AbstractAmynthas Kinberg, 1867 species belonging in family Megascolecidae s. stricto are sketched, dissected and described. Amynthas daeari Blakemore sp. n. has spermathecae in 6/7/8 complying with an Amynthas tokioensis spp-group, whilst Amynthas jinburi Blakemore sp. n. has spermathecal pores in 5 & 6 strictly complying with Amynthas canaliculatus-group. A definitive COI gene barcode is provided for the holotype of Amynthas daeari but the age since collection or preservation of the Amynthas jinburi type in 2000 precluded its mtDNA extraction at this time.Two Korean endemic pheretimoid  Pheretima auct.) group of Oriental origin that provides approximately 970 valid species from 1,200 nominal taxa .Specimens, now in 80% Ethanol, are lodged in the NIBR facility. Small tissue samples taken for mtDNA COI barcoding as proposed 10 yrs ago by Discussion is confined to remarks after each species’ description. For brevity, not all taxonomic authorities are cited in References as these may be sought elsewhere.urn:lsid:zoobank.org:act:E6B103F4-2FFE-4A05-BCD8-356A90754AC5http://species-id.net/wiki/Amynthas_daeari35.9801N, 127.2981E); collected 27th July, 2012 by Dr Hong-Yul Seo. DNA tissue sample code – w53.IV0000261261, mature specimen complete but broken in two at clitellum after being figured and dissected. Collected from small valley at Jeollabuk-do, Wanju-gun, Dongsang-myeon, Daea-ri  with GMs anterio-median and shallow clefts laterally .Female pores. Single on 14.Spermathecal pores. 6/7/8 ca 0.3 C apart at edge of puckered area and lateral to GMs.Genital markings. Paired discs just median to male and spermathecal pores as noted; composite glands on spermathecal pore GMs but none found for GMs near male pores although the body here is macerated and they may well have broken off and dissipated.Septa. Nephridial forests on septa 5 & 6; 7/8 thin, 8/9/10 aborted.Dorsal blood vessel (dbv). Single.Hearts. Last hearts in 13 (preceding vascularization unclear/damaged).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. Indeterminate as specimen macerated; caeca ventrally incised from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, seminal vesicles in 11 & 12.Ovaries. In 13 as usual.Prostates. Racemose glands in 17-19, duct short and muscular.Spermathecae. Two pairs in 7 and 8; that in 7lhs inflated, that in 8lhs deflated .Gut contents. Coarse organic debris, i.e., a litter diet suggesting superficial feeding.Amynthas daeari Holotype.>w53 PageBreakGCTCATGCATTTTTAATAATCTTCTTTCTTGTAATACCAGTATTTATTGGTGGGTTTGGAAATTGACTTCTACCTCTAATACTAGGTGCCCCAGATATAGCTTTCCCGCGACTTAACAATATAAGATTCTGATTACTGCCCCCATCACTAATTTTACTAGTATCGTCTGCAGCAGTAGAAAAAGGTGCCGGAACAGGATGGACAGTGTACCCCCCACTTGCGAGAAACATTGCACATGCCGGCCCTTCAGTAGATCTTGCAATTTTTTCTCTCCATCTAGCCGGAGCATCATCAATTCTCGGTGCCATCAACTTCATTACTACCGTAATTAATATACGATGATCTGGGCTACGCTTAGAACGAATTCCTCTATTTGTATGAGCAGTTGTAATTACTGTAATTCTTTTACTTCTATCTTTACCAGTCTTAGCCGGTGCTATTACAATATTACTAACAGACCGAAACCTAAATACATCATTTTTTGATCCAGCGGGAGGAGGTGATCCAATTCTATATCAACACTTATTTCTATATTTCATTTTAGGAATTTGAGCTGGAATAATTGGGGCAGGAATAAGACTGCTTATTCGAATTGAGCTAAGACAGCCGGGCTCTTTTCTAGGAAGGGATCAACTCTATAATACAATTGTAACAAmynthas tappensis” (AB542547.1) from Japan max. identity <88% this then is a different and likely new taxon. The closest match from current Korean studies with BLASTn identity 565/653 (87%) is WO49, an immature Amynthas sp. from Jeju that itself comes closest to the Amynthas tokioensis/Metaphire hilgendorfi spp. complexes ; about twenty other species, many placed in this group after 1972, have simple intestinal caeca. Only four have simple incised caeca as here, but they all differ in characteristics of their GMs, at least, and none of these latter are known from Korea (Blakemore unpublished). The incised caeca is assumed to be a characteristic transitional or intermediate from simple to complex/manicate. The GMs in 7-8 obviously correspond to those in 18 during amphimixis but it is not known whether they interlock serially. The shape of the spermathecae and spermathecal pores are further distinguishing characteristics of Amynthas daeari that, along with its objective DNA barcode data, now serve to define this taxon.Of urn:lsid:zoobank.org:act:9BA5299B-0BA0-4E50-8EAC-C76C2D66B2EAhttp://species-id.net/wiki/Amynthas_jinburi38.2961N, 128.3546E) just north of Seoraksan Park on East coast; collected 1st – 2nd June, 2000 by unknown person(s) and deposited in NIBR. DNA tissue sample w61b (unsuccessful at this time).IV0000213690, sub-mature specimen, figured and dissected. From Gangwon-do, Goseong-gun, Ganseong-eup, Jinbu-ri .Length. 210 mm.Width. ca. 10 mm at male pore level.Segments. 143 with some secondary annulation (from preservation?).Colour. Bleached pale yellow in aged alcohol, possibly darker in life.Prostomium. Open epilobous.First dorsal pore. 11/12.Setae. >100 per segment; e.g. 100+ on 11 and 112 counted on segment 12; approximately 16 setae intervene between male pore pads that are asetal on 18.Nephropores. Not found.Clitellum. Slightly darker at 14-16.Male pores: On 18 on small, rounded and flat porophores.Female pores. Single on 14.Spermathecal pores. At posterior of 5 and 6 approximately 0.3 C apart.Genital markings. None (sub-adult?).Septa. Nephridial forests on septa 5 & 6; 5/6/7/8 thick, 8/9 thin to base of gizzard, 9/10 aborted.Hearts. Seen in 11-13 (aborted in 10?).Gizzard. Single in 8-9.Calciferous glands. Absent.Intestine. From 15; caeca simple elongate from 27; typhlosole not noted.Nephridia. Meroic.Male organs. Holandric, testes small in 10 &11; seminal vesicles in 11 & 12.Ovaries. Compact in 13; ovisacs not found in 14.Prostates: Racemose glands not fully developed in 18 on short, muscular duct.Spermathecae. Two pairs in 6 & 7 exiting to anterior of 5/6 and 6/7 in 5 & 6  or denatured by pH.Amynthas serenus  from Pahang, Malaysia that also lacks GMs, and Amynthas? breviclitellatus  from Vietnam that differs, at least, in its GMs in 7, 18 and 19. From “Kôryô” Korea (about 30 Km from Seoul), Amynthas fibulus fibulus  is superficially similar but has spermathecal pores anteriorly in 6 & 7 (rather than posteriorly in 5 & 6) plus its caeca are incised ventrally (rather than smooth); ditto for Amynthas fibulus ranunculus  that further has slits lateral to male pores. Interestingly, Amynthas fibulus closely resembles the current specimen’s gland . It appears that many of Spermathecal pores in 5/6 and 6/7...at or near leading edge of vi, vii” and no useful figures are provided for the reader to decide.It should be here noted that morrisi-group’s possible nearest relatives from Korea would likely be Amynthas koreanus  that, however, has manicate caeca; or Amynthas kobayashii  and Amynthas geojeinsulae  that both have male fields from 17-19 but differ in simple or incised caeca, respectively; or Amynthas assimilis Hong & Kim, 2002 that, like many of its similar cited taxa, has seminal grooves on 18.If spermathecal pores were in 5/6/7 in any of the above taxa, then the The current species has simple, superficial male pores on large disc-like pads on 18. Although not fully mature, it appears unique in the Korea fauna on its combination of this aspect of its male field, spermathecal pores in 5 & 6 and its profusion of setae that number more than 100 per segment, combined with simple elongate intestinal caeca.Fresh topotypic material is required to confirm these conclusions and to provide definitive DNA data, unless refinement of techniques allows extraction from older types.


Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.We discovered that domain D of the The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in  E. coli remains an attractive host for the production of recombinant proteins, even though more complex proteins with posttranslational modifications, such as glycosylation patterns require alternative host systems ; SOD [BT008028.1]; Staphylococcal Protein A[P38507]:Domain D:GFPmut3.1 [proEDDIE [ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK NproEDDIE DNA sequence sSpAD (codon optimized):GCAGACGCACAACAGAATAAGTTTAACAAAGACCAGCAGAGCGCATTCTACGAAATTCTGAACATGCCGAATCTGAATGAGGAACAACGTAATGGCTTTATTCAGTCTTTAAAAGACGACCCATCTCAGAGCACCAACGTTCTGGGCGAAGCAAAGAAACTGAACGAATCTCAGGCACCAAAA


Exposure of neurons to 2 µM Aβ1–42 resulted in significant viability loss and cell apoptosis. Accumulation of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and activation of caspase-9 and caspase-3 were also observed after Aβ1–42 exposure. All these effects induced by Aβ1–42 were markedly reversed by DG treatment. In addition, DG could alleviate lipid peroxidation and partially restore the mitochondrial function in Aβ1–42-induced AD mice. DG also significantly increased the PGC-1α expression in vivo and in vitro, while knocking down PGC-1α partially blocked the protective effects, which indicated that PGC-1α contributed to the neuroprotective effects of DG. Furthermore, DG significantly decreased the escape latency and search distance and increased the target crossing times of Aβ1–42-induced AD mice in the Morris water maze test. Therefore, these results demonstrated that DG could attenuate Aβ1–42-induced neuronal injury by preventing mitochondrial dysfunction and oxidative stress and improved cognitive impairment in Aβ1–42-induced AD mice, indicating that DG exerted potential beneficial effects on AD.Mitochondrial dysfunction is a hallmark of beta-amyloid (Aβ)-induced neurotoxicity in Alzheimer's disease (AD), and is considered an early event in AD pathology. Diammonium glycyrrhizinate (DG), the salt form of Glycyrrhizin, is known for its anti-inflammatory effects, resistance to biologic oxidation and membranous protection. In the present study, the neuroprotective effects of DG on Aβ Alzheimer's disease (AD), with typical pathological abnormalities including amyloid plaques, neurofibrillary tangles and neuron death, is the most prevalent neurodegenerative disease in vivo and CREB-dependent gene expression played critical roles in the neuroplasticity associated with cognitive function The peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) are a small family of transcriptional coactivators which play a critical role in the control of glucose, lipid, and energy metabolism 1–42-induced oxidative stress and mitochondrial dysfunction partially via induction of PGC-1α and alleviated Aβ1–42-induced cognitive impairment, suggesting DG might be developed into a promising drug for treatment of AD.Glycyrrhizin (GL), which is extracted in liquorice root, has a wide range of pharmacological actions including anti-virus, anti-allergenic and anti-immune-mediated cytotoxicity 1–42  was dissolved in 1% NH3·H2O at a concentration of 1 µg/µl and incubated at 37°C for 5 days to allow for fibril formation. DG was purchased from Jiangsu Chia-Tai Tianqing Pharmacy Company. The male ICR mice (weight range: 15–20 g) were anesthetized and Aβ1–42  was injected to bilateral hippocampus by infusion cannulae. DG was co-injected intraperitoneally with Aβ1–42. The mice were randomly assigned into four groups: the normal mice with saline or DG , and Aβ1–42-induced AD mice with saline or DG . All animal experiments were approved by the Animal Care Committee in Nanjing University and performed according to institutional guidelines. We made every effort to minimize the number of mice used and their suffering.The Aβ5 cells/ml on poly-D-lysine-coated plates. Cells were maintained in Neurobasal media supplemented with B27  and 25 nM glutamine at 37°C in a humidified 5% CO2 incubator. The purity of neurons was over 95%. The cells at day 8 were incubated with 2 µM Aβ1–42 with DG or saline for 24 h.Primary cortical neurons were prepared from E15–17 mouse embryo. Cortexes were dissected and plated at 4×102 incubator.HEK293T, BV-2 and RAW264.7 cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM containing 10% of heat-inactivated fetal bovine serum (FBS), 2 mmol/L of L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in a humidified 5% COSmall hairpin RNAs (shRNAs) were synthesized and subsequently cloned into pCMV-U6 vector using Bbsl and BglII . Five PGC-1α shRNAs sequences (shP1–shP5) were designed to target mouse PGC-1α gene. The plasmid expressing scrambled shRNA (sh-con) was used as a negative control. ShRNA sequences were as follows:5′-TTTGGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGCTTTTTG-3′, Reverse: 5′-GATCCAAAAAGCCATTGTTAAGACCGAGAATCTCGAGATTCTCGGTCTTAACAATGGC-3′;shP1: Forward: 5′-TTTGCCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGGTTTTTG-3′, Reverse: 5′-GATCCAAAAACCCATTTGAGAACAAGACTATCTCGAGATAGTCTTGTTCTCAAATGGG-3′;shP2: Forward: 5′-TTTGCGGAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTTTTTTTG-3′, Reverse: 5′-GATCCAAAAAAAGACTATTGAGCGAACCTTAACTCGAGTTAAGGTTCGCTCAATAGTCTCCG-3′;shP3: Forward: 5′-TTTGCGGTAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTATTTTTTG-3′, Reverse: 5′-GATCCAAAAAATAACTATGCAGACCTAGATACCTCGAGGTATCTAGGTCTGCATAGTTACCG-3′;shP4: Forward: 5′-TTTGTCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGATTTTTG-3′, Reverse: 5′-GATCCAAAAATCCAGTAAGCACACGTTTATTCTCGAGAATAAACGTGTGCTTACTGGA-3′.shP5: Forward: 5′-TTTGGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGCTTTTTG-3′, Reverse: 5′-GATCCAAAAAGCATTGCTTCTGTGTAAATTACTCGAGTAATTTACACAGAAGCAATGC-3′.sh-con: Forward: The oligonucleotides were synthesized by Biocolor BioScience and Technology Company . shRNAs were transfected into neurons using Lipofectamine 2000  according to the manufacturer's instructions. Cells were harvested for RT-PCR and western blotting at 24 h after the transfection.Apoptosis was determined by Annexin V-FITC apoptosis detection kit . After treatment, the cells were rinsed with PBS twice, centrifuged at 600 g for 10 min and resuspended in 0.5 ml binding buffer containing 5 µl Annexin V and 5 µl propidium iodide (PI), and then incubated for 15 min at 37°C in the dark. The apoptotic rate was examined by flow cytometry.Cell viability was determined using the conventional MTT assay. After treatment, primary cortical neurons were treated with 0.5 mg/ml MTT for 4 h at 37°C. The formazan crystals were dissolved in 100 ml of DMSO and the absorbance was measured at 570 nm in a plate reader. Cell survival rates were expressed as percentages of the value of normal cells.LDH is the most widely used marker in cytotoxicity study. At the end of incubation, the supernatant was collected from plates and the LDH content was determined using an LDH assay kit according to the manufacturer's instructions . LDH cytotoxicity was calculated by the following formula: LDH cytotoxicity = (sample OD−blank OD)/(standard solution OD−blank standard solution OD )×2000.Change of the mitochondrial transmembrane potential in neurons was quantified by JC-1 . Briefly, neuronal cells were centrifuged at 600 g for 10 min, and resuspended in 0.5 ml medium containing 5 µM JC-1. After 20 min of incubation at 37°C in the dark, the cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.To monitor intracellular accumulation of ROS, flow cytometry was used with commercial kit  according to the manufacturer's instructions. After treatment, the cells were harvested, rinsed with PBS twice, centrifuged at 600 g for 10 min, and then resuspended in 10 µM DCFH-DA solutions. After 20 min of incubation at 37°C, cells were washed with PBS twice and resuspended in 0.5 ml PBS. Samples were analyzed by flow cytometry.The levels of 4-HNE from hippocampus and serum were measured by the ELISA kits  according to the manufacturer's instruction. Briefly, supernatant from hippocampus or serum were added into the 96-well plate coated with purified anti-4-HNE antibody, and then HRP-labeled 4-HNE antibody was added. The absorbance was measured at 450 nm and the concentration of 4-HNE was determined by comparing the O.D. of the sample to the standard curve.For measurement of cytochrome c release, the mitochondrial and cytosol fractions were prepared according to the manufacturer's instructions . Briefly, mice hippocampus were washed twice with cold PBS, resuspended in fresh cytosolic extract buffer and incubated for 30 min on ice with frequent tube tapping. Tissues were homogenized on ice, and then nuclei, unbroken cells, and cell debris were pelleted at 600 g for 10 min at 4°C. The supernatant was spun again at 13,000 g for 20 min at 4°C. The supernatant was carefully transferred and the final pellet was used as the mitochondrial fraction. The cytochrome c levels were determined using a monoclonal antibody to cytochrome c by western blotting as described below.Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dry milk for 1 h and incubated overnight at 4°C with rabbit anti-cleaved caspase-3 , rabbit anti-caspase-3 , rabbit anti-caspase-9 , rabbit anti-PGC-1α , mouse anti-cytochrome c , or mouse anti-GAPDH  antibody. GAPDH was used as a loading control. The proteins were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies and visualized with chemiluminescence reagents provided with the ECL kit  and exposure to film. The intensity of the blots was quantified with densitometry.Caspase-3 and caspase-9 activities of primary cortical neurons were measured by means of colorimetric assay kits , according to the manufacturer's instructions. In brief, harvested cells were incubated with 50 µl lysis buffer on ice for 30 min, followed by centrifugation at 10,000 g for 1 min at 4°C. Then, cells were suspended in 50 µl 2×reaction buffer and 5 µl caspase-3 or caspase-9 substrate incubating for 4 h at 37°C. Later, the absorbance was read in a microplate reader at 400 nm.Real-time PCR was performed as described previously 5′-TGACACAACGCGGACAGAA-3′, Reverse: 5′-GGTAGGTGATGAAACCATAG-3′;PGC-1α: Forward: 5′-GCCAAGGCTGTGGGCAAGGT-3′, Reverse: 5′-TCTCCAGGCGGCACGTCAGA-3′.GAPDH: Forward: The PCR products were analyzed on 1.5% agarose gels and visualized by ethidium bromide. The gel was visualized with UV-transilluminator and photographed.5′-ATAAACGCGTAATGTGTGGCCGAACACACTGT-3′, Reverse: 5′-CGCCGAGATCTAAAGCTATTAAAAAGTAGGCT-3′) to facilitate directional cloning. The PCR products were cloned into the pGL3 basic in sense orientation (designated as p-PGC-3K). The truncated constructs were made using the following primers:The promoter regions of mouse PGC-1α (−3000 to 0 bp) were amplified using PCR, DNAs of primary cortical neurons as templates, and specific primers with MluI and BglII restriction enzyme  cut sites engineered on the ends : Forward: 5′-ATAAACGCGTAATGTGTGGCCGAACAC-3′, Reverse: 5′-GTCGAGATCTTCTACTTTCCACACAGTC-3′;−3000–2000 bp (named as p-PGC-1K): Forward: 5′-ATAAACGCGTAATGTGTGGCCGAACAC-3′, Reverse: 5′-CCGCCGAGATCTTCTGACTTTATATAGTC-3′;−3000–1500 bp (named as p-PGC-1.5K): Forward: 5′-ATAAACGCGTAATGTGTGGCCGAACACACT-3′, Reverse: 5′-GCCGAGATCTTCCAACCCTAGTGCCTTG-3′;−3000–1000 bp (named as p-PGC-2K): Forward: 5′-ATAAACGCGTAATGTGTGGCCGAACACACT-3′, Reverse: 5′-GCCGAGATCTGATTTTCTTTCTCTCTCTCCT-3′;−3000–500 bp (named as p-PGC-2.5K): Forward: 5′-AAATAAACGCGTGGGGGTGTTGCCTTCAAAC-3′, Reverse: 5′-GCCCCGAGATCTAAAGCTATTAAAAAGTAGG-3′.−500–0 bp (named as p-PGC-500 bp): Forward: The sequence of −100–0 bp in the PGC-1α promoter (named as p-PGC-100 bp), CREB binding site mutation sequence (named as p-PGC-100 bp mutate) and deletion sequence (named as p-PGC-100 bp delete) were synthesized with MluI and BglII restriction enzyme cut sites as followers:5′-CGCGTGAGGGCTGCCTTGGAGTGACGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3′, Reverse: 5′GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACGTCACTCCAAGGCAGCCCTCA-3′;p-PGC-100 bp: Forward: 5′-CGCGTGAGGGCTGCCTTGGAGTGTGGTCAGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3′, Reverse: 5′-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCTGACCACACTCCAAGGCAGCCCTCA-3′;p-PGC-100 bp mutate: Forward: 5′-CGCGTGAGGGCTGCCTTGGAGGGAGTTTGTGCAGCAAGCTTGCACAGGAGAAGGGAGGCTGGGTGAGTGACAGCCCAGCCTACTTTTTAATAGCTTTA-3′, Reverse: 5′-GATCTAAAGCTATTAAAAAGTAGGCTGGGCTGTCACTCACCCAGCCTCCCTTCTCCTGTGCAAGCTTGCTGCACAAACTCCCTCCAAGGCAGCCCTCA-3′.p-PGC-100 bp delete: Forward: Renilla were cotransfected to cells followed by DG treatment for 24 h. The Luciferase activity was assayed by using the Promega Bright-N-Glo system as previously described All transfection experiments in this study were performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. pPGCs and phRL-CMV The Morris water maze test was conducted as previously described post hoc test with day and treatment as the sources of variation. Otherwise comparison between two groups was statistically evaluated by Student's t-test and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey post hoc test. Values of P<0.05 were considered statistically significant.The data were expressed as means ± SEM and analyzed by SPSS12.0 statistical analytical software . Group differences in the escape latency, searching distance and swimming speed during the Morris water maze test were analyzed using two-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni 1–42 (2 µM) and different concentrations of DG or saline. As expected, the viability of cortical neurons exposed to Aβ1–42 was reduced by 34.1% in comparison with the control group . To further investigate DG's ability to inhibit Aβ-induced oxidative stress, a marker of lipid peroxidation, 4-HNE was examined. As shown in 1–42-induced AD mice were significantly increased by 40.4% and 67.3% compared to control mice respectively (P<0.05), while 4-HNE levels were reduced 24.2% and 33.2% in the serum and hippocampus after DG treatment respectively (P<0.05).Emerging evidence suggests that mitochondrial dysfunction and oxidative stress are involved in Aβ1–42 (P<0.01). However, the decrease of Δψ induced by Aβ1–42 was greatly alleviated after DG treatment (P<0.01), indicating that DG protected mitochondrial against Aβ1–42-induced injury. Meanwhile, the activities of caspase -9 and caspase-3, were assessed. As shown in 1–42-treated neurons, while DG-treated neurons exhibited lower caspase-9 and caspase-3 activities compared to Aβ1–42-treated neurons (P<0.05).Mitochondrial membrane potential (Δψ) is widely recognized as an indicator of mitochondrial functionality, which is measured by JC-1, a cationic lipophilic fluorescent. The results showed that there was a significant loss of Δψ in neurons treated with Aβ1–42 , P<0.01.reatment , P<0.01,in vivo, the release of cytochrome c from the mitochondrial membrane as well as the subsequent activation of caspase-9 and caspase-3 was investigated by western blotting. The levels of cytosolic cytochrome c expression in Aβ1–42-induced AD mice were significantly increased, which were significantly reversed by the treatment with DG , 6 h (1.47±0.08-fold), 12 h (1.70±0.11-fold), 24 h (2.11±0.48-fold) and 48 h (1.24±0.38-fold)  , with si38-fold) . PGC-1α pression . To demopression  and the  neurons .P<0.01). Also DG treatment significantly up-regulated the transcriptional activity of PGC-1α by 4.08-fold in HEK293T cells, 1.98-fold in BV-2 cells and 1.62-fold in RAW264.7 cells at a concentration of 0.001 µg/µl, indicating that induction of transcriptional activity of PGC-1α by DG may not be cell type specific (P<0.01), which indicated that −500–0 bp in the promoter of PGC-1α might be involved in the protective effects of DG  = 3.620, P = 0.016; group x day: F = 0.915, P = 0.516). In addition, the search distance was also significantly decreased by DG treatment compared to AD mice  = 41.688, P = 0.014; days: F = 11.536, P = 0.01; group x day: F = 1.942, P = 0.067, P<0.05, 1–42-induced AD mice.To explore whether DG could improve cognitive impairment in Aβ1–42-induced AD model in vitro and in vivo, this study for the first time shows: 1) DG exerts neuroprotective effects and improves cognitive impairment; 2) DG rescues mitochondrial dysfunction and inhibits oxidative stress; and 3) DG increases the expression of PGC-1α, which might contribute to the neuroprotection of DG.In the Aβ1–42-induced neuronal toxicity, and is considered as an early event in AD pathology. Several evidences indicated that Aβ triggered mitochondrial dysfunction through a number of pathways such as increase of ROS, interaction with ABAD, impaired mitochondrial biogenesis, and alteration of mitochondrial dynamics 1–42-treated neurons. In addition, DG decreased lipid peroxidation and release of cytochrome c from the mitochondria, and the activation of caspase-9 and caspase-3 in Aβ1–42-induced AD mice. Furthermore, this anti-oxidation function of DG could refrain neurotoxicity mediated by Aβ1–42, that is, increased cell viability, decreased apoptosis and LDH release in Aβ1–42-treated neurons.Mitochondrial dysfunction is a hallmark of Aβ1–42-treated neurons.Regardless of the possible mechanisms of DG restraining oxidative stress, it is clear that PGC-1α is a major regulator of mitochondrial biogenesis and is protective against oxidative damage 1–42-treated neurons and CREB might play an important role in induction of the transcriptional activity of PGC-1α. PGC-1α was a direct target of CREB induction of gluconeogenesis in vivoIt is intriguing that DG treatment increased the expression of PGC-1α in Aβ1–42-induced toxicity in vitro and in vivo. DG significantly increased the viability of Aβ1–42-treated neurons by inhibiting oxidative stress and reversing mitochondrial dysfunction. Furthermore, PGC-1α upregulated by DG treatment might play an important role against Aβ1–42-induced neurotoxicity. Findings of current study revealed new function and mechanism of DG on neurotoxicity induced by Aβ1–42, suggesting that DG may be developed into a new drug for treatment of AD.Taken together, DG exerted neuroprotective effects against Aβ


Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by  Methanocaldococcus jannaschii (M. jannaschii) tyrosyl-tRNA synthetase (TyrRS) to charge an amber codon-suppressing tRNA with an UAA Transfer ribonucleic acids (tRNAs) serve as an adaptor molecule, bridging between genetic information and amino acid sequences during protein biosynthesis. Aminoacyl-tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to a corresponding tRNA and have evolved active sites that are highly specific for a given substrate M. jannaschii TyrRS can only be used in prokaryotic cells; they are not orthogonal in eukaryotic systems M. jannaschii TyrRS can thus be used in a eukaryotic system if they are manipulated into ignoring endogenous tRNAs.All of the existing unnatural aaRSs derived from M. jannaschii TyrRS can be made orthogonal in mammalian cells by using peptide transplantation involving the connective polypeptide 1 (CP1) domain of E. coli TyrRS M. jannaschii that is not orthogonal in mammalian cells can be made orthogonal by switching C1:G72 to G1:C72 thus generating 1bp-tRNACUAM. jannaschii TyrRS (aaRS) specific to an unnatural amino acid in E. coli allowing it to charge 1bp-tRNACUA with the same unnatural amino acid in mammalian cells We have previously shown that M. jannaschii in mammalian cells, the tRNA was previously flanked with sequences taken from a human tyrosyl-tRNA gene. Six tandem repeats of the resulting genes were cloned into plasmid pZeoSV2 (+) to create 6x_wt-tRNACUA  of green fluorescent protein (GFP) gene in vector p-EGFP-N1 to afford p-GFP_39TAG. Mammalian HEK 293T cells were co-transfected with p-GFP_39TAG and the plasmid expressing the amber-suppressor tRNA. Suppression of the 39th amber codon TAG led to the expression of a full-length GFP in vivo  region in recognizing its corresponding tRNA acceptor stem M. jannaschii TyrRSs (E. coli (pdb.org 1X8X) and M. jannaschii TyrRS (pdb.org 1J1U) were used to design six E. coli CP1 swapped mutants: TyrRS_36CP1, TyrRS_39CP1, TyrRS_39CP1_2, TyrRS_42CP1, TyrRS_44CP1, and TyrRS_fullCP1. Amino acid sequence alignment  of the two TyrRS was used to design another three CP1-transplanted mutants: TyrRS_29RED, TyrRS_34RED, and TyrRS_38RED. The N-terminal regions of these swaps are similar to the structural homology designs, although the C-terminal region is based on a RED sequence that is shared by both TyrRSs. The crystal structure of Thermus thermophilus (T. thermophilus) TyrRS is also available, and it is known to charge a G1:C72-containing tyrosyl-tRNA T. thermophilus TyrRS and the M. jannaschii TyrRS, two additional CP1-substituted mutants were designed: TyrRS_39tt and TyrRS_45tt. All CP1-swapped TyrRSs also contained an Arg286 mutation that has previously been shown to increase recognition of the anticodon region of a suppressor tRNA by M. jannaschii TyrRSs Our next focus was manipulating the i TyrRSs Figure 4CUA, and with a plasmid containing a gene for a CP1-swapped TyrRS, harvested 72 hours later, and analyzed by FACS analysis. Since the tRNA is not recognized by any endogenous aaRS, full-length GFP could only be detected when the 1bp-tRNACUA was charged by the CP1-swapped TyrRS. Wild type E. coli TyrRS was used as a positive control and offered a 13.5-fold increase in fluorescence over that seen when cells were transfected with a plasmid harboring 1bp-tRNACUA only  orthogonality in the mammalian host system; b) stop codon-suppression efficiency; c) expression of a protein, reaching yields up to five-fold higher than realized with previously used systems that rely on the amber suppression approach in mammalian cells. These improvements pave the way for efficient and specific incorporation of UAA into recombinant mammalian proteins.E. coli TOP10 cells and isolated using a Qiagen Miniprep Kit. HEK293T cells (ATCC® CRL-11268TM) were cultured in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% (v/v) fetal bovine serum  in a 37°C humidified incubator containing 5% CO2. Transfections were performed using FuGENE HD according to product manual . Full-length GFP was excited at 460-500 nm and detected using a Nikon Eclipse TE2000-S microscope equipped with a FITC HyQ filter (Chroma).All plasmids were amplified in The commercially available plasmid p-EGFP-N1 (Clontech) was used to express a gene for EGFP using a CMV promoter. The 39th codon in the EGFP gene was mutated from TAC to TAG by site-directed mutagenesis (SDM) using primers designed by the Stratagene online primer design program5′-GCGAGGGCGAGGGCGATTAGACCTACGGCAAGC-3′5′-GCTTGCCGTAGGTCTAATCGCCCTCGCCCTCGC-3′A QuikChange II Site-Directed Mutagenesis Kit was purchased from Stratagene , and site-directed mutagenesis was completed according to the provided protocol. Plasmids were sequenced by the University of Texas at Austin ICMB DNA Sequencing Facility. The new plasmid created was termed plasmid GFP_39TAG.The MCS-1 region from a commercially available plasmid, pTRE-TIGHT-BI (Clontech) was removed by digesting the plasmid with the KpnI and EcoRI restriction enzymes . Oligonucleotides, containing a sequence for the T7 promoter:5′-GGGGTACCCCTAATACGACTCACTATAGGGGGAATTCC-3′5′-GGAATT-CCCCCTATAGTGAGTCGTATTAGGGGTACCCC-3′were annealed and digested with KpnI and EcoRI. The digested insert was then ligated using DNA T4 Ligase into the pre-digested p-TRE-TIGHT-BI plasmid to form plasmid p-TRE-TIGHT-T7. Next, oligonucleotides containing the sequence for the human H1 promoter were annealed and extended using a Klenow enzyme. The oligonucleotide sequences were:5′-CAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAAC ACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAA-3′5′-GAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTG ATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCC-3′The sequence was then amplified by PCR using the primers:5′-GGAATTCCAATTCGAACGCTGACGTCATCAACCCGCTCCAAGG AATC-3′5′-GAAGATCTGTGGTCTCATACAGAACTTATAAGATTCCCA-3′The resulting product was digested with the KpnI and BglII restriction enzymes and ligated into a pre-digested p-TRE-TIGHT-T7 plasmid to generate plasmid pT7-H1.M. jannaschii TyrRS (amino acids 110-148) with the CP1 region of E. coli TyrRS (amino acids 129-172). The 5′ end of the gene (containing sequences for amino acids 1-109) was amplified using the following primers:TyrRS_44CP1 was created by replacing the CP1 region of 5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3′The 3′ end (containing amino acids 149-306) was amplified by PCR using primers:5′-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3′5′-CCCGAATTCTAATCTCTTTCTAATTGGCT-3′E. coli CP1 region (amino acids 129-172) was amplified from a wild type E. coli TyrRS gene by PCR using the following primers:Finally, the 5′-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′All three PCR fragments were combined, denatured for 15 minutes at 85°C and elongated with a Klenow enzyme for 30 minutes at room temperature. The Klenow product was again amplified by PCR, purified, and digested with the HindIII and EcoRI restriction enzymes. Finally, TyrRS_44CP1 was created by ligating the digested product into a pre-digested pEF6-V5-His6-TOPO plasmid  using T4 DNA ligase (NEB). All other CP1-transplanted mutants were created by PCR in a process similar to that described in the previous section. The chimeric gene was divided into three segments, and each was amplified by PCR. Finally, a joint gene was also amplified by PCR, digested, and ligated into plasmid pEF6-V5-His6-TOPO. Primers are listed for each of the segments, based on the size and sequence of the CP1 swap.TyrRS_36CP1:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-CGCAGGAAGGTCAGCACATTCATCTGGAATTCACTTCCATAAACAT-3′5′-ATGTTTATGGAAGTGAATTCCAGATGAATGTGCTGACCTTCCTGCG-3′5′-TGGATAGATAACTTCAGCAACAATCCCCTGATCTTCACGGTTGAGA-3′5′-TCTCAACCGTGAAGATCAGGGGATTGTTGCTGAAGTTATCTATCCA-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_39CP1:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3′5′-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3′5′-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3′5′-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_39CP1_2:5′-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3′5′-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3′5′-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3′5′-ATAACTTCAGCAACCTTTGGCCCCTGATCTTCACGGTTGA-3′5′-TCAACCGTGAAGATCAGGGGCCAAAGGTTGCTGAAGTTAT-3′5′-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3′TyrRS_42CP1:5′-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3′5′-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3′5′-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3′5′-TTCAGCAACCTTTGGATTACGGTTGAGACGCTGCTTAACC-3′5′-GGTTAAGCAGCGTCTCAACCGTAATCCAAAGGTTGCTGAA-3′5′-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3′TyrRS_44CP1:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-ACATTCATATTGCCGAACCACTGGAATTCACTTCCAT-3′5′-AGGGGATTTCGTTCACTGAGGTTATCTATCCAATAATGCA-3′5′-CCCGAATTCTAATCTCTTTCTAATTGGCT-3′5′-ATGGAAGTGAATTCCAGTGGTTCGGCAATATGAATGT-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_fullCP1:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3′5′-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3′5′-CCAACTGCAACATCAACGCCACCGTACTGTTTGTTCAGAC-3′5′-GTCTGAACAAACAGTACGGTGGCGTTGATGTTGCAGTTGG-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_29RED:5′-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3′5′-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3′5′-AAGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3′5′-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3′5′-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3′5′-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3′TyrRS_34RED:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-ACATTCATATTGCCGAACCAATAAACATATTTTGCCTT-3′5′-AGGCAAAATATGTTTATTGGTTCGGCAATATGAATGT-3′5′-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3′5′-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_38RED:5′-GCGGATCCGCCACCATGGACGAATTTGAAATGATAAAGAGAA-3′5′-TCATAGTTGTTCGCCGCGATTGCCTTTAACCCCATTGCTT-3′5′-AAGCAATGGGGTTAAAGGCAATCGCGGCGAACAACTATGA-3′5′-TTGGATTTTCATCCTCTCTGTTGAGACGCTGCTTAACCGC-3′5′-GCGGTTAAGCAGCGTCTCAACAGAGAGGATGAAAATCCAA-3′5′-GCTCTAGAGCTTATAATCTCTTTCTAATTGGCTCTAAAATCTTTATA AGTTCTTCAGCTACAGCATTTTTTAACCTCATTGGATGCAATTCCTT-3′TyrRS_39tt:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-GTGAGGCCCTCCAGCCACTCGGAATAAACATATTTTGCCTTTAAC-3′5′-GTTAAAGGCAAAATATGTTTATTCCGAGTGGCTGGAGGGCCTCAC-3′5′-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3′5′-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′TyrRS_45tt:5′-AAGGATCCACCATGGACGAATTTGAAATGAT-3′5′-CTCGGAGTTGTAGCGGAGCTCAAATAACCCCATTGCTTCAAAAAC-3′5′-GTTTTTGAAGCAATGGGGTTATTTGAGCTCCGCTACAACTCCGAG-3′5′-ATAACTTCAGCAACCTTTGGGGGAATCCCCGCCTCGTACCGCTTC-3′5′-GAAGCGGTACGAGGCGGGGATTCCCCCAAAGGTTGCTGAAGTTAT-3′5′-TGCATTATTGGATAGATAACTTCAGTGAACGAAATCCCCT-3′.CUA gene were annealed, extended using a Klenow enzyme, and amplified by PCR. The sequences of oligonucleotides were:Two oligonucleotides for the 1bp-tRNA5′-GAAGATCTGCGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCT AAATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCGCAGACAAGTGCGGTTTTTTT-35′-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGCGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3′CUA plasmid.The PCR product was then digested with the BglII and PstI restriction enzymes and ligated into a pre-digested pT7-H1 (described above) plasmid to create the 1bp-tRNACUA was created in a similar fashion using the following oligonucleotides to create the tRNA gene:H1_2bp-tRNA5′-GAAGATCTGGGGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCCCCAGACAAGTGCGGTTTTTTT-3′5′-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTGTC TGGGGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3CUA was generated using the following oligonucleotides:Finally, H1_3bp-tRNA5′-GAAGATCTGGTGCGGTAGTTCAGCCTGGTAGAACGGCGGACTCTA AATCCGCATGTCGCTGGTTCAAATCCGGCCCGCACCAGACAAGTGCGGTTTTTTT-3′5′-CCAATGCATTGGTTGCCCGCTCGAGTAGAAAAAAACCGCACTTG TCTGGTGCGGGCCGGATTTGAACCAGCGACATGCGGATTTAGAGTCCGCCGTTCTA-3′.E. coli TyrRS was amplified from a plasmid by PCR using the following primers:A gene for 5′-GCTCTAGATTATTTCCAGCAAATCAGACAGTA-3′5′-CGGGATCCATGGCAAGCAGTAACTTGATTAAA-3′E. coli TyrRS in HEK293T cells.The PCR product was digested with the XbaI and BamHI restriction enzymes  and ligated using T4 ligase into a pre-digested pEF6-V5 plasmid to express All new sequences of TyrRSs have been deposited in GenBank with the following accession numbers:TyrRS_36CP1: KF050826TyrRS_39CP1: KF050827TyrRS_39CP1_2: KF050828TyrRS_42CP1: KF050829TyrRS_44CP1: KF050830TyrRS_fullCP1: KF050831TyrRS_29RED: KF050832TyrRS_34RED: KF050833TyrRS_38RED: KF050834TyrRS_39tt: KF050835TyrRS_45tt: KF050836A BD FACSCalibur flow cytometer was used to gate approximately 10,000 cells, based on forward and side scatter. Each cell was then excited at a wavelength of 488 nm and the resulting emission was detected with an FL-1 filter (515–545 nm). Positive fluorescent populations were gated based on negative or positive controls and analyzed using Cyflogic v.1.2.1 computer software .


The aim of this study was to investigate the presence of nerve growth factor (NGF) and its receptors tyrosine kinase A (TrkA) and p75 in the ovaries of the wild ground squirrels during the breeding and nonbreeding seasons. In the breeding period, NGF, TrkA and p75 were immunolocalized in granulosa cells, thecal cells, interstitial cells and luteal cells whereas in the nonbreeding period, both of them were detected only in granulosa cells, thecal cells and interstitial cells. Stronger immunostaining of NGF, TrkA and p75 were observed in granulosa cells, thecal cells and interstitial cells in the breeding season compared to the nonbreeding season. Corresponding for the immunohistochemical results, immunoreactivities of NGF and its two receptors were greater in the ovaries of the breeding season then decreased to a relatively low level in the nonbreeding season. The mean mRNA levels of NGF, TrkA and p75 were significantly higher in the breeding season than in the nonbreeding season. In addition, plasma gonadotropins, estradiol-17β and progesterone concentrations were significantly higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, and TrkA and p75 were correlated with changes in plasma gonadotropins, estradiol-17β and progesterone concentrations. These results indicated that NGF and its receptors, TrkA and p75 may be involved in the regulation of seasonal changes in the ovarian functions of the wild ground squirrel. The nerve growth factor (NGF) belongs to a family of related proteins required for the survival, maintenance, and development of discrete neuronal populations in the central and peripheral nervous systems -3. It isIt is now well known that NGF and its receptors are expressed in the mammalian ovary, including women -14, ratsCitellus dauricus Brandt) is a typical seasonal breeder which has a strict and extremely compressed breeding period  from April to May and a long period of sexual dormancy from June to the following March including a 6-month hibernation period [The wild ground squirrel . Wild female ground squirrels that were regarded as adults according to their body weights (242–412 g) were captured on April 13 (10.2 hours of daylight) after emergence from hibernation in the breeding period (n = 10) and on June 9 (12.6 hours of daylight) in the nonbreeding period (n = 8) of 2009 in Hebei Province, PR China.Animals were anesthetized with 4% isoflurane and blood samples were rapidly collected from leg vein. Plasma samples were frozen and stored at -20C, after the blood samples were added heparin sodium and centrifuged . An overdose of pentobarbital was applied afterwards for euthanasia. Ovary and brain were quickly removed and dissected. Length, width and weight of each ovary were measured. A part of the tissues were fixed in 0.05 M phosphate-buffered saline  containing 4% paraformaldehyde for histological and immunohistochemical observation, while the rest were immediately frozen in liquid nitrogen and stored at -80C for RNA isolation and protein extraction.Ovarian samples were dehydrated in ethanol series and embedded in paraffin wax. Serial sections (4 μm) were mounted on slides coated with poly-L-lysine . Sections were stained with hematoxylin-eosin (HE) for observations of general histology. The sections were screened using an Olympus photomicroscope with a × 20 objective lens and imaged with software Image-Pro Plus 4.5 . Every one in ten serial sections, and altogether 50 and 30 sections for the breeding and nonbreeding season ovary respectively were selected for follicle identification [2O2. Finally, the sections were counterstained with hematoxylin  and NGF, TrkA and p75 were detected, respectively. The immunostained slides were scanned using the software Image-Pro Plus 4.5  at 20× magnification. The specificity of NGF and its receptors, TrkA and p75 antibodies have been described previously [Ovarian sections were blocked with 10% normal goat serum to prevent the non-specific binding of the second antibody. The sections were then incubated with polyclonal primary antibody against NGF , TrkA  or p75   for 12 h at 4C, and incubated with the second antibody, goat anti-rabbit IgG conjugated with biotin and peroxidase with avidin for 1 h at room temperature. The sections were visualized using a rabbit ExtrAvidin™ staining kit  in 150 ml of 0.05 M Tris–HCl buffer containing 30 mg 3,3-diaminobenzidine  plus 30 μl Heviously . The imm2O2 and visualized with Odyssey infrared imaging system. Brain tissue of wild ground squirrel was used as a positive control and water, instead of primary antisera, was used as a negative control. β-actin was selected as the endogenous control. The intensities of the bands were quantified using Quantity One software  and expression ratios were calculated.Ovarian tissues were weighed and dissected into small pieces using a clean razor blade. The tissues were homogenized in a tissue homogenizer containing 300 μl of 10 mg/ml PMSF and incubated for 30 min on ice. Homogenates were centrifuged at 12,000 g for 10 min at 4C. Protein extracts (25 μg) were mixed with equal volumes of 2 × Laemmli sample buffer. Equal amounts of proteins from each sample were loaded onto a 12% SDS-PAGE gel and electrophoretically separated at 18 V/cm and transferred to nitrocellulose membrane using a wet transblotting apparatus . The membrane was blocked in 3% BSA for 1 h at room temperature. Primary incubation of the membrane was carried out using NGF, TrkA or p75 antibody (1:1000 dilution) for 1 h at room temperature. Secondary incubation of the membrane was then carried out using an IRDye  for 1 h at room temperature. Finally, the membrane was washed in 25 ml Tris-Buffered Saline with Tween-20  plus 3 μl HTotal RNA from each sample was extracted using ISOGEN . Approximately 1 g of ovarian tissues were thawed and immediately homogenized in 10 ml of ISOGEN™. The homogenate was incubated for 5 min at room temperature to allow the complete dissociation of nucleoprotein complexes. After the addition of 2 ml of chloroform, the mixture was vigorously shaken for 3 min at room temperature and centrifuged at 12,000 g for 10 min at 4C. The aqueous phase was then transferred to a fresh tube and washed with an equal volume of chloroform. An equal volume of isopropanol was added, and the sample was kept for 10 min at room temperature. RNA was precipitated by centrifugation at 12,000 g for 10 min at 4C. The RNA pellet was washed twice with 75% ethanol, briefly dried under air, and dissolved in 100 μl of diethylprocarbonate-treated water.12–18 according to the manufacturer’s protocol. The 20 μl of reaction mixture contained 4 μg of total RNA, 0.5 μg of oligo (dT)12–18, 2.5 mM MgCl2, 0.5 mM dNTP, 10 mM dithiothreitol, 20 mM Tris–HCl (pH 8.4) and 200 U of Superscript II enzyme. The first-strand cDNA was used for PCR amplification with the appropriate primers previously proved  and 2.5 U of Taq polymerase . The amplification was under the following condition: 94C for 5 min for the initial denaturation of the RNA/cDNA hybrid, 30 cycles of 94C for 1 min, 52C for 1 min, and 72C for 2 min for amplification. The PCR product was electrophoresed in the 2% agarose gel and individual bands were visualized by ethidium bromide staining. Brain tissue of wild ground squirrel was used as positive control and water, instead of cDNA, was used as negative control. The housekeeping gene, RpL7, was selected as the endogenous control as it is an estrogen-independent gene. The bands were quantified using Quantity One software  and expression ratios were calculated.The first-strand cDNA from total RNA was synthesized using Superscript II Reverse Transcriptase  and oligo (dT)E. coli using TOPO TA Cloning Kit . Plasmids were extracted from the bacteria and positive clones containing the proper insert were sequenced in both directions using Thermo Sequenase II Dye Terminator Cycle Sequencing Premix Kit  with an automatic sequencing system . After obtaining the sequence of each PCR product, we blasted with the known mRNA sequences of mouse , rat , bovine  and human , find the homologous sequence fragments in each species and compare for homology using DNAman.The purified PCR products were ligated into pCR 2.1-TOPO  and the ligation products were used to transform the competent 125I-labeled radioligands as described previously [Plasma concentrations of estradiol-17β and progesterone were determined by double-antibody RIA systems using eviously . Antisereviously  and progeviously  was kindMeans and standard deviations were calculated. Data were analyzed using a one-way ANOVA and the means were compared for significance using Duncan’s Multiple Range Test (P = 0.05) using the SPSS computer software package.Ovaries of breeding and nonbreeding seasons were observed morphologically and histologically Figure . In lineImmunohistochemistry was performed to detect the localization pattern of NGF and its receptors in the wild ground squirrel ovary and representative stainings were shown in Figure We then moved on to detect the protein and mRNA expression levels of NGF, TrkA and p75 using Western blot and PCR, and representative bands were shown in Figure To further confirm the nature of the PCR signals, cDNA fragments of NGF, TrkA and p75 in ovarian tissues were sequenced and compared to the corresponding fragments in mouse, rat, bovine, and human. The partial mRNA sequences in the wild ground squirrel are as below:NGF#GGGGGACTCAGTGTGTGTGCTGGTGTCAGTGTGTGGGTTGGAGATAAGACCACAGCCACAGACATCAAGGGCAAGGAGGTGACAGTGCTGGCCGAGGTGAACATTAACAACAGTGTATTCAGACAGTACTTTTTTGAGACCAAGTGCCGAGCTrkA#GGAAGTGACATCTCTACCGCAGTTCAGCACCGAGAGCGATGTGTGGAGCTTTGGGGTGGTGCTCTGGGAGATCTTCACCTATGGAAAGCAGCCCTGGTACCAGCTCTCTAACACTGAGGCGATCGAGTGTATCACGCAGGGCCGGGAGCTGGAGCGGCCGCGCGCCTGCCCTCCTGATGTCTACGCCATCATGCGAGGCTGCTGGCAGCGAGAACCGAATCAACGCCTP75#CCAGTAGGGCAGTGTGGCGGAGCCTTGCGGAGCCATCCAGACCGTGTGTGAACCCTGCCTGGACAGTGTTACGTTCTCTGACGTGGTGAGCGCCACCGAGCCGTGCAAGCCGTGCACCGAGTGCCTGGGCCTGCAGAGTATGTCCGCTCCCTGTGTGGAGGCAGACGATGCCGTGTGCCGATGCTCCTATGGCTACTACCAGGACGAGGAGACTGGCCGCTGCGAGGCTTGCAGCGTGTGCGGGGTGGGCTCAGGATCGGTGTCCTCCCRpL7#AAGGATCTGCTGCTGCTTCTGTTCCAGATCTCAATGGCACCTTTGTTAAGCTCAACAAGGCTTCAATTAACATGCTGCGGATTGTGGAGCCATACATTGCATGGGGGTACCCCAACCTGAAGTCAGTAAACGAGCTCATCTACAAGCGAGGCTACGGCAAAATCAACAAGAAGCGGATTGCCTTGACAGATAATTCCTTGAATGCACGGTCTCTTThe 176-bp NGF cDNA nucleotide sequence identity was 92.11%, 90.20%, 81.70% and 86.18%, respectively; the 261-bp TrkA cDNA nucleotide sequence identity was 94.32%, 90.83%, 84.72% and 83.41%, respectively; the 295-bp p75 cDNA nucleotide sequence identity was 94.42%, 88.10%, 84.01% and 83.64%, respectively; the 207-bp RpL7 cDNA nucleotide sequence identity was 94.55%, 84.65%, 74.23% and 76.87%, respectively  that involves both sex steroids and growth factors.The authors declare that they have no competing interests.BL participated in performing the experiments, analyzing the data and drafting the manuscript. XS, LB, SH, QL and YL assisted with sample collection, all experiments and helped revising the manuscript. YH and QW designed, supervised the study, and revised manuscript. GW and KT provided some reagents and revised the manuscript. All authors read and approved the final manuscript.


There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA.Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition. Regions were defined by using SICER (v.1.03). Only regions that were called by using both input and 'no antibody' as a background control with Benjamini corrected false discovery rate < 0.05 were kept. Finally, only regions called in both antibody hmeDIP-seq experiments were kept and analyzed. Gene ontology analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT) . PublishFully hydroxymethylated DNA was produced by endpoint PCR using Phusion polymerase  and hm-dCTP  followed by PCR purification . Unmethylated and fully methylated control DNAs were produced in the same manner with dCTP and m-dCTP , respectively. Various amounts of DNA were denatured, snap cooled and dotted onto positively charged nylon membranes . Membranes were crosslinked, blocked with 5% milk, and incubated with Active Motif  5-hmC antibody for 1 hour. Membranes were washed and then incubated with anti-rabbit secondary horseradish peroxidase-linked antibody  for 1 hour, washed, and developed with ECL reagent (CST) and Biomax MS film (Kodak). DNA sequences were (primer sequences in bold):TACTCTATACTCTACTCATCATTACACGCGCGATATCGTTAACGATAATTCGCGCGATTACGATCGATAACGCGTTAATATGAGATATGAGATGTGTATG; 6 CG-TACTCTATACTCTACTCATCATTACAATATATATATCGTTAACGATAATTCGCGCGATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 3 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTCGCGAAATTACGATTTATAATTAATTAATATGAGATATGAGATGTGTATG; 1 CG-TACTCTATACTCTACTCATCATTACAATATATATATAATTAATTATAATTAACGAAATTATAATTTATAATTAATTAATATGAGATATGAGATGTGTATG.12 CG-Human stem cell genomic DNA 5 to 10 μg) was treated with the EpiMark 5-hmC and 5-mC Analysis Kit as per the included protocol (NEB). Briefly, DNA was either glucosylated with beta-glucosyltransferase and UDP-Glc or mock treated with beta-glucosyltransferase and no UDP-Glc for 12 to 18 hours. These reactions were then split into three and mock digested, digested with MspI, or with HpaII for at least 4 hours. Samples were treated with proteinase K that was then heat inactivated. All DNA were diluted to a final concentration of 16 ng/μl to be used for PCR analysis. Quantitative PCR was completed with iQ SYBR Green Supermix  using a CFX384 Real-Time PCR Detection System (Biorad). Primers used for quantitative PCR are listed in Table S1 in Additional file 0 μg was 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; bp: base pair; ESC: embryonic stem cell; H3K4me1: histone H3 mono-methylated at lysine 4; H3K27ac: histone H3 acetylated at lysine 27; hESC: human embryonic stem cell; hmeDIP: hydroxymethyl-DNA immunoprecipitation; RPKM: reads per kilobase per million mapped reads; TFBS: transcription factor binding site.HS, SP and SEJ designed the study. SF and SMK performed the experiments. HS and SEJ analyzed the data. HS wrote the paper.Supplementary figures and table. Figure S1: 5hmC dot blots on oligos with varying amounts of 5hmC. Figure S2: characterization of defined 5hmC peaks. Figure S3: correlation of 5hmC and gene expression. Figure S4: 5hmC and different classes of putative enhancers [nhancers . Figure nhancers . Figure nhancers ,14,15. FClick here for file


L26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the UL26 protein that contribute to viral infection, we created a panel of site-directed UL26 mutant viruses and assessed their impact on phenotypes attributed to UL26. We find that the C-terminal 38 amino acids of the UL26 protein are absolutely necessary for UL26 function. A stop-insertion mutant that produced a truncated UL26 protein lacking this region behaved identically to UL26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of UL26 packaged into the virion resulting in reduced delivery of UL26 to newly infected cells. Further, this C-terminal truncated UL26 exhibited substantially reduced nuclear localization compared to wildtype UL26. Translation of UL26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of UL26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of UL26, are also important for the function of the UL26 protein. A viral mutant that produces only the short isoform of UL26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short UL26 isoform in the presence of the long UL26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the UL26 protein that contribute to HCMV infection.The human cytomegalovirus (HCMV) U Human cytomegalovirus (HCMV), a betaherpesvirus, is a widespread opportunistic pathogen. HCMV causes severe disease in various immunosuppressed populations including the elderly, cancer patients receiving immunosuppressive chemotherapy, transplant recipients, and AIDS patients L32 and UL99 HCMV is a relatively large virus, with a ∼240-kb DNA genome that encodes >200 open reading frames. The viral particle is enveloped and its genome is encased within a protein capsid. Packaged in between the capsid and the viral envelope is a protein layer called the tegument, a structural feature unique to herpes viruses L26 gene, which has been found to be critical for high-titer viral replication L26 protein is expressed with early kinetics, and synthesis of the protein initiates at one of two start codons resulting in 21- or 27-kDa products L26 deletion grow to lower final titers, with slower growth kinetics, and exhibit a small plaque phenotype L26 has been implicated in transcriptional activation of the immediate early promoter L26 also impacts the structural characteristics of virions. These mutants are less stable than wildtype virions and contain hypophosphorylated tegument constituents L26 protein localizes to the nucleus at early times post infection, and to viral assembly compartments at late time points While many HCMV tegument proteins are known to be important for HCMV replication, the mechanisms through which many of these proteins contribute to the infectious cycle are unclear. One such tegument protein is encoded by the UL26 that contribute to UL26-dependent phenotypes through the creation of a panel of mutant UL26 viruses. Site-directed mutagenesis was employed to target both of UL26’s initiation methionines and to introduce stop codons throughout the UL26 ORF. Analysis of these mutant viruses indicates that the UL26 short isoform is dispensable in vitro when in the presence of the UL26 long isoform. In contrast, the extra N-terminal 34 amino acids of the long UL26 isoform was found to be important for UL26-dependent phenotypes, exhibiting intermediate defects in plaque size and virion stability in comparison to wildtype and UL26-null viruses. Lastly, the carboxy terminal 38 amino acids were found to be critical for wildtype replication as deletion of this domain phenocopied UL26-deletion viruses. Deletion of these of 38 amino acids reduced the nuclear localization and tegumentation of the resulting UL26 protein product. These studies indicate that distinct domains of UL26 contribute to different UL26-dependent phenotypes and shed light on how these domains contribute to HCMV replication.Here, we analyzed specific domains of UBAdwt, a bacterial artificial chromosome (BAC) clone of Ad169 4 cells per cm2. Prior to infection, cells were serum starved for 24 hours. In all infections, viral innocula were added to cells for a 2 hr adsorption period and then aspirated. For experiments involving measurement of viral titers via plaque assay, unbound virus was inactivated through a sodium citrate wash  followed by a DMEM wash immediately following viral adsorption.MRC5 fibroblasts (passages 23–29) were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum. The wild type HCMV strain used in this study was L26 mutants were derived from the BAdwt clone of Ad169 (Genebank accession number: FJ527563) L26 mutants constructed are: BAdUL26 double methionine deletion (referred to as DBmetΔ in the text); BAdUL26 1st Methionine deletion (referred to as 1stmetΔ in the text); BAdUL26 2nd Methionine deletion (referred to as 2ndmetΔ in the text); BAdUL26 double methionine deletion rescue (referred to as DBrescue in the text); BAdUL26 #68 stop codon mutant (referred to as #68stop in the text); BAdUL26 #107 stop codon mutant (referred to as #107stop in the text); BAdUL26 #146 stop codon mutant (referred to as #146stop in the text); BAdUL26 #185 stop codon mutant (referred to as #185stop in the text). Wild type BAdwt is referred as WT in the text and BAdUL26 transposon insertion virus L26 flanking sequences was recombined into BADwt through electroporation into E. coli (strain SW105) containing BAdwt. Recombination was screened by growth in kanamycin. The Kan/Isce I cassette containing BAC was then electroporated into GS1783 cells, which contain an arabinose-inducible I-Sce 1 restriction site used for negative selection L26 gene. The UL26 DBmetΔ mutant was created by deletion of the 2nd Methionine from the UL26 1stmetΔ mutant. The primers for generating mutant viruses via two sequential PCR reactions (first reaction to introduce Kan/Isce I cassette and second reaction to insert the point mutation) were as follows (5′ to 3′): UL26 1stmetΔ insertion:F-GGCCCTCGGTGCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCAll UTTCATAGGATGACGACGATAAGTAGGG; R-GCGGCTTCATGTGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACAACCAATTAACCAATTCTGATTAG;stmetΔ Met to ATCUL26 1: F -GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATCTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA;R–TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTAGATGAAGACGCTCAAACTTTTGAATGTGGGCCC GGTAGCGC; stmet to ATC negative controlUL26 1:F-GCGCTACCGGGCCCACATTCAAAAGTTTGAGCGTCTTCATGTACGCCGTTTTCGGCCTCACGAGGTCGGAGGTCACGCCA; R- TGGCGTGACCTCCGACCTCGTGAGGCCGAAAACGGCGTACATGAAGACGCTCAAACTTTTGAATGTGGGCCCGGTAGCGC;ndmetΔ insertionUL26 2:F-GCGGCGCGTTATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATAGGATGACGACGATAAGTAGGG;R- CGCATAAAATCGTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCAACCAATTAACCAATTCTGATTAG; ndmetΔ Met to ATCUL26 2: F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATCACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTGATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; ndmet to ATC negative controlUL26 2:F-ATAAGCACCGTGGGGTCATCGACCGACAAGGCGCGGCGATGACGAGTAGGCGCGCACCCGACGGCGGTTTGAATTTAGAC;R-GTCTAAATTCAAACCGCCGTCGGGTGCGCGCCTACTCGTCATCGCCGCGCCTTGTCGGTCGATGACCCCACGGTGCTTAT; UL26 DBmetΔ: F-CGCCGTTTTCGGCCTCACGAG; R-GGTGCCGATGACGCGCAACTG. The single step PCR process employs only a single set of primers that contain homology sufficient for both recombination events UL26#185stop:F-CACGGTGACGTAGCAGCACGCGGCTCACGTAGCAGGCCGATTAGCGGATGACCTGGCCGTCGGAGGATGACGACGATAAGTAGGG;R-CTCGGGCCTGCGACGCGACGCCGACGGCCAGGTCATCCGCTAATCGGCCTGCTACGTGAGCCGCAACCAATTAACCAATTCTGATTAG; UL26#146stop:F-GCTCCACGTCTTCAAAGTAGCTGTGTAGCAGGCCGCGCTCTTACAGCTGCGGCAGCGAGTCGGAGGATGACGACGATAAGTAGGG; R-GAACTTTGTAGTGCGCGCCGCCGACTCGCTGCCGCAGCTGTAAGAGCGCGGCCTGCTACACAGCAACCAATTAACCAATTCTGATTAG; UL26#107stop:F –CGGCCGCCACGCCGGCCACGCTGCGGTCCCAACTGAAAAGTTAGGCGAGTCCGATGGTGCCGAAGGATGACGACGATAAGTAGGG; R-CCAGGGTCAGTTGCGCGTCATCGGCACCATCGGACTCGCCTAACTTTTCAGTTGGGACCGCAGCAACCAATTAACCAATTCTGATTAG; UL26#68stop:F-GCGGGGTGAGGATGGTCTCCTCCACGTCGCAGACAAACAATTAGTAGCCGCGCGGATAGGGCAAGGATGACGACGATAAGTAGGG; R-GCGCGGTCGCCACCTGGATCTGCCCTATCCGCGCGGCTACTAATTGTTTGTCTGCGACGTGGACAACCAATTAACCAATTCTGATTAGL26 [7H19] , PP28 [10B4–29], UL83-coded pp65 (8F5) and pUL44  and cellular protein tubulin [Epitomics]. A rabbit UL26 N-terminal specific antibody was generated by Biomatik (http://www.biomatik.com/) using the following underlined sequence: MTSRRAPDGGLNLDD. The methionine preceding this sequence is the 2nd UL26 initiation methionine. The secondary antibodies were rabbit polyclonal  and mouse monoclonal [Abcam].Protein accumulation was assayed by Western blotting. Protein from cell lysates was solubilized in disruption buffer , separated by either 10% or 15% SDS-PAGE, and transferred to nitrocellulose in Tris-glycine transfer buffer. Blots were then stained with Ponceau S to visualize protein bands and ensure equal protein loading. The membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 (TBST), followed by incubation in primary antibody. After subsequent washes, blots were treated with secondary antibody and protein bands were visualized using the enhanced chemiluminescence (ECL) system (Pierce). The primary antibodies were specific for viral proteins UL123-coded IE1 [1B12], UViral DNA accumulation was monitored by real-time PCR. At various times post infection, medium was aspirated from cells and viral DNA was harvested in lysis buffer . The extracted nucleic acid was quantified and checked for purity through 260∶280 absorbance by NanoDrop. Quantitative PCR (qPCR) was performed using Fast SYBR green master mix, a model 7500 Fast real-time PCR system, and Fast 7500 software (Applied Biosystems) according to manufacturer’s instructions. Viral DNA was quantified with specific primer pairs targeting UL83 (pp65), 5′-CAG-GAA-GAT-TTG-CTG-CCC-GTT-CAT-3′ (forward) and (5′-GGC-TTT-ACG-GTG-TTG-TGT-CCC-AAA-3′ (reverse).For immunofluorescence, MRC5 fibroblasts were grown on glass coverslips. At various time points post infection, cells were washed once with PBS, fixed with 2% paraformaldehyde in PBS for 20 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 and 0.1% SDS for 15 min, then washed twice with PBS containing 0.05% Tween-20. Cells were subsequently blocked by overnight incubation in PBS containing 2% bovine serum albumin (BSA), 5% goat serum, 5% human serum, and 0.3% Triton X-100. Cells were incubated with anti-UL26 sera [7H19] that had been diluted 1∶2 in PBS containing 0.05% Tween-20 for 1 hr. Slides were subsequently washed with PBS containing 0.01% Tween-20 three times, incubated with fluorochrome-conjugated anti-mouse secondary antibody for 1 hr, and washed three times in the same buffer lacking antibody. Coverslips were mounted in slow-fade Gold antifade reagent (Molecular Probes) and DAPI . Confocal images were captured with FV1000 Olympus laser scanning confocal microscope. All images were captured under identical confocal settings.L26 and anti-pp65-specific western to ensure the linearity of UL26 and pp65 detection. Quantitation of UL26 and pp65-specific protein bands was performed using ImageLab software tools from BioRad.To produce partially purified virions for the analysis of their constituent proteins, WT and #185stop virus stocks were first clarified by low speed centrifugation and then centrifuged through a sorbitol cushion at 26K rpm for 1 hr. The virion pellet was then resuspended in T.N. buffer containing 20 mM Tris-HCl, PH 7.4, 100 mM NaCl, and 1.5% BSA and purified by centrifugation through a glycerol tartrate gradient as previously described Replicate cultures of MRC5 fibroblasts were infected with 25 PFU of the indicated recombinant virus. Representative plaques at day 15 post infection for each virus are shown. Areas of representative plaques for each virus were quantified by Image J and normalized to the WT plaque size. To investigate the stability of virion infectivity, an equivalent number of plaque forming units from freshly thawed viral stocks were incubated at 37°C for 0, 4, 8, or 20 hours. After the indicated incubation period, confluent MRC5 fibroblasts were infected. The percentage of plaques remaining relative to the 0 h control was plotted.To investigate virion stability after trypsin exposure, infected MRC5 cells (MOI = 3.0) were harvested when the CPE reached 80%. The media containing infectious virus was reserved, and cells were scraped in a small volume of media and sonicated. The sonicated cells and reserved culture media were combined and centrifuged at 6,000 rpm for 30 min. The supernatants were then sedimented at 38,000×g for 60 min. The pellets containing virus were resuspended in serum-free minimal essential medium. Two hundred µl of either 2.5% trypsin (Invitrogen) or media was mixed with 1.8 ml of the resuspended virus, and incubated at 37°C for 30 or 60 minutes for the trypsin treated, or 0 minutes for the media control. To inactivate the trypsin, at the end of the prescribed intervals, calf serum was added to a final concentration of 10%. The suspension was then tittered by plaque assay. The percentage of plaques remaining relative to the media control was plotted.t-test unless otherwise indicated. A probability of value (p) <0.05 was considered statistically significant. For comparison of the viral growth between wildtype and the #185stop mutant from 48–120 hpi a homoscedastic paired two-tailed ttest of viral titers at each time point was performed. Averages are plotted with either standard deviation (SD), or standard error of the mean (SE) as indicated.Statistical significance was assessed by a non-paired two tailed homoscedastic student’s L26 gene have been shown to be growth attenuated L26 protein is expressed from a spliced mRNA transcript that is also responsible for the expression of the UL29, UL28, UL27, and UL29/28 open reading frames L26-null viruses contain large deletions within the UL26 open reading frame which could impact the expression of the other open reading frames that are expressed from this mRNA transcript. Further, it is unclear how the two separate isoforms of the UL26 protein contribute to HCMV infection. To address these issues, and to map the domains of UL26 that impact viral replication, we employed BAC-mediated recombineering to create a panel of viruses containing site-directed UL26 mutations. This panel included viruses containing a mutation ablating one or both initiating methionines as well as viruses containing stop codon insertions throughout the UL26 open frame.Viruses containing deletions in the UL26 protein is illustrated in L26 reading frames that could be stably expressed, stop insertions were made at locations that approximated transitions between these predicted domains , nor did the previously described transposon-deleted UL26 virus (UL26TI)  and were not analyzed in further detail. The specific mutations of the remaining recombinant viruses, and the UL26 ORFs they produce, are illustrated in A plot of manavalan hydrophobicity  domains . To veri isoform . Further(UL26TI) . The repisoforms . The stoL26 protein impacts production of viral progeny, we wanted to elucidate how specific domains of the UL26 protein contribute to viral replication. We first wanted to assess whether the double methionine deletion mutant (DBmetΔ), grew with similar kinetics as the transposon insertion mutant (UL26TI). As shown in L26 ORF does not substantially impact the in vitro viral growth over and above what is observed with less disruptive targeting of the UL26 initiation methionines.As it has previously been found that deletion of the UL26 protein contribute to viral growth, we analyzed the replication of the individual methionine mutants and the #185 stop insertion mutant. At high MOI, the 2ndmetΔ virus, which only expresses the long isoform of UL26   . The #18(DBmetΔ) . Similarl titers . Interesl titers . The comL26 is important for wildtype levels of IE1 accumulation L26 domains on viral gene expression, we analyzed the accumulation of viral proteins during infection. Upon infection at an MOI = 3.0, both UL26-null viruses, UL26TI and DBmetΔ, accumulated less IE1 at 4 hpi, but recovered by 24 hpi , viral DNA accumulated similarly between the WT and 2ndmetΔ  from our panel of mutants, overlaid with agarose and incubated at 37°C for 15 days. Images of the resulting plaques were captured with subsequent analysis of the area of each plaque. Images of representative plaques are shown in L26-null viruses. The plaques produced by the 1stmetΔ virus were intermediate in size between the UL26-null viruses and WT HCMV, whereas the 2ndmetΔ virus produced plaques of WT size. These results indicate that the C-terminal 38 amino acids of the UL26 protein, and to a lesser extent, the N-terminal 34 amino acids are important for WT plaque size.We and others have observed that deletion of the UL26 protein is important for virion stability, inasmuch as prolonged incubation at 20°C causes UL26-defective virions to lose their ability to initiate infection faster than WT virions do. L26 mutations impact virion stability, viral supernatants containing equivalent PFUs were incubated at 37°C for various times and then plated. The percentage of plaques remaining after incubation in comparison to control was plotted in L26-null viruses demonstrated a ∼50% drop in infectivity, a statistically significant difference . This suggests that large differences in virion protein constituents are likely not responsible for the decreased stability of mutant UL26 viruses. Another major question is the function of UL26 in the nucleus at early times. It seems likely that this nuclear UL26 is responsible for impacting IE1 gene expression at early times, although the potential mechanism involved still needs to be elucidated. Our identification of the important UL26 N-terminal and C-terminal domains will facilitate addressing these questions. The C-terminal 38-amino acids of UL26 are important for proper UL26 tegumentation, nuclear localization, and viral replication. Our studies highlight the importance of these C-terminal 38-amino acids for future study. Further functional analysis will distinguish how the specific residues within this domain contribute to UL26 nuclear localization and proper tegumentation, and subsequently to HCMV replication. Given its importance to HCMV infection, elucidating the mechanisms through which UL26 domains contribute to high-titer replication may shed light on possibilities for therapeutic intervention.A number of questions remain about U


Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects. RNA interference (RNAi) is not only a potent cellular pathway to silence endogenous or exogenous genes but is also a widely used tool for sequence-specific gene knockdown . The triThe core protein component of RISC and direct interaction partner of siRNAs is a member of the Argonaute (Ago) protein family. Ago proteins are characterized by PAZ, MID and PIWI domains . The PAZAfter an initial hype and the hope that RNAi develops into a potent novel strategy for therapy, it became clear that several problems associated with RNAi experiments have not been solved. Besides delivery issues in RNAi-based therapeutic approaches, one of the major problems of siRNAs are off-target effects ,21. MostHere, we report the development of a new generation of siRNA tools without off-target effects. Using an enzymatic approach, we generate complex pools of accurately defined siRNAs. While synergistically silencing one single on-target gene, each individual siRNA is present at very low concentrations, effectively diluting off-target effects below detection limits. The novel enzymatic strategy allows for the free combination of individually selected siRNA sequences to obtain optimal siRNA pools. We refer to our novel siRNA pools as siPools and overcome off-target effects, the major shortcoming of classical siRNA reagents.in vitro transcribed using an integrated T7 promoter sequence followed by RNA precipitation with 2.5 M LiCl. Sense and antisense strands were annealed by incubation at 95°C for 5 min followed by slow cooling to room temperature. Annealed dsRNAs were subsequently digested with 10 U/μg RNase T1 to obtain ds siRNAs with 2 nt 3′ overhangs. RNase T1 digested siRNAs were purified by native 20% polyacrylamide gel electrophoresis and eluted from the gel overnight at 4°C in elution buffer  followed by ethanol precipitation. To obtain siPools with 30 siRNAs, siPool #1 was mixed with siPool #2, and to generate siPool 45 with 45 different siRNAs, siPool #1 was mixed with siPool #2 and #3. siPool 60 containing 60 different siRNAs was mixed by the use of all four different siPools #1, #2, #3 and #4. TNRC6A, TNRC6B, TNRC6C and unspecific control siPools (neg. pools) each comprised of 30 individual siRNA sequences. For all template and siRNA sequences, see Supplementary Material. Ready-to-use siPools against various genes were purchased from siTools Biotech, Munich, Germany.DNA templates were synthesized by GeneArt (Life Technologies). SiPool templates were Antisense strands of siRNA sequences were depicted: PolG siRNA off-T: 5′-AAUAUCCAGCGCUUCACCC-3′, Scyl1 siRNA off-T: 5′-ACAUUGUUGUGGAUGAGGC-3′; Mad2 siRNA: 5′-CCAAUCUUUCAGUUGUUCC3′; neg. ctrl. siRNA: 5′-UUGUCUUGCAUUCGACUAA-3′. esiRNAs (Sigma) were delivered as RNase III digested double-stranded products derived from the following sequences, PolG esiRNA: 5′-GGAAGAAGTGGGAGGTGGTTGCTGAACGGGCATGGAAGGGGGGCACAGAGTCAGAAATGTTCAATAAGCTTGAGAGCATTGCTACGTCTGACATACCACGTACCCCGGTGCTGGGCTGCTGCATCAGCCGAGCCCTGGAGCCCTCGGCTGTCCAGGAAGAGTTTATGACCAGCCGTGTGAATTGGGTGGTACAGAGCTCTGCTGTTGACTACTTACACCTCATGCTTGTGGCCATGAAGTGGCTGTTTGAAGAGT-3′, Scyl1 esiRNA: 5′-CAGCCGAGAAGCAAAAATTCTTCCAGGAGCTGAGCAAGAGCCTGGACGCATTCCCTGAGGATTTCTGTCGGCACAAGGTGCTGCCCCAGCTGCTGACCGCCTTCGAGTTCGGCAATGCTGGGGCCGTTGTCCTCACGCCCCTCTTCAAGGTGGGCAAGTTCCTGAGCGCTGAGGAGTATCAGCAGAAGATCATCCCTGTGGTGGTCAAGATGTTCTCATCCACTGACCGGGCCATGCGCATCCGCCTCCTGCAGCAGATGGAGCAGTTCATCCAGTACCTTGACGAGCCAACAGTCAACACCCAGATCTTCCCCCACGTCGTACATGGCTTCCTGGACACCAACCCTGCCATCCGGGAGCAGACGGTCAAGTCCATGCTGCTCCTGGCCCCAAAGCTGAACGAGGCCAACCTCAATGTGGAGCTGA-3′.To generate low-complexity pools, the following siRNAs were used: PolG siRNA 1: 5′-UCAUCCGACAGCCGAUACC-3′, PolG siRNA 2: 5′-AAUUCUUGCAGGUCCCACU-3′, PolG siRNA 3: 5′-GCUAUUACCAUCCUUGUGA-3′, PolG siRNA 4: 5′-UUAAACUGCAUUAGUAAGC-3′. Scyl1 siRNA 1: 5′-UUUCUCAGGAUCUACAGUGAG-3′, Scyl1 siRNA 2: 5′-UUGAGGUAUAUUCCCAACGGG-3′, Scyl1 siRNA 3: 5′-UUGGUUUCUACAAAGCGGUUG-3′, Scyl1 siRNA 4: 5′-UUGUACAAUAAAUACAUCUGU-3′. Three siRNAs were mixed in different combinations with the corresponding siRNA off-T to obtain four different siRNA-containing low-complexity pools with possible Mad2 off-target effects. For pool #1, siRNA 1, 2, 3; for pool #2, siRNA 1, 2, 4; and for pool #3, siRNA 1, 3 and 4 were mixed with off-T siRNAs. Low-complexity pools consisting of siRNA 1–4 were used as non-Mad2 off-target controls (pool #4).HeLa cells were cultivated in Dulbecco's modified Eagle's medium substituted with 10% FCS  and penicillin/streptomycin. siRNA transfections were done using Lipofectamine RNAiMax (Life Technologies) according to the manufacturer's protocol. Cells were harvested 24 h or 48 h after transfection.RNA was isolated 24 h after transfection followed by cDNA synthesis and quantitative polymerase chain reaction (qPCR). Following primers were used: PolG for: 5′-TTCCAGGACCTGATGCAGTA-3′, PolG rev: 5′- ACAGGCAGGTAGGAGACACC-3′, Scyl1 for: 5′-CTGGAGGAAGTGGAGAAGGA-3′, Scyl1 rev: 5′-TCAGCTTGGAGGTGAGTGAG-3′, Mad2 for: 5′-AGATGACAGTGCACCCAGAG-3′, Mad2 rev: 5′-TCCAACAGTGGCAGAAATGT-3′ GAPDH for: 5′-ATGGGTGTGAACCATGAGAA-3′, GAPDH rev: 5′-GTGCTAAGCAGTTGGTGGTG-3′, TNRC6A for: 5′-CCCTCAGGTTCCAGTTTCAT-3′, TNRC6A rev: 5′-GCTGGTTTAGCTGGGATAGC-3′, TNRC6B for: 5′-ATGGTTCTGGCTTCAGCTCT-3′, TNRC6B rev: 5′-CATATTGGCTTCCTGTGTGG-3′, TNRC6C for: 5′-TAGCAAGCATGGTGCTATCC-3′, TNRC6C rev: 5′-GTACCGGCCATAGGAGTCAT-3′, OAS1 for: 5′-TGCCATTGACATCATCTGTG-3′, OAS1 rev: 5′-GAGCCACCCTTTACCACCT-3′, INFB1 for: 5′-AGTAGGCGACACTGTTCGTG-3′, IFNB1 rev: 5′-GAAGCACAACAGGAGAGCAA-3′, IL6 for: 5′-AATGAGGAGACTTGCCTGGT-3′, IL6 rev: 5′-GCAGGAACTGGATCAGGACT-3′, STAT1 for: 5′-AATTGACCTCGAGACGACCT-3′, STAT1 rev: 5′-CACATGGTGGAGTCAGGAAG-3′.For western blot analysis cells were harvested and lysed in NET buffer  48 h after transfection. Proteins were separated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by semi-dry electroblotting. Following antibodies were used: polyclonal anti-MAD2 (Bethyl Laboratories) at a dilution of 1:5000 and a monoclonal mouse anti beta actin antibody (clone AC15 from Abcam) at a dilution of 1:5000 in TBS-Tween with 5% milk powder. Fluorescently labeled IRDye 800 CW antibodies were used as secondary antibodies (Li-COR). Western blots were imaged with an Odyssey Fluorescence scanner (Li-COR).To generate an off-target reporter construct, a modified pMIR dual luciferase reporter plasmid  was usedHeLa cells were transfected with 10 nM siPools and lysed in NET buffer  48 h after transfection. Lysates were used for Ago2-siRNA co-immunoprecipitation. Protein-G sepharose beads (GE) were pre-incubated with monoclonal anti-Ago2 (11A9) antibody . LysatesHeLa cells were transfected with 3 nM Scyl1 siRNA, Scyl1 siPool 15, Scyl1 siPool 60, or mock transfected in three biological replicates. RNA was isolated 48 h after transfection and further processed for Affymetrix Human Gene 1.0 ST array analysis. The Human Gene 1.0 ST array platform from Affymetrix  was used to assess transcriptome-wide expression profiles. Normalization of raw intensity values from CEL files was performed using variance stabilization , and thelimma , or mock transfected in three biological replicates. RNA was isolated 48 h after transfection. Transcription profiles were analyzed using the Human Gene 2.0 ST array platform (Affymetrix). Raw intensity values (from CEL files) were normalized using RMA (oligo ). The nsanalyzeR . All logAnalyses were performed within the statistical programming environment R and using Bioconductor  packagesP-value for the number of repressed transcripts with a seed match for the Scyl1 siRNA to belong to the same distribution. For the pool 15 and pool 60, three additional siRNA seed sequences were drawn from the pools and tested analogously.Human 3′-UTR sequences were retrieved from Ensembl version 68 for the transcripts represented on the cartridge microarray. The reverse compliment of the siRNA seed sequences (7 nt) was searched for in the 3′-UTRs of the transcripts and 2292 transcripts with a seed sequence match were found. To analyze whether there is significant enrichment of transcripts with a seed-binding site among the repressed transcripts after siRNA transfection, the number of repressed transcripts with a seed match was compared to the mean number of transcripts with a random seed match. Seven thousand five hundred random seed sequences were drawn and from these, 2000 with similar numbers of transcripts with a seed match in the 3′-UTR (range 1692–2892 transcripts per sequence) were drawn and the number of repressed transcripts with a seed match were recorded for each random seed sequence. The distribution of these values was used to calculate the We hypothesized that siRNA pools containing up to 60 individual siRNAs against one target possess very low off-target effects because of the very low concentration of each individual siRNA. However, chemical synthesis of high numbers of siRNAs is cost-intensive and therefore not practicable. Therefore, we set out to generate complex siRNA pools enzymatically. For our experiments, we chose the human target genes PolG and Scyl1. siRNAs with strong off-target effects against the gene MAD2 had been reported for both genes . These sFor siRNA pool production, we selected siRNA sequences using standard siRNA prediction tools, which are connected by non-complementary linker sequences Figure . The DNAThe two strands were transcribed, annealed and analyzed on a native polyacrylamide (PAA) gel , but not in the siPool transfections   or siRNASince siPools and esiRNAs derive from longer dsRNA precursors and such precursors might cause interferon response, we tested the expression of interferon response genes after siRNA transfection Figure . siPoolsOff-target effects are a severe but often ignored error source in RNAi experiments. This is particularly important for genome-wide screening approaches, in which only a number of hits can be validated individually . In factO-methylation of the guide strand leads to a reduction of miRNA-like off-target effects presumably due to less efficient binding to the seed sequence of the off-target mRNA. For siRNA on-target activity, this modification seems to be tolerated  and theobserved  highlighEscherichia coli RNase III generates more discrete size products thus improving RNase III generated siRNAs  have been discovered in various organisms and tissues ,59. Theshttp://www.ncbi.nlm.nih.gov/geo/) under accession GSE57674.Raw intensities and normalized expression data of the microarrays are publicly available at the NCBI Gene Expression Omnibus (GEO, Supplementary Data are available at NAR Online.


CCND1) and a 3′UTR consisting of sequences from both the CCND1 3′UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3′UTR with siRNA results in decreased CCND1 levels.The t translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3′UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (The online version of this article (doi:10.1186/s13045-016-0260-7) contains supplementary material, which is available to authorized users. CCND1) transcript [CCND1 mRNA has a long 3′UTR (~3.1 Kb) that contains numerous destabilizing elements [CCND1 transcripts with truncated 3′UTRs correlating with reduced survival [Mantle cell lymphoma (MCL) is considered incurable upon relapse . The halanscript . The CCNelements , 5. MCL survival . In somesurvival . In othesurvival , has beesurvival . Aside fsurvival .CCND1 mRNA. We observed 3′rapid amplification of cDNA end (3′RACE) products in all the MCL lines that would indicate that 3′UTR shortening has occurred  gene  from MRCK, which triggers the subsequent addition of the poly(A) tail, creating a chimeric 3′UTR. The observed CCND1/MRCK fusion gene likely is formed by a second translocation event between chromosomes 11 and 14, which positions the full open reading frame of CCND1 and a truncated 3′UTR within intron one of MRCK  or the truncated CCND1 3′UTR from Jeko-1 (Tr-CCND1) and co-transfected them with three miRNA mimics known to repress CCND1. The FL-CCND1 reporter was downregulated in response to each mimic tested  and 5% penicillin/streptomycin. All three MCL cell lines: Jeko-1, Granta-519, and SP-53 contain the t11;14) translocation [;14 per the manufacturer’s protocol. HF Phusion buffer was used together with MgCl2. Primers were designed to provide the largest DNA PCR product possible, and were validated to work with genomic DNA. The primer sequences used were CCND1 forward 5´TCCGGAGCATTTTGATACCAG and MRCK reverse 5´TCCAATTCTGCTAGACCTTTGTGATA.Total mRNA was extracted using TRIzol reagent (Life Technologies). After DNase (Promega) treatment, cDNA synthesis was carried out using M-MLV Reverse Transcriptase (Life Technologies). For 3´RACE, oligo(dT25)T7 primer was used in cDNA synthesis, and the first round of PCR was performed using the CCND1 primer 5´ TGGTGAACAAGCTCAAGTGG and oligo(dT25)T7. The second round of PCR was performed using a nested CCND1 primer 5´TGGCATTTTGGAGAGGAAGTG and a T7 primer. All PCR was performed using pfu polymerase. The resulting PCR product was cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Life Technologies) and sequenced . The qRT-PCR protocol used, as well as the amplicons used to measure 7SK and CCND1, were previously described . The priThree different CCND1 3´UTR constructs were cloned downstream of the Renilla luciferase gene of psicheck2 (Promega) between Xho1 and Not1. To clone the gene fusion, the CCND1-MRCK sequence: CTCGAGGGGCGCCAGGACGGCGGGCGCCACCGCCACCCGCAGCGAGGGCGGAGCCGGCCCCAGGTGCTCCCCTGACAGTCCCTCCTCTCCGGAGCATTTTGATACCAGAAGGGAAAGCTTCATTCTCCTTGTTGTTGGTTGTTTTTTCCTTTGCTCTTTCCCCTAAAAAAGCATAATAGGTTAGCTGACCAATGATTAAAACATTATAGATCCGGAACGAACTAGAGAAGGCATGCAAAATTCAGATGAGAAAGTTTCTAAGTGATAGCTCCAGCTACCCCTCCAAATATCACAAAGGTCTAGCAGAATTGGAATTAAAAAAAAAAAGTCCTCTTGCACGGTTTATTTAAAATAAAGCCTTAACCTTAGGTGGTGCACCAAGTTGAACCTGACAGTGGAACTGTGTGGGTTTCAAGATCGAGTGATCAGAAAGGAACGGTAAACAAGCTGGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACCCGAGGTCAGGAGTACAAGACCAGCCTGGCCAACACTGTGGCGGCCGC was synthesized by GenScript and ligated into pUC57-Kan. After Xho1 and Not1 Restriction enzyme digestion, the sequence was directly cloned into the psicheck2 plasmid. The Jeko-1 specific truncated 3´UTR was cloned from Jeko-1 cDNA using the following primers: forward 5´GGCCCTCGAGGGGCGCCAGGCAGGCGGGCGC and reverse 5´GGCCGCGGCCGCTGCCTAGAACCCCACTACAGCTGTGC. Full length CCND1 3´UTR was cloned from the CCND1-pLightSwitch 3´UTR plasmid (S813994) from SwitchGear Genomics. The primers used were: forward 5´ GGCCGTCGACGGGCGCCAGGCAGGCGGGCGC and reverse 5´ GGCCGCGGCCGC CGTCTTTTTGTCTTCTGCTGGA.Either control siRNA , CCND1-sCells were lysed using RIPA buffer and 20ug of each protein sample was resolved on a 12% SDS-PAGE gel. After transfer to a PVDF membrane, blocking was done for 1hr in 5% non-fat milk resuspended in PBS (+0.001 % Tween 20). The membranes were probed with the monoclonal cyclin D1 antibody  and polyclonal alpha tubulin antibody (Abcam). After probing with HRP-conjugated secondary antibody, proteins were detected by luminol.We used predicted CCND1-miRNA interactions using starBase software, which consolidates TargetScan, PicTar, PITA, Miranda and RNA22 miRNA prediction softwares, and overlaps the data with CLIP-Sequencing (CLIP-Seq.) data. We used a high stringency cutoff, where only miRNAs supported by >/=3 CLIP-Seq. experiments were selected, to reduce false positives .