C genotypes, nucleotide similarities were calculated . Among tAmong the detected 43 GBV-C isolates, genotype 7 was the most frequent accounting for 93%. In contrast, only one and two isolates belonged to genotype 3 and 4, respectively, accounting for 2.3% and 4.7%. This GBV-C genotypes distribution pattern is vastly different from those previously reported from China, in which genotype 3 was most predominant 133–156, E2289–306 and E2325–342), which have been proved to inhibit HIV entrance into target cell 133–156 region, only one in E2289–306 region, and no one in E2325–342 region. The more mutation sites of E2133–156 may confer its impact on the interaction of GBV-C and HIV-1. In alignment, the consensus amino acid sequence (U45966) has been recently demonstrated to block HIV-1 entry by interference with gp41-mediated cell-cell fusion It has been confirmed that certain E2 domains of GBV-C may interfere with HIV-1 cellular entry by interaction with fusion peptide-Vesicle In this study, the infection with HIV-1, HCV and GBV-C was investigated among 120 IDUs who were recruited in five prefectures of Yunnan province, China. We found the overall GBV-C RNA positive rate  was agreed with previous reports that GBV-C infection rate among IDUs ranged from 17.9% to 58.0% For GBV-C genotyping, different methods have been used. These included RT-PCR with specific primers, restriction fragment length polymorphism, and direct DNA sequencing Of this study, the GBV-C genotype distribution pattern is vastly different from that previously reported in China where genotype 3 is predominant In this study, GBV-C genotype 7 was designated by following the system Mueroff and his colleagues had recommended + cell count and HIV-1 viral load. In addition, there is a need of investigating the correlation between GBV-C genotypes and HIV-1 subtypes or circulating recombinant forms (CRFs). As cousins of Flaviviridea, GBV-C and HCV have similar genome organization. A potential association between the two viruses in the context of genetic diversity and evolution also need to be investigated.A benefit of GBV-C infection on disease progression of AIDS or HIV-1 replication has been documented A total of 120 IDUs were recruited, from whom anti-coagulated blood samples were collected. These IDUs were retained in six drug detoxification centers in five prefectures of the Yunnan province, China, during 2005–2008. Among them, 30 were from Kunming, 46 from Honghe, 13 from Wenshan, 15 from Dehong, and 16 from Dali . This stViral RNA was extracted from 200 µl plasma using High Pure Viral RNA Kit (Roche Applied Science). This was followed by RT-nested-PCR using the primers listed in From three GBV-C RNA + samples, full-length GBV-C genomes were characterized. To amplify the GBV-C complete genome, 51 primers were designed based on the aligned reference sequences. Their sequences were listed in http://www.ncbi.nlm.nih.gov) and aligned with reference sequences using CLUSTAL_W program. Further adjustments to the alignments were manually made using BioEdit program. Bootstrap resampling was performed with 1000 neighbour-joining replicates. Phylogenetic analysis of 5′UTR, E2 region, and full-length sequences was conducted using Mega 4 software, which employed Jukes-Cantor distances of neighbour-joining tree.All the amplicons were directly sequenced, and sequence information was analyzed using the BLAST, CLUSTAL_W, BioEdit and Mega 4 software. In more detail, the obtained sequences were confirmed as GBV-C using the NCBI  BLAST searching program Click here for additional data file.Figure S2Strategies used to amplify the KY117, DL185 and DH028 complete genomes. The bar at top represents the genomic organization of GBV-C and shows the seven protein encoding regions of various lengths, flanked by the 5′ and 3′ UTRs. Nucleotide numbering is according to the U36380 genome. Arrows represent the overlapping fragments amplified for the three GBV-C isolates.(TIF)Click here for additional data file.Table S1The infection rates of GBV-C/HCV/HIV-1 among recruited IDUs in Yunnan province.(DOC)Click here for additional data file.Table S2Primers used for amplification and sequencing of the GBV-C genome.(DOC)Click here for additional data file.