Nicotiana tabaccum L.) and rustica tobacco (Nicotiana rustica L.) were analysed between the third intron and the fourth intron of the propylene alcohol dehydrogenase gene. The results showed that the alcohol dehydrogenase gene is a low-copy nuclear gene. The intron sequences have a combination of single nucleotide polymorphisms and length polymorphisms between common tobacco and rustica tobacco, which are suitable to identify the different germplasms. Furthermore, there are some single nucleotide polymorphism sites in the target sequence within common tobacco that can be used to distinguish intraspecific varieties.A pair of primers was designed to amplify the propylene alcohol dehydrogenase gene sequence based on the cDNA sequence of the tobacco allyl-alcohol dehydrogenase gene. All introns were sequenced using traditional polymerase chain reaction (PCR) methods and T-A cloning. The sequences from common tobacco ( Nicotiana sp.) is one of many plants currently used as models for molecular studies and is an economically important crop. The interspecific phylogenetic relationships within tobacco are complicated due to complex hybridization events within the tobacco lineage. DNA molecular markers have been used to identify germplasms and to accelerate the breeding cycle. The second generation of molecular markers that include random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) has been employed broadly to differentiate tobacco varieties,[Tobacco .The specific primers were designed to target the boundary between the third intron and the fourth intron of the tobacco propylene alcohol dehydrogenase gene, and the IP sequences were identified as follows, IP-S: GTGCTGGAAGCAAAGAAAAG, IP-A: CAATTCCATCAGGGAAGTAC. The PCR materials included 5 μL of 10 × PCR buffer, 4 μL of dNTP (2.5 mmol/L each), 1 μL of each primer (10 μmol/L), 1 μL of the DNA template and 1.5 U of PCR was performed at 94 °C for 5 min, then 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min for 30 cycles, with a hold at 72 °C for 20 min. The PCR products were ligated to a pGEM-T vector  and sequenced on an ABI 3730 sequencer after electrophoretic detection in a 1.0% agarose/EB gel.The propylene alcohol dehydrogenase gene of sample C1 contained four introns and five exons that were 225, 178, 167, 83 and 379 bp long . The boundary sequence between the intron and exon segments were the sites of interest for which a primer was designed to target.Nicotiana rustica) exhibited two bands when both primers, IP-S and IP-A, were used, while those of common tobacco (sun-shined tobacco and flue-cured tobacco) exhibited only one band on the 1.0% agarose/EB gel .In the common tobacco, the shorter fragment was 559-bp long in the target region, and the two introns found were 223 and 253 bp. There were no length differences in this fragment among the 12 samples sequenced, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATCTTCATAGTGTTGAATCTATCTTCATTCTTCGTTATGTTAATCATTGTGCTCCGTCCACCATCAAACCCAAAGGTTGGGGTTGGGGTTGGGGTTAGGGTTGGGTGGGTTATGCCTTTGGGGGGAAAGAGAGGTTTTAGTTTAATAGACCATGATGTTTAAAACTCTTGTTATTTAATGTCTCTTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAGGCTTTTAACTATAAAGAGGAGCAGGATTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGCTACAAAACAATCTACCTATTGTACAAAAGCAGTTAAAGATGTTCTGTGAAAAGTATGATGATATTATGTAGATAGTGTGAAACTTATAGAAAGACTTTAAGATATGCCTACGAAAACCGAGGAACATGGATCATTTCAAATCTGGCTTTTCATGAATATGGAACCCCATTCACTAGATTTTAGTGCGAAATTCCTAGACTTGAAGTGACTGGAAATACTGGTTTGTTATTAGwww.ebi.ac.uk/Tools/clustalw2/). The results indicated that there were two transitions and one transversion in addition to three gaps within the third intron. The two transitions and one transversion were also present within the fourth exon, but no amino acid replacement had occurred. There were four transitions and one transversion within the fourth intron as well . These polymorphic sites within the third and fourth intron may function as references to distinguish the varieties of common tobacco studied here.In the common tobacco, the longer sequence obtained was between 588 and 591-bp long in the target region, and the two introns were either between 194 and 197 bp or 311 bp in length. The sequences from 13 samples were analysed with the software ClusterW2 (In the rustica tobacco, the shorter sequences were 442-bp long in the target region, and the two introns found were 195 and 164 bp in length. No length polymorphisms were found in this fragment among the 12 samples sequenced, and the consensus sequence obtained was as follows:GTAAGTTCCTTCATAGTGTTGAAGCTCTCTTCATTCTTCGTTATGTTAATTATTGTGCTCCGTCCTCCATCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGGGTATACCTTTGGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTCTCTTTTTATATTCATTTCCAAAGGTTGATCTGTTGAGGAACAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATGCAGCTTTGAAGAGGTGAGTAATCTTACTACTTGGCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCATCAATATTTCCATAGAATGATATTTATAATGGAAGTCCCAAAGTAAAGATGTTCAGTGAAAAGTATGCCTGTGAAAACTGAGGGAAATAATACTGGTTTCTTATTAGIn the rustica tobacco, the longer sequence was 583-bp long in the target region, and the two introns found were 220 and 280 bp in length. No polymorphisms were found in this fragment among the 12 samples sequenced as well, and the consensus sequence was as follows:GTAAGTTCCATTGCGTTGGATGTTCATAGTGTTGAAGCTATCTTCATTCTTCGTTATGTTAATCATTGTGTTCTGTCCACCGTCAAACCCAAAGGTTGGGGTTTTGTGGGTTATGAGGAGGAGAAAGGTATACCTTTGGGGGGAAGAGATGTTTTAGTTTAATAGACCATGATGTTTAACTATCTTTTTATTTAATGTCTCTTTTTATTCATTTCCAAAGGTTGATCTGTTGAAGAGCAAATTTGGTTTTGACGAAGCTTTTAACTATAAAGAGGAGCAGGACTTAAATTCAGCTTTGAAGAGGTGAGTAATCTTACTTCCTGTTAGAAAACAATCTACCTATTGTAACAAACTTGCCCTGGTGTTAGAGAAGCAGTTAGCAACAGCTTTTCCTTTTCATGTAAACGGAATCATCGATATTTCCATAGAATGATATTTATTCCTTGAACTGAAAAATAGAAGTCCCAAAGTAAAGATGTACAGTGAAAAGTATGAAGATTGTGTGAAAATTATAGAAAAGACTTTAGGATATGCGTATGAAAACTGAGGGAAATTCCTCCCTGGAATACTGGTTTCTTATTAGIn this study, the tobacco propylene alcohol dehydrogenase gene proved to be a low-copy nuclear gene. It could have formed as a result of divergent evolution after gene duplication, or by a parallel evolutionary process between the two sets of genomes, as the tobacco plants tested were all allotetraploid. Although the PCR product for the allyl-alcohol dehydrogenase gene of sample C1 was sequenced successfully, this does not mean that only one copy of the target gene is present in common tobacco. There were obvious intron length polymorphisms between common tobacco and rustica tobacco, which were mostly due to different modes of evolution of the two kinds of tobacco.Nicotiana sylvestris.[N. tomentosiformis,[N. otophora or from introgressive hybridization of N. otophora and N. tomentosiformis within the subgenus of common tobacco.[N. rustica originated from a natural chromosome duplication event after ancestral hybridization of N. paniculata and N. undulate.[N. rustica L. group and Nicotiana tabaccum L. group, after an analysis that used AFLP methods coupled with unweighted pair group method with arithmetic mean analysis, which was consistent with our results in this work. Furthermore, the two categories of tobacco can be distinguished using only one pair of primers according to the results of this work.It is thought that the maternal ancestor of common tobacco was lvestris. It is alsiformis, N. otoph tobacco. It has bundulate. Yang et undulate. divided The two copies of the propylene alcohol dehydrogenase gene found in tobacco each contained four introns and five exons. The length polymorphisms of the intron existed intraspecifically as well as interspecifically. The primers produced here, IP-S and IP-A, were able to easily differentiate common tobacco from rustica tobacco in a 1% agarose gel. Some SNPs of the intron within common tobacco were found, and may be useful in the identification of varieties of this species.http://dx.doi.org/10.1080/13102818.2014.907651.Supplemental data and research materials for this article can be accessed at


Mycobacterium avium and Mycobacterium abscessus for potential antiviral activity. Both of these IFITMs conferred a moderate level of resistance to influenza virus in human cells, identifying them as functional homologues of IFITM3. Analysis of sequence elements shared by bacterial IFITMs and IFITM3 identified two hydrophobic domains, putative S-palmitoylation sites, and conserved phenylalanine residues associated with IFITM3 interactions, which are all necessary for IFITM3 antiviral activity. We observed that, like IFITM3, bacterial IFITMs were S-palmitoylated, albeit to a lesser degree. We also demonstrated the ability of a bacterial IFITM to co-immunoprecipitate with IFITM3 suggesting formation of a complex, and also visualized strong co-localization of bacterial IFITMs with IFITM3. However, the mycobacterial IFITMs lack the endocytic-targeting motif conserved in vertebrate IFITM3. As such, these bacterial proteins, when expressed alone, had diminished colocalization with cathepsin B-positive endolysosomal compartments that are the primary site of IFITM3-dependent influenza virus restriction. Though the precise evolutionary origin of vertebrate IFITMs is not known, our results support a model whereby transfer of a bacterial IFITM gene to eukaryotic cells may have provided a selective advantage against viral infection that was refined through the course of vertebrate evolution to include more robust signals for S-palmitoylation and localization to sites of endocytic virus trafficking.Interferon induced transmembrane proteins (IFITMs) found in vertebrates restrict infections by specific viruses. IFITM3 is known to be essential for restriction of influenza virus infections in both mice and humans. Vertebrate IFITMs are hypothesized to have derived from a horizontal gene transfer from bacteria to a primitive unicellular eukaryote. Since bacterial IFITMs share minimal amino acid identity with human IFITM3, we hypothesized that examination of bacterial IFITMs in human cells would provide insight into the essential characteristics necessary for antiviral activity of IFITMs. We examined IFITMs from  Interferon-induced transmembrane proteins (IFITMs) encoded by a variety of vertebrates, including pigs, chickens, bats, fish, mice, and humans, have been shown to restrict cellular infection by specific viruses ,3,4,5,6.The essentiality of mouse IFITM3 in restricting influenza virus infection was demonstrated by showing that IFITM3 knockout mice succumb to sublethal doses of virus and exhibit higher lung titers and more severe lung pathology than wild type mice ,15. SimiAn extensive evolutionary study of IFITMs posited that vertebrate IFITMs originated from a horizontal gene transfer from bacteria to a common ancestor of choanoflagellates and metazoans . WhetherExpression constructs in the pCMV-HA vector or pCMV-myc vectors  encoding mouse and human HA-IFITM3, human myc-IFITM3, and mouse HA-IRGM1 have been previously described ,20,21. UMAV IFITMGAATTCCCATGACCCAACCACCTCCTCCCCCGCCACCACCAGGATATCCCCCACAACAGCCAGCCGCTCAGGCTCCTAATAACTATCTGGTGTGGTCTATCCTTGTCACTCTCTTCTGCTGCCTGCCGTTTGGCATTGTCGCTATCGTAAAGAGCTCTCAAGTGAACGGACTTTGGGCACAGGGTAGATATGCTGAGGCACAGGCCTCCGCAGACAGTGCCAAGAAATGGGTGATATGGAGCGCAGTTATAGGCGTCGTGGTGGGAATAATCTATGGAATCCTTATGGCCGTAGGCGCCCTCAACACAAATACAAACGCGGCCCTCGCCGCGATGTTTTAGTAAGTCGAC        MAB IFITMGAATTCCCATGAGTGATGAAACCAAAAGCGACGAGCCTACAGGCGCTATCACCACACCGACCCCTCCTCCCCCACCGGCTCCTGCCTCTGTGACTGGCCCACCCAAACCCCCACCCACTAACGTGGGTTGGGCCGTCGCTAGCGTGATTTTTTTCTGGCCTCTGGCATTTAGCGCATTCACCAATGCACTGAATGTGACTCAGTTTTGGCTGACGGGGCAGTATGATCGGGCCCAGGAGTCTAGCGATCGGGCCAAGCTCCTGGGAAAGATTGCCCTCCTGACCGGGTTGGTACTGCTGTTCCTGTTCATCACCCTCCGCATTGCCTGCGCCATCTGGTGGCACTCACATGGTGGGGGATGGGGTCATCATGGCGGATGGCATAGGAGTTGGGACGACGGCGGGTGGGATGGCCCTGGCCCCATCGGGCCTATGGGTAGGCCGGGTCGCGACAACTAGTAAGTCGAC2 at 37 °C. For imaging experiments, cells were grown on sterilized glass coverslips in 12-well plates. For all other experiments, cells were grown in 12-well or 6-well plates. Cells were transfected overnight using LipoJet  according to the manufacturer’s instructions. For biochemical experiments, 2 ug/well of each plasmid was transfected into cells grown in 6-well plates. For infection experiments, 1 ug/well of each plasmid was transfected into cells grown in 12-well plates, with the exception of plasmid encoding HA-MAB IFITM for which 0.5 ug/well was transfected. For examination of palmitoylation, transfected cells were labeled with 50 uM alk-16 for 1 h as previously described in detail [HEK293T cells were obtained from the ATCC and were grown in DMEM supplemented with 10% FBS  in a humidified incubator with 5% COn detail ,22,23. In detail ,22,23. In detail . Sendai n detail . Cells wFor palmitoylation experiments, cells were lysed with 1% Brij Buffer , pH 7.4) containing EDTA-free protease inhibitor cocktail. For co-immunoprecipitation experiments, cells were lysed with 1% digitonin in PBS containing EDTA-free protease inhibitor cocktail. Immunoprecipitations were performed with either anti-HA or anti-myc EzView Affinity Gel (Sigma). For palmitoylation experiments, immunoprecipitations were washed 3 times with standard RIPA buffer , and for co-immunoprecipitation experiments, the immunoprecipitations were washed 5 times with 0.1% digitonin in PBS. All detergents were purchased from Sigma. Western blotting was performed with anti-HA antibody  or anti-myc antibody . For immunofluorescence and flow cytometry, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X100 in PBS for 10 min, and blocked with 2% FBS in PBS for 10 min. For immunofluorescence, cells were stained with the anti-HA or anti-myc antibodies described above that were directly labeled with Alexafluor 488 or 555 using 100 ug antibody kits from Life Technologies. Slides were mounted with Prolong Gold Antifade Reagent containing DAPI from Life Technologies and images were taken using an Olympus Fluoview FV10i confocal microscope. Quantification of Manders overlap coefficients was performed using the Just Another Colocalization Plugin (JACoP) for ImageJ software. For flow cytometry, cells were stained with anti-IFITM3 antibody  and anti-rabbit-Alexafluor-488 secondary antibody (Life Technologies), or with anti-HA antibody directly conjugated to Alexafluor-488 and anti-influenza NP antibody  directly conjugated to Alexafluor-647 as previously described [Mycobacterium abscessus (MAB) was significantly divergent from the majority of the remaining mycobacterial IFITMs (Mycobacterium avium (MAV) IFITM as a representative of the more typical mycobacterial IFITMs (Evolutionary studies of the IFITM gene family have identified numerous IFITM genes in different vertebrates ,26,27. Ol IFITMs A. For ful IFITMs A. Clustal IFITMs B, 65.6%,l IFITMs B. Howevel IFITMs D. One iml IFITMs B. We, anl IFITMs B.S-palmitoylated cysteines, including an S-palmitoylated di-cysteine motif, within the first hydrophobic domain [S-palmitoylation site on murine IFITM1 [Despite the major differences between the amino acid sequences of IFITM3 and mycobacterial IFITMs, a similar fundamental domain structure could be identified in each of these proteins. Two hydrophobic domains separated by approximately 30 amino acids are defining characteristics of IFITMs and were present in both MAV and MAB IFITMs B. Most vc domain ,31,32 (Fe IFITM1  (Figure e IFITM1 . InteresGiven the important differences and striking similarities between the mycobacterial IFITMs and IFITM3 B, we souWe synthesized codon optimized MAB and MAV IFITM genes for insertion into the pCMV-HA mammalian expression vector in-frame with the HA tag of the vector, and transfected these constructs into HEK293T cells. After transfection, we observed high expression of MAB IFITM, far greater than IFITM3 expression from the same vector, and a more similar expression level of MAV IFITM A. Thus, S-palmitoylation of IFITM3 is important for its proper membrane targeting and maximal antiviral activity [S-palmitoylation sites on IFITM3 (S-palmitoylation site present on murine IFITM1 [S-palmitoylation of these IFITMs (S-palmitoylation (S-palmitoylation of IFITMs is a driver of maximal antiviral activity [activity ,12,36. Uactivity ,23,45,46n IFITM3 B. MAB IFn IFITM3 B that ise IFITM1 . Using ce IFITMs B. We nexe IFITMs C. Similae IFITMs C. Consisoylation A,B, mutaoylation C. Overalactivity .IFITM3 was reported to homodimerize and also to heterodimerize with other IFITMs ,47. ThisN-terminus that interacts with endocytic adaptor complexes, resulting in the effective trafficking of IFITM3 from the plasma membrane to endosomes [S-palmitoylation that we observed for mycobacterial IFITMs compared to IFITM3 (IFITM3 has been reported to co-localize with early and late endosomal markers and with markers of lysosomes and multi-vesicular bodies ,29,36,48ndosomes ,29. Thisndosomes  (Figure o IFITM3 A,B, thiso IFITM3 C.Monosiga brevicollis [Monosiga brevicollis. IFITMs were also identified in multiple ancient bacteria species, and given the identifiable sequence similarities between bacterial IFITMs and metazoan IFITMs (S-palmitoylation (Though the exact evolutionary origin of vertebrate IFITMs is unknown, an IFITM with a splicing pattern that is conserved in all metazoans was identified in vicollis , a unicen IFITMs B, it wasn IFITMs C,D. Confn IFITMs D, and thoylation , which iS-palmitoylation level and that positioning of this domain evolved toward optimal modification in vertebrates. Alternatively, other determinants may affect interaction with the as yet unidentified palmitoyltransferase enzyme that modifies IFITMs. Further interrogation of MAV IFITM may shed light on these important questions.Many aspects of IFITM3 cell biology that are not fully understood may possibly be clarified by study of the mycobacterial IFITMs. For example, IFITM3 is known to be optimally active when expressed at high levels in cells, and its turnover is promoted by post-translational ubiquitination . HoweverThe precise mechanism of antiviral action of IFITMs is also currently unknown. Two potentially complementary models have been proposed for the anti-influenza virus activity of IFITM3. One, IFITM3 may directly alter fluidity of endolysosomal membranes ,10,49 th


AbstractHarpacticella Sars, 1908 is described from a tidal pool on Jeju Island, Korea. Harpacticellajejuensissp. n. is closely related to Harpacticellaitoi Chang & Kim, 1991, with regard to the structure of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the setal number of the P5 exopod in males. However, the new species is clearly distinguishable from Harpacticellaitoi by the combined following characters: six setae on the P5 exopod in females, one naked seta on the inner margin of P1 exp-2, the short endopod of P1 compared to the exopod, and a naked long seta on the proximal inner margin of the P5 exopod of males. The mtCOI partial sequence is provided as a DNA barcode for the new species.A new species of the genus  Harpacticella Sars, 1908 is a genus of harpacticoid copepods, family Harpacticidae Dana, 1846. The genus has been reported from various habitats , mostly in Asian waters , and Harpacticellaamurensis Borutzky, 1952 were described from freshwater, Harpacticellalacustris Sewell, 1924 and Harpacticellaitoi Chang & Kim, 1991 from brackish water, and Harpacticellaoceanica Itô, 1977 from the marine environment.The first reported . Among tHarpacticella from a tidal pool. Here, we describe the new species and provide a key to species in the genus Harpacticella. Partial mtCOI sequence was also obtained as a DNA barcode for the new species.During a study of the harpacticoid community along the coast of Jeju Island of Korea, we collected a new species of Samples were collected by hand net (63 µm mesh-size) from a tidal pool on the coast of Jeju Island, Korea. Specimens were preserved in 99% ethanol. Specimens were dissected in lactic acid, and the dissected parts were mounted on slides with lactophenol mounting medium. Preparations were sealed with transparent nail varnish. All drawings were prepared using a drawing tube attached to an Olympus BX51 differential interference contrast microscope., antennuleA1; , antennaA2; , aesthetascae; , exopodexp; , endopodenp; , first to sixth thoracopodP1-P6; , proximal  segment of a three-segment ramusexp (enp)-1; , caudal ramiCR. Specimens were deposited in the National Institute of Biological Resources, Incheon, Korea (NIBR). Scale bars in figures are indicated in µm.Descriptive terminology is adopted from Molecular analysis. For DNA extraction, fixative materials (99% Et-OH) were removed from specimens by washing with distilled water, and DNA was extracted using a tissue DNA purification kit . Amplifications were performed in 20 µl reactions volumes containing extracted tissue DNA, primers LCO-1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and HCO-2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) , and PCRPageBreakTaxon classificationAnimaliaHarpacticoidaHarpacticidaehttp://zoobank.org/77BE96F4-597C-44D0-A07E-03A4D993A5F233°13.949'N; 126°30.653'E) on Beophwan beach, Seoguipo-shi, Jeju Island, Korea.A tidal pool (Holotype: 1♀ (NIBRIV0000304111) in 70% ethanol from the type locality. Paratypes: 5♀♀ (NIBRIV0000304112) in 70% ethanol, 2♀♀ (NIBRIV0000304113 – NIBR0000304114) dissected on 11 and 10 slides, respectively, and 2♂♂ (NIBRIV0000304115 – NIBR0000304116) dissected on 11 and 2 slides, respectively. All from the type locality and collected by R. Jeong on 3 March 2013.KM272559, 619 bp).DNA-barcode (mtCOI) sequence and trace were submitted to GenBank . Maximum width at posterior margin of cephalosome . Urosome gradually tapering posteriorly. Body surface armed with some sensilla Figs .Prosome Fig.  4-segmenRostrum Fig.  well devUrosome Figs , 2C 5-seGenital double somite Figs , 2C, 7A Anal somite Fig.  without PageBreakPageBreakPageBreakPageBreakouter corner; seta III as long as lateral seta II; apical seta IV unipinnate, slightly longer than urosome; apical seta V bipinnate, as long as whole body; apical seta VI similar in length to seta III; dorsal seta VII bare and bi-articulate at its base.Caudal ramus Figs , 2D wideAntennule 7-segmented Fig. . AesthetAntenna Fig.  3-segmenMandible Fig.  with larMaxillule Fig.  with praMaxilla Fig. : syncoxaMaxilliped Fig. : syncoxaSwimming legs 1–4 Figs , 5A–B biP1 Fig. : coxa wiPageBreakP2 Fig. : coxa wiP3 Fig. : coxa wiP4 Fig. : coxa wiArmature formulae as follows:PageBreakP5 Fig. : exopod Male. Total body length of examined samples 631–650 µm . Greatest width at posterior margin of cephalosome. Cephalosome with sensilla along lateral margin. Other prosomites also with sensilla along lateral margin Fig. .Prosome Fig. , 4-segmeUrosome Figs , 7E 6-seAntennule Fig.  6-segmenP5 Figs , 7D, basPageBreakPageBreakPageBreakP6 Fig. : small fmitochondrial oxidase subunit I; partial cds; 619 bpPageBreakGATTTTTGATGCCCTCTCTTATATTAATAATTATTAGAAGAGTTGTTGAAGGCGGGGCAGGGACAGGGTGAACTGTTTACCCCCCTTTAAGAAGAAATTTAGCACATGCAGGAGGCTCGGTGGATTTAGTAATTTTTTCTTTACATTTAGCAGGAGTTTCTTCCTTATTAGGGGCTGTAAATTTTATTAGGACTTTAAGAAATCTTCGAGTATTCGGGATGTATTTTGACCAAGTGCCGTTATTTTGTTGATCTGTCTTGGTTACAGCTGTTCTATTACTTTTATCACTGCCTGTATTAGCGGGGGCAATTACTATATTGTTGACCGATCGAAACATTAATTCAAGCTTCTATGATGTTAACTTTATATCTTTTAAGGGGGATATGAGCGGGAGTTATGGGGGCGGCAATAAGAGTTATTATTCGGCTTGAATTAGGACAGCCTGGGACTTTAATTAAGGATGAGCAAATTTATAATGTTTTAGTGACTTCGCATGCTTTTATTATAATTTTCTTTATGGTTATACCAATTTTAATTGGGGGGTTTGGAAACTGGTTAGTTCCTTTAATATTAGGAGCTCCTGATATGGCCTTTCCTCGATTAAATAATTTGAGATTCTThe specific name refers to the type locality of Jeju Island, Korea.Harpacticella based on the combination of following character sets: a) 7-segmented antennule in the female, b) 2-segmented antennary exopod, c) 3-segmented P1 endopod and exopod, d) only one seta on the inner edge of P2 enp-2 and e) spiniform outer spine of exp-3 P3 and P4  six setae on the P5 exopod of females  P1 see Fig. ; (4) naksee Fig. , but plusee Fig. ; (5) naksee Fig. , but it see Fig. .PageBreakPageBreakHarpacticella species have a wide distribution ranging from freshwater to true marine environments, and have been found in Asian waters, American waters, and the Aldabra Atoll in the Indian Ocean  is the most ubiquitous species; it has been recorded in China, Japan, and the northwest coast of the USA  for use in future studies; no sequences have been obtained from congeners to date, even though it would be interesting to determine the phylogenetic relationships among congeners based on analysis of mtCOI sequences.DNA barcoding is an efficient tool to identify species, especially morphologically similar species . This ba


Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO).The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Navβ2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Navβ2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Navβ2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navβ2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Navβ2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO.We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navβ2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH. Since voltage-gated sodium channel (VGSC) is necessary in the initiation and propagation of action potentials in neurons, it is a valuable therapeutic target for neurological disorders, such as epilepsy and chronic neuropathic pain –3. Recen+ channels (VGSCs) are macromolecular protein complexes, which are composed of α-subunits (Nav1.1–Nav1.9) and β-subunits , in which α-subunits are necessary for forming a functional ion-selective channel and β-subunits affect ion channel gating and trafficking to regulate the voltage-dependency and density of VGSC on the cell membrane. It has been reported that mutation of Nav1.1, a remarkable feature of Dravet syndrome, could induce higher seizure activity and cognitive dysfunction [BACE1-transgenic mice, accompanied with disturbed Navβ2 [hAPP) transgenic mice [BACE1-null mice [BACE1) in BACE1-deficient or over-expressing mice [APP) mice [Voltage-gated Nafunction . Interesed Navβ2 . And resnic mice . Furtherull mice . These sull mice –17, and ull mice . Interesing mice , and thePP) mice . HoweverChronic brain hypoperfusion (CBH)-mediated chronic cerebral ischemia and consequent cognitive impairment , 22 is mMicroRNAs are small non-coding RNA, which regulate protein synthesis. MicroRNA-9 (miR-9), enriched in central nerve system (CNS) , contribIn this study, our data provide strong evidence that miR-9 regulates Nav1.1/Nav1.2 trafficking by post-transcriptional regulating SCN2B gene under CBH status.P < 0.01 vs sham). In order to explore whether the decreased surface protein expression of Nav1.1/Nav1.2 was due to the reduction of total Nav1.1/Nav1.2 or the impairment of trafficking, the total protein levels of Nav1.1/Nav1.2 were evaluated. The data showed that the total protein levels of Nav1.1 and Nav1.2 were significantly increased in the hippocampi and cortices of 2VO rats  rather than reduced. These results suggested that the decreased expression of Nav1.1/Nav1.2 on the cell membrane were due presumably to the impaired trafficking of Nav1.1 and Nav1.2 protein from cytoplasm to cell membrane in 2VO rats rather than the changes of their total protein levels.Previous studies have reported that Nav1.1/Nav1.2 trafficking was changed after dementia , 20, 40.P < 0.01). However, their mRNA expression of Navβ2 (SCN2B) was not changed consistently with the protein expression of Navβ2 .MicroRNAs have long been known to control post-transcriptional gene regulation and are essential for neuronal function . MiR-9 ihttp://www.targetscan.org/), we found that SCN2B has a poorly conservative ‘seed’ sequence of miR-9 in its 3’UTR and the length of 3’UTR of SCB2B in rat was very short based on the database of the University of California Santa Cruz (UCSC) and the National Center for Biotechnology Information (NCBI) Genome Browsers. Since recent studies have reported that microRNAs regulate protein expression by binding to the coding regions of target protein [http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and found that the CDS domain of SCN2B is likely to serve as potential targets for miR-9. We next identified whether there are binding sites for miR-9 on the CDS domain of SCN2B gene at the position of 336–358 and 575–597 with highly conservative regions , however, scrambled negative control and inhibitor of miR-9 had no effect on luciferase activity compared with control group , and the difference of luciferase activity between inhibitor of miR-9 and scramble negative inhibitor control was also not detected . For each of the two binding sites, we generated and tested several variants by introducing silent mutations designed to disrupt the putative base pairings between the microRNAs and the corresponding predicted targets. Silent mutations introduced into the predicted targets at the position of 336–358 of SCN2B CDS disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS . However, simultaneous introduction of silent mutations at the position of 575–597 of SCN2B CDS did not abolish the downregulation of SCN2B by miR-9 . Silent mutations introduced into both two predicted targets at the positions of 336–358 and 575–597 of SCN2B CDS, MutSCN2B-1 & 2 disrupted the ability of miR-9 to repress the translation of the SCN2B-CDS, strongly suggesting that miR-9 uniquely targets at the position of 336–358, rather than 575–597, of SCN2B CDS.Since the protein level of Navβ2 was reduced in both hippocampi and cortices in 2VO rats, which is discrepancy with the unchanged SCN2B expression, the alternation of miR-9 is highly expected in this process. By searching the database of microRNA targets , implying that the SCN2B shows a great potential as the target for miR-9.To observe the influence of miR-9 on the protein translation of SCN2B, we analyzed the protein levels of Navβ2 in primary cultured neonatal rat neurons (NRNs) co-transfected with miR-9 mimics. The successful transfection of miR-9 was identified Fig.  by qRT-PP < 0.05) and Nav1.2  total protein. And they were prevented in the presence of AMO-9  was injected directly into CA1 region of bilateral hippocampus of each rat and significantly higher expression of miR-9 in both hippocampi and cortices was observed at 8 weeks after injection compared with the negative control oligonucleotide . The surface expressions of both Nav1.1 and Nav1.2 proteins were markedly reduced in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which was reversed by lenti-pre-AMO-miR-9 , even though the total protein levels of Nav1.1 and Nav1.2 were increased in both hippocampi and cortices of rats with lenti-pre-miR-9 treatment, which were reversed by lenti-pre-AMO-miR-9 . These data suggested that miR-9 plays an important role in trafficking and cellular distribution of Nav1.1/ Nav1.2.To verify the functional role of miR-9 on Nav1.1/Nav1.2 trafficking NC, Fig. . ImportaP < 0.01). And furthermore, the surface protein expression of Nav1.1 was increased and the total protein of Nav1.1 level was decreased by lenti-pre-AMO-miR-9 compared with that in 2VO control rats . Similarly, lenti-pre-AMO-miR-9 effectively improved the impaired trafficking of Nav1.2 from cytoplasm to cell membrane under the same experimental condition .Our data displayed that 2VO results in miR-9 increase, and over-expression of miR-9 then induces the trafficking defects of Nav1.1/Nav1.2 in vitro. In order to evaluate the protective effect of AMO-miR-9 on the onset of the trafficking defects of Nav1.1/Nav1.2 in 2VO rats, lenti-pre-AMO-miR-9 was injected into the hippocampus area. At 8 weeks after injection, miR-9 level was significantly decreased in both hippocampi and cortices compared with 2VO rats Fig. . To our In this study, our observations have demonstrated, for the first time that CBH induces the trafficking defects of Nav1.1/Nav1.2 in hippocampi and cortices areas in 2VO rats, leading to the decrease in the expression of Navβ2 protein. Further study has shown that the increased miR-9 negatively regulated the expression of Navβ2 protein by binding to the target in CDS region of SCN2B gene. This observation provides a novel mechanism to modify the reduction in Nav1.1/Nav1.2 membrane trafficking, and careful monitoring the changes in miR-9 level and the expression for Nav1.1/Nav1.2 and targeted gene are considerably necessary during CBH.BACE1-trangenic mice although total Nav1.1 and Nav1.2 levels are elevated [BACE1-null mice [BACE1-trangenic mice [It has been known that Nav1.1, Nav1.2, and Nav1.6 are abundant in the central nervous system, whereas Nav1.3 is mostly present during embryonic stage . Nav1.6 elevated , 14, andull mice . In the nic mice .BACE1 affects Nav1.1 and Nav1.2 surface trafficking differentially [BACE1 activity leading to decrease in surface levels of Nav1.1 in neuronal cells [APP and BACE1 expression [APP and BACE1 is unknown and need to be elucidated further.Previous studies have demonstrated that Navβ2 subunit, an auxiliary subunit of Nav channel, participates in channel trafficking, re-localization, and interaction of both Nav1.1 , 47 and entially . Furtheral cells . Interespression , 41. WheAs far as we know, microRNAs are newly discovered and commonly considered as modulators of protein expression at post-transcriptional level, which are associated with the pathogenesis in multiple kinds of diseases –53. In tOur study provides strong evidences that miR-9 increases in both hippocampi and cortices, and inhibited the expression of Navβ2, which in turn blocked the trafficking of Nav1.1 and Nav1.2 from cytoplasm to plasma membrane Figs. , 4 and 5In addition, our important finding here is that miR-9 regulates endogenous Navβ2 expression by targeting its coding sequence (CDS) region rather than not 3’UTR of SCN2B Fig. . Additioin vivo study supports the data collected from our in vitro observations that the upregulation of miR-9 induced by both CBH and lenti-pre-miR-9 could also disturb the trafficking of both Nav1.1/Nav1.2 by downregulation of Navβ2 expression. On the contrary, lenti-pre-AMO-miR-9 injection into hippocampus markedly prevents the abnormal trafficking of both Nav1.1 and Nav1.2 following either CBH or lenti-pre-miR-9 treated normal rats accompanied by increased Navβ2 expression. These results combination with our in vitro data suggested that the inhibition of miR-9 in hippocampi and cortices in CBH model rats would be a way to prevent sodium channel dysfunction after CBH. An understanding of miR-9-Navβ2-Nav1.1/Nav1.2 trafficking pathway could yield to the potential therapeutic targets for the prevention of abnormal electrical activation induced by CBH.More importantly, our in vitro and in vivo, we did not provide evidence whether these changes could induce abnormal sodium channel currents and its dynamics characteristics in hippocampi and cortices of 2VO rats. These need to be studied further.In the present study, though we have demonstrated the regulation effect of miR-9 on the trafficking of Nav1.1/Nav1.2 by inhibiting the expression of Navβ2 both MiR-9 plays a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navβ2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially-valuable approach to prevent the Nav1.1/Nav1.2 trafficking disturbance induced by CBH.ad libium. Rats used for operation of permanent, bilateral common carotid artery occlusion (2VO) and stereotaxic injection of the lentiviral vectors were anesthetized with chloral hydrate  and maintained by administrating 0.5-1.0 % isoflurane. The depth of anesthesia was monitored by detecting reflexes, heart rate and respiratory rate. Samples for qRT-PCR, and Western blot assay were obtained from the hippocampi and cortices of rats after anesthetized with chloral hydrate  following by confirmation of death by exsanguination. Tissues for primary neuron culturing were from neonatal SD rats after administration of 20 % isoflurane and confirmation of death by cervical dislocation. All animal procedures were approved by the Institutional Animal Care and Use Committee at Harbin Medical University (No.HMUIRB-2008-06) and the Institute of Laboratory Animal Science of China (A5655-01). All procedures were conformed to the Directive 2010/63/EU of the European Parliament.Male Sprague–Dawley rats  were housed at 23 ± 1 °C with 55 ± 5 % of humidity and maintained on 12 h dark–light artificial cycle (lights on at 07:00 A.M.) with food and water available The method for preparation of 2VO rat was according to the previous study , 62. BriThe hippocampi and cortices regions were removed from the postnatal day 0 (P0) rat pups. After tissues were dissected and triturated, they were plated onto cell plates precoated with 10 μg/mL poly-D-lysine  and cultured in the culture media containing neurobasal medium  with 2 % B27 supplement  and 10 % fetal bovine serum . After 3 days, the neurons were treated with 5 μM cytosine arabinoside  to inhibit astrocyte proliferation. For all experiments, the neurons were used at 14 days after plating .MiR-9 mimics  and AMO-miR-9 (5’-UCAUACAGCUAGAUAACCAAAGA-3’) were synthesized by Shanghai GenePharma Co., Ltd . AMO-9 contains 2’-O-methyl modifications at every base and a 3’ C3-containing amino linker. Additionally, a scrambled RNA was used as a negative control . The Navβ2-masking antisense oligodeoxynucleotides (ODNs) were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. Navβ2 masking antisense-ODN-1 was 5’-ATGCCTTCGTCTTCTAGCTGC-3’, which masks the binding sites of miR-9, located in the position 336–358 of SCN2B CDS (coding sequence) region; Navβ2 masking antisense-ODN-2 was 5’TCCTCTTCGGTCTTCAGGTCA-3’ , which masks the binding sites of miR-9, located in the position of 575–597 of SCN2B CDS region. Five nucleotides or deoxynucleotides at both ends of the antisense molecules were locked by a methylene bridge connecting between the 2’-O- and the 4’-C atoms.Thirty pmol/mL miR-9 and/or AMO-9, ODNs or NC siRNAs were transfected into neonatal hippocampal and cortical neurons with X-treme GENE siRNA transfection reagent  according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were collected for total RNA isolation or protein purification.miR-9 (“top strand” oligo: TGCTGTcTTTggTTaTcTagcTgTaTgaGTTTTGGCCACTGACTGACTcaTacagagaTaaccaaaga) and its complementary chain (“bottom strand” oligo: CCTGTcTTTggTTaTcTcTgTaTgaGTCAGTCAGTGGCCAAAACTcaTacagcTagaTaaccaaagaC); (2) pre-AMO-miR-9 (“top strand” oligo: TGCTGTcaTacagcTagaTaaccaaagaGTTTTGGCCACTGACTGACTcTTTggTTcTagcTgTaTga) and its complement (“bottom strand” oligo: CCTGTcaTacagcTagaaccaaagaGTCAGTCAGTGGCCAAAACTcTTTggTTaTcTagcTgTaTgaC); (3) negative control (“top strand” oligo: tgctgAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTT) and its complement (“bottom strand” oligo: cctgAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTc). We then cloned the double-stranded oligonucleotides (ds oligo) generated by annealling the top and bottom strand oligos into the pcDNA™6.2-GW/± EmGFP-miR vector and transformed the ligated mixture into competent E. coli. After colony was purified and identified as the correct expression clone, the pre-microRNA expression cassette was transferred to other Gateway® adapted destination vectors utilizing PolII promoters and formed a new miRNA expression clone containing attR substrates. The vector was identified after analyzing the plasmid sequence . The titers of the vectors used for experiments were 9.25 × 108 transducing U/ml. Virus suspensions were stored at −80 °C until use and were briefly centrifuged and kept on ice immediately before injection.Using the BLOCK-iT polII miR-RNAi expression vector with the EmGFP kit from invitrogen, three single-stranded DNA oligonucleotides were designed as follows: (1) pre-After anaesthetized, rats were placed onto a stereotaxic frame  described as previous study . InjectiBefore the luciferase activity assay, plasmid design and construction was shown as in the below figure: a 707 bp fragment from the coding region of SCN2B containing the putative binding sequences for miR-9 (position 336–358 and position 575–597 of SCN2B CDS) was amplified by PCR, cloned into the pSICHECK-2-control vector.GACGAAGGC)ATTTACAACTGCTAATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAGACCGAAGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGT; mutSCN2B CDS (bold nucleotide): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAAGTACGACGTGTCAGTGACTCTAAAGAA(CGTGCAGCTAGAAATCGATCGC)ATTTACAACTGCTACATCACCAACCCTCCAGACCGCCACCGTGGCCATGGCAAGATCTACCTGCAGGTCCTTCTAGAAGTGCCCCCAGAGCGGGACTCCACGGTGGCAGTCATCGTGGGTGCCTCAGTGGGGGGTTTCCTGGCTGTGGTCATCTTGGTGCTGATGGTGGTCAAATGTGTGAGGAGGAAAAAAGAGCAGAAGCTGAGC(ACGGATGACCTGAAATCGATCGA)GGAAGGCAAGACGGATGGCGAGGGCAACGCGGAAGATGGCGCCAAGTAACCGGAAGCTTGCCCTGAAGCCCCTTCCTGTGTCCTGTCTCCTCTCACTCTCTGCCCTGTMutagenesis nucleotides were carried out using direct oligomer synthesis for the CDS region of Navβ2-binding site 1 and Navβ2-binding site 2. Point mutations were introduced into a possible miR-9 binding site located in the coding region of SCN2B (position 336–358 and position 575–597 of SCN2B CDS). MutSCN2B-1 represents that “GACGAAGG” was mutated “ATCGATCG” in the position 336–358 of SCN2B CDS, MutSCN2B-2 represents that “GACCGAAG” was mutated “ATCGATCG” in the position 575–597 of SCN2B CDS. MutSCN2B-1&2 represents that both sites were mutated. All constructs were sequence verified. Rat SCN2B CDS and mutSCN2B CDS sequences were shown as following: SCN2B CDS sequences (bold nucleotide showed the putative binding sequences for miR-9): ATGCACAGGGATGCCTGGCTACCTCGCCCTGCCTTCAGCCTCACGGGGCTCAGTCTGTTTTTCTCTTTGGTGCCCTCGGGGCGGAGCATGGAAGTCACAGTCCCCACCACTCTTAGTGTCCTCAACGGGTCTGATACCCGCCTGCCCTGTACCTTCAACTCCTGCTATACCGTGAACCACAAGCAGTTCTCTCTGAACTGGACTTACCAGGAGTGTAGCAATTGCTCAGAGGAGATGTTCCTCCAGTTCCGAATGAAGATCATCAACCTGAAGCTGGAGCGGTTTGGAGACCGCGTAGAGTTCTCGGGGAACCCCAGTAGTACGACGTGTCAGTGACTCTAAAGAA as well as 0.5 μg of psi-CHECKTM-2-target DNA (firefly luciferase vector) and 1 μl blank plasmid using lipofectamine 2000  transfection reagent according to the manufacturer’s instructions. After 48 h of transfection, Firefly and renilla luciferase activities, as indicated by relative luminescence units (RLU) were determined using luciferase assay kits  and luminometer  according to the manufacturer's instructions.The sequence of miR-9 mimic is 5’-UCUUUGGUUAUCUAGCUGUAUGA-3’ (synthesized based on the sequence of rno miR-9 (miRBase Accession No. MIMAT0000781)); that of miR-NC is 5’-UUCUCCGAACGUGUCACGUAA-3’; the sequence of the antisense 2’-O-methyl (2’-O-Me) oligonucleotide for miR-9 is 5’-UCAUACAGCUAGAUAACCAAAGA-3’, that of inhibitor-NC is 5’UUCUCCGAACGUGUCACGUTT-3’; HEK293T cells (plated at 40 % ~ 50 % confluence) were transfected with 20 μmol/l miRMiR-9 level was quantified by the TaqMan® MicroRNA Reverse Transcription Kit  and the TaqMan® Gene Expression Master Mix . The TaqMan qRT–PCR probes and primers for miR-9, were designed by Applied Biosystems [SCN2B forward: CTCTCTGAACTGGACTTACC and SCN2B reverse: GGTTGGTGATGTAGCAGTTG; β-actin forward: GGAAATCGTGCGTGACATTA and β-actin reverse: AGGAAGGAAGGCTGGAAGAG. All reactions were performed in triplicate, and the expressions of microRNAs data were shown as Delta-Delta Ct method.Total RNA was purified with the Trizol Reagent , according to the manufacturer’s instructions as described previously . MiR-9 losystems . U6 was Both the total protein and surface protein samples were extracted from hippocampi and cortices of rats or primary cultured neurons for immunoblotting analysis. For the total protein analysis, frozen tissue was homogenized with 1000 μl solution contained 40 % SDS, 60 % RIPA and 1 % protease inhibitor in each 200 mg brain tissue. The homogenate was then centrifuged at 13,500 rpm for 30 min and the supernatants (containing cytosolic and membrane fractions) were collected. The method of surface protein extraction was using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit  according to the manufacturer's instructions. Protein concentrations were measured spectrophotometrically using a BCA kit . Protein samples were fractionated by SDS-PAGE (10 % polyacrylamide gels for sodium channels) then transferred to PVDF membrane. The primary anti-Nav1.1, Nav1.2, Navβ2 antibodies  were used and β-actin  β-actin  was selected as an internal control of total proteins, mouse anti-human transferrin receptor (TfR)  was selected as an internal control of surface proteins. Western blot bands were captured on the Odyssey Infrared Imaging System  and quantified with Odyssey v1.2 software by measuring the band intensity (area × OD) in each group and normalizing to the internal control.The cultured neonatal rat neurons were transcardially perfused by 4 % buffered paraformaldehyde, pH7.4. After blocking, cultured neonatal rat neurons were incubated with the anti-β-Tubulin III  antibody  or anti- Nav1.1, Nav1.2, Navβ2 antibodies  overnight at 4 °C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594  for 1 h at room temperature.t-test was applied for comparisons between the two groups. Multi-group’s comparisions were performed by One-way ANOVA. SPSS19.0 software was used for all statistical analyses. P < 0.05 was considered significant.Data were described as mean ± s.e.m for experimental data. The two-tailed Student’s


There is an error in the third sentence of the “16S library preparation” subsection of the Materials and Methods. The first primer listed is incorrect. The correct sentence is: The V6 region was amplified with custom barcoded primers that consist of a region that anneals to the V6 region of interest, followed by a unique five basepair barcode, and the Illumina sequencing adapter [44]:V6-L [5’- ACACTCTTTCCCTACACGACGCTCTTCCGATCTnnnnnCWACGCGARGAACCTTACC-3’]V6-R [5’-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTnnnnnACRACACGAGCTGACGAC-3’]


Traditional fermented cheese whey (TFCW), containing probiotics, has been used both as a dairy food with ethnic flavor and a medicine for cardiovascular disease, especially regulating blood lipid among Kazakh. We therefore investigated anti-atherosclerotic effects of TFCW in atherosclerotic rabbits and identified lactic acid bacteria (LAB) and yeasts in TFCW.Atherosclerotic rabbits were induced by administration of atherosclerotic diet for 12 weeks and divided randomly into three groups and treated for 4 weeks with Simvastatin (20 mg/kg) or TFCW (25 mg/kg) and (50 mg/kg). In addition, a normal control group and an atherosclerotic group were used for comparison. All drugs were intragastrical administered once daily 10 mL/kg for 4 weeks. Body weight (BW), lipid profiles, C-reactive protein (CRP), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) were tested and theromatous plaques and the number of foam cells and infiltrating fibroblast cells in the thoracic aorta endothelium was evaluated by hematoxylin and eosin stainin. LAB and yeasts were isolated and purified by conventional techniques and identified using morphological and biochemical properties as well as gene sequences analysis.P < 0.05) compared to atherosclerotic group, and increased HDL-C (P < 0.05) compared to normal controls. Histological analysis showed TFCW reduced VCAM-1 expression and formation of atheromatous plaques on the aortic endothelium of atherosclerotic rabbits.After 4 weeks of treatment, high and low dose TFCW decreased serum TC, TG, LDLC, CRP, VCAM-1 and ICAM-1 (Seven classes of LBA from two different genera including Lactobacillus brevis, Lactobacillus kefianofaciens, Lactobacillus helveticus, Lactobacillus Casei, Lactobacillus plantarum, Lactobacillus kefiri and Lactococcus lactic as well as 2 classes of yeasts from two different genera including Saccharomyces unisporus and Issatchenkia orientalis were isolated and identified from TFCW. In summary, TFCW, containing 7 classes of LBA and 2 classes of yeasts, has significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium. The public health burden of cardiovascular disease (CVD) is substantial, as CVD remains the leading cause of mortality and morbidity worldwide and atherosclerosis is the major cause of CVD , 2.Traditional fermented cheese whey (TFCW), by-product of cheese-making, has been widely used as a traditional dairy medication for regulating blood lipid among Kazakh people . Indeed,Lactobacillus and Bifidobacterium and a few yeast species including Saccharomyces boulardi [S. boulardii, one of the probiotic yeasts, provides anti-inflammatory and host immunity stimulatory effects [Probiotics mainly include boulardi . Lactic boulardi  and atheboulardi  in anima effects  and lowe effects .However, anti-atherosclerotic effects of TFCW have not been experimentally demonstrated and no LAB or yeast has been found in TFCW. The aims of this study were to investigate anti-atherosclerotic effects of TFCW in a rabbit model of atherosclerosis and to identify LBA and yeast in TFCW.Traditional fermented cow’s milk is the source of the cheese whey. Experimental TFCW samples were manufactured by standard procedures in 10 L vats in Altay Kanas Dairy Co. Ltd., . Fresh cow’s milk samples were obtained from Jimunai Saur farm  and skimmed in centrifuging at 3000 × g for 30 min, homogenized under the pressure of 1.5 ~ 1.7 Mpa and pasteurized by high temperature short time (HTST) then cooled to about 30 °C and fermented by inoculation with traditional home made Kazak yogurt purchased from Jimunai Saur farm  at 37 °C for 12 h. After ferment, the whey was filtered in sterile gauze and dialyzed in cellulose membrane  under constant magnetic stirring at 8 °C, also performed lactose removal by periodic water exchange. The experimental TFCW was stored at −20 °C until further use.Sodium pentobarbital was purchased from Merck & Co., (Germany). Simvastatin was purchased from Merck Sharp & Dohme  Pty Ltd., . VCAM-1, ICAM-1 and CRP ELISA kits were purchased from Shanghai Senxiong Technology Co. Ltd., . Man Rogosa Sharpe (MRS) was purchased from Merck Sharp & Dohme  Pty Ltd., . All media for cultivation of zymocytes were purchased from Hangzhou Microbial Reagent Co. Ltd., .ad libitum access to food and water. After a week of adaptive feeding, all the rabbits were randomly divided into 5 groups with 12 in normal group and 12 in atherogenic group, the normal control groups were given regular die and the atherogenic models were developed using an atherogenic diet for 12 weeks. The atherogenic diet consisted of 3 % cholesterol, 0.5 % sodium taurocholate, 0.2 % propylthiouracil, 5 % sugar, 10 % lard, and 81.3 % standard laboratory rabbit chow, which were provided by Experimental Animal Center of Xinjiang Medical University, China. After developing atherogenic models, Group 1  was treated with saline in a matched volume; Group 2 (atherogenic group) had atherogenic rabbits treated with saline in a matched volume; Group 3 (positive control) had atherogenic rabbits administered with simvastatin 20 mg/kg; Group 4 and Group 5 were treated with TFCW 25 mg/kg and 50 mg/kg, respectively (low and high doses). Simvastatin and TFCW were intragastrical administered once daily 10 mL/kg for 4 weeks. All animals received care in compliance with the Chinese Convention on Animal Care, and the study was approved by the Institutional Ethics Committee of Xinjiang Medical University.Sixty male white New Zealand rabbits, weighing 1.95-2.05 kg, specific pathogen free (SPF), were provided by Experimental Animal Center of Xinjiang Medical University, China and placed in separate cages and maintained on a 12-h day/night cycle at an ambient temperature, with At the end of experiments, all rabbits were fasted for 12 h, weighed, anesthetized with sodium pentobarbital  and continually monitored until total loss of consciousness as indicated by a total lack of response after a foot pinch. Blood samples were collected from abdominal aorta, allowed to clot on ice and subsequently subjected to centrifugation (3500 rpm at 4 °C for 10 min), where after serum aliquots were stored at −80 °C for further analysis. Serum TC, TG, LDL-C and HDL-C were examined via an automatic biochemical analyzer . CRP was determined by rate nephelometry . Serum ICAM-1 and VCAM-1 were determined using commercially-available ELISA kits according to manufacturer instruction.Aorta was harvested from rabbits, placed immediately in formaldehyde 10 %, embedded in paraffin 24 h later, cut at 5 μm, stained with hematoxylin and eosin (H&E), and then scanned to assess pathological changes. For immunohistochemical staining, sections were incubated with anti-VCAM-1  and anti-F4/80  at 37 °C for 1 h, color developed with 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Samples in the absence of the primary antibodies were used as negative controls. Slides were observed under a light microscope, and images were subjected to statistical evaluation of positively stained cells in 10 random fields of view at a magnification of × 400. The average numbers of positively stained cells were counted per high power field (HPF).2) production were analyzed. All isolates were presumptively identified as LAB strains based on their ability to grow on MRS agar plates, Gram-positive staining, and a catalase activity-negative phenotype [Agar plates with Man Rogosa Sharpe (MRS) broth suitable for lactobacillus growth were used for initial isolation of LAB single colonies. Single bacterial colonies were initially separated based on their morphological differences on agar plates. Cell morphology was observed under light microscopy after Gram staining. Catalase activity, carbohydrate fermentation, acidogenicity, aciduricity , and gas . Significance was defined as p < 0.05), whereas Simvastatin group and low and high dose TFCW group showed significantly lower serum TC, TG and LDL-C than did atherogenic group (p < 0.05). TFCW significantly increased HDL-C levels compared to normal control group (p < 0.05), but no significant difference was observed in treated groups with TFCW, compared with atherogenic group. Data indicated that TFCW affects lipid metabolic parameters and TFCW treatment could effectively improve lipid metabolism in atherogenic rabbits.Table P < 0.05). Outstandingly, the simvastatin and low and high dose TFCW groups had significantly lower CRP (P < 0.05) at values 3.43 ± 0.80 mg/L, 3.33 ± 0.50 mg/L and 1.34 ± 0.90 mg/L, respectively, compared with the atherogenic group. CRP in low and high dose TFCW was significantly decreased, compared to atherogenic group (P < 0.05). Data indicate that TFCW affects CRP and TFCW treatment could effectively improve inflammatory status in atherogenic rabbits. No significant differences were recorded in treated groups with TFCW on body weight compared with atherogenic group.Serum CRP was measured to determine inflammatory status of experimental groups as shown in Table p < 0.05), while Simvastatin and low and high dose TFCW group showed significantly decreased ICAM-1 and VCAM-1, compared to atherogenic group (p < 0.05). Data indicated that TFCW might affect ICAM-1 and VCAM-1 and TFCW treatment might effectively inhibit adhesion of circulating inflammatory cells to endothelial cell walls in atherogenic rabbits.Adhesion molecules ICAM-1 and VCAM-1 play important roles in perpetuation of inflammation. Table Histopathological examination of aorta sections from experimental groups were shown in Fig. Immunohistochemical evaluation of VCAM-1 expression in aorta section and histomorphometric analysis from experimental groups were shown in Fig. Lactobacillus species was rod-shaped, slender short or long, and the majority of Lactobacillus exhibited chain-like arrangement. We successfully isolated and purified seven isolates of LBA and identified them as L. brevis, L. kefianofaciens, L. helveticus, L. casei, L. plantarum, L. kefiri and Lactococcus lactic based on morphological characteristics, physiological tests, biochemical tests, and 16S rDNA and 16S rRNA sequence homology with NCBI Reference Sequences NC 008497.1, NC 015602.1, NC 006814.3, NC 008526.1, NC 004567.2, NC 015428.1 and NC 017486. All the test results and sequence homology were shown in Table L. brevis:GCTGACTCCCGAAGGTTATCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTAGCCTCACGACTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTGAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGTCTTATCTCTAAGATTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACTAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAACCCTTGAACAGTTACTCTCAAAGGTGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCATTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATCGTCTTGGTGGGCCTTTACCTCACCAACTAACTAATACGCCGCGGGATCATCCAGAAGTGATAGCCGAANCCACCTTTCAAACAAAATCCATGCGGATTTTGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTGCTTCTGGGCAGATTTCCCACGTGTTACTCACCAGTTCGCCACTCGCTTCATTGTTGAAATCAANGCAAGCACGTCATTCAACGGAAGCTCGTTCGACTL. kefianofaciens:CTGCTTAGACGGCTCCTTCCTTGCGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTAATAGCTATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGAATTGCTTGCCTTTGCAGGCTCGCTGCTCGTTGTGCTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAGCCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTATTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCTACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAATTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGGTCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCACTGCCTTGGTAGGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGTCCATCCTTTAGCGACAGCTTGCGCCGCCTTTTAAAAGCTGTTCATGCGAACTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTTAAGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCCAGCGTCCTCACCGAAGTGATTCTGCTGGTTCCGCTCGCTCGACTTGCATGTATTAGL. helveticus:TTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTATAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCGTCCCCAGCATCATTACCGAAGTAAATCTGCTGGTTCTGCTCGCTCGACTGCATGTATL. casei:TAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTANNAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTCGTTCGACTGCATGTATAGCL. plantarum:GGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTGGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCACCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAGGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGL. kefiri:CTTAGACGGCTGGTCCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTGAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCTGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAAGATGTCAACAGTTACTCTGACACCTGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTAGGCCATTACCTTACCAACAAGCTAATACGCCGCGGGTCCATCCTAAAGTGATAGCCGAAGCCATCTTTTAAACCAAAACCATGTGGTTTTGGTTGTTATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTTTCGTGTTAAATCATTTAAATGCAAGCATCTAAAATCAATAACGGAAACGCGTTCGACTTGCATGTATLactococcus lactic:GGTCTTACCTTAGGAAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTANGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAATACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAAAACTTATGTTTAAAGTTGTTATGCGGTATTAGCATTCGTTTCCAAATGTTGTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCTTCAAATTGGTGCAAGCACCAATCTTCATCGCTCAACTTGCATGATTAGSeven gram-positive, catalase-negative bacilli were isolated from TFCW. Most of the bacilli had yellow or white colonies with uneven edges and a rough and dull surface. Some of the colonies in the center were translucent, with neat edges. The type colony size was approximately 0.5-3.0 mm. Morphology of the separated S. unisporus and I. orientalis based on morphological characteristics, physiological and biochemical tests and 26S rDNAD1/D2 sequence homology with sequences AY 707865 and EU 019220 in Genbank. All the test results and sequence homology were shown in Table S. unisporus:TGCATATTCAATAAGCGGAGGAAAAGAAACCAACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTAGTACCTTCGGTGCTCGAGTTGTAATTTGTAGAGGGATACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTATGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGCGCCCTCTGCTCCTTGTGGGTGGGGGAATCTCGCAGCTCACTGGGCCAACATCAGTTTTGGTGGTCGGATAAATCCGTAGGAATGTGGCTTGCCTCGGCAAGTGTTATAGCCTGCGGGAATACGGCCAGCTGGGACTGAGGACTGCCACTTTTGTCAAGGATGTTGGCATAATGGTTATATGCCGCCCGTCTTGAAACACGGACCAAI. orientalis:GCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTGAAGCGGCAAGAGCTCANATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTTGGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGAGCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAATGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAGGGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTCGTGGGCGGCGCTCTGGGCTTTCCCTGGGCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGTTCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGACCGAGGACTGCGGCCGTGTAGGTCACGGATGCTGGCAGAACGGCGCAACACCGCCCGTCTTGAAACACGGACCAWe identified two yeast isolates to be Major finding of the current study is that treatment with TFCW significantly modified lipid profile and reduced CRP, ICAM-1 and VCAM-1 in atherosclerotic rabbit model. Preventive effects of TFCW in atherogenic rabbits were also demonstrated by reduction in VCAM-1 expression and formation of atheromatous plaques on aortic endothelium. In fact, accumulation of cholesterol and lipids leads to foam cell formation, which is regarded as a critical process in development of atherosclerosis . OverwheL. casei [L. helveticus [L. plantarum [L. lactis [In this study, 7 potential probiotic lactobacillus species, including L. casei , L. helvlveticus , L. planlantarum  and L. l. lactis , which a. lactis  and redu. lactis , 23 both. lactis , 25 and . lactis . LAB or . lactis .Lactocillus fermenterum ME-3 improves antioxidant activities in human blood, thus providing antiatherogenic activity [In addition, goat milk fermented with activity . Consumpactivity , 29.S. unisporus is ubiquitously present in fermented milk, cheese and kefir-based milk products and may produce vitamins and interact with LAB, which may enhance LAB growth [S. unisporus contains middle chain fatty acids up to C 14:0 to 18:1 and produces a high percentage of palmitoleate. Palmitoleic acid, an omega-7 monounsaturated fatty acid, is a major constituent of human adipose tissues and is considered antioxidant [I.orientalis exhibits a higher tolerance for pH, bile, and heat stress for survival in gastrointestinal environment as a probiotic [I. orientalis commonly exists in cheeses and other fermentation milk products and exhibits ability to scavenge 1,1 diphenyl-2-picrylhydrazyl and to inhibit lipid peroxidation, thus presenting antioxidant activity as a potential probiotic in fermented milk products [We also identified two probiotic yeasts in TFCW. B growth . S. unisioxidant . I.orienrobiotic . I. orieproducts .Notably, oxidized LDL in vascular wall seems to be a key factor in atherosclerosis, because oxidized LDLs might recruit monocytes and favor their transformation into foam cells through a receptor-mediated intake (scavenger pathway). Moreover, cytotoxic oxidized form of LDLs are likely responsible for endothelial cell damage and macrophage degeneration in atherosclerotic human plaque . PolyunsIn conclusion, current study indicates that 7 LAB and 2 yeasts identified from TFCW and TFCW have significant anti-atherosclerotic potential in atherosclerotic rabbits and may modulate lipid metabolism and protect aorta in the atherosclerotic condition, which might be related to various probiotics acting through reducing the CRP, VCAM-1 and ICAM-1 levels and protecting the aortic endothelium.


Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial’s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time . Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy  were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. To date, mitochondria have been thought to play an important role in myocardial ischemia-reperfusion (I/R) injury . It is wIn contrast, heme oxygenase-1 (HO-1), which belongs to the low-molecular weight heat shock protein HSP family, induction and expression are important protective mechanisms during cell stress –6. In raTherefore, in this study, we chose to use genetic engineering techniques to construct an Lv-HO1-H9c2 cell line that enables a steady increase in the expression of exogenous HO-1. After myocardial hypoxia-reoxygenation injury, the RFP-GFP-LC3 double-labeled adenovirus was used to detect autophagy, and flow cytometry was used to measure mitochondrial membrane potential and mitochondrial ROS levels to investigate the mechanism by which HO-1 protects mitochondrial function following myocardial hypoxia-reoxygenation injury.The effect of different durations of hypoxia/reoxygenation on myocardial cytotoxicity was measured. As shown in Cell viability was compared between the four groups . SignifiCell viability was compared between the four groups . An Lv-HCell viability in the four groups was compared . After ZHO is the initial and rate-limiting enzyme that catalyzes the oxidative metabolism of heme. Three HO isozymes exist, and HO-1 is the only isoenzyme that exhibits inductive expression. During cardiac ischemia-reperfusion injury, ROS generation can increase the expression of HO-1 , consistThe autophagic dysfunction of myocardial cells has attracted attention as one of the mechanisms underlying ischemia-reperfusion injury ,24,25. IIn addition, in this study, flow cytometry detection showed that during autophagy, the mitochondrial membrane potential was significantly higher  and the Therefore, clearing damaged mitochondria is extremely important. Autophagy, the major degradation pathway involved in mitochondrial quality control, has been reported as a cellular adaptive response to oxidative stresses . PreviouInterestingly, in our studies, rapamycin could up-regulate the expression of the HO-1 protein . It is aTo define the role of HO-1, most studies have used methods such as employing adeno-associated viruses to achieve instantaneous HO-1 over-expression ,14 or knWith HO-1 overexpression, LC3II levels were significantly increased after reoxygenation, and p62 protein expression was significantly decreased . MoreoveRecent studies have shown that an HO-1 activator can activate transcription factor EB (TFEB), a regulator that induces lysosome and autophagosome formation, to protect against LPS-induced oxidative stress in heart tissue . MeanwhiIn summary, we based our conclusions on evidence that showed that during myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect against myocardial hypoxia-reoxygenation injury. By studying the molecular and regulatory mechanisms of mitochondrial function protection, we will obtain further knowledge regarding the mechanism underlying the development of ischemic heart disease, information that will aid in for the development of new disease prevention and treatment strategies.Dulbecco’s Modified Eagle’s Medium (DMEM) and other cell culture supplies, such as trypsinogen, were purchased from Invitrogen. Fetal calf serum was purchased from Gibco BRL . A Cell Counting Kit-8 was purchased from Dojindo . HO-1 and pAbR rabbit antibody were purchased from Enzo Life Sciences Inc. . p62 and LC-3 antibody were purchased from Abcam (USA), and GAPDH antibody was obtained from CST (USA). AntiRabbit was obtained from Sigma (USA). BSA was obtained from Invitrogen (USA). TritonX-100 was obtained from Sigma (USA). A JC-1  Mitochondrial Membrane Potential Detection Kit was purchased from Beyotime (China). Confocal microscopy and imaging systems (ZEISS-Axio) were used at the Wuhan University School of Basic Medical Sciences.34H32N4O4Zn and a molecular weight of 626.03 g/mol.Zinc protoporphyrin (ZnPP), a HO-1 inhibitor, was supplied by Sigma. The purity of ZnPP (≥95%) was determined by HPLC; the inhibitor was soluble in water and has the molecular formula C2. The medium was replaced every 2–3 days, and the cells were subcultured or subjected to experimental procedures at 80–90% confluence. In all experiments, the cells were rendered quiescent by serum starvation for 24 h before treatment. For the H/R experiments, hypoxic conditions were created by incubating the cells in an anaerobic chamber equilibrated with 2.5% O2, 5% CO2 and 92.5% N2 at 37°C for 24 h. The cells were then reoxygenated under normoxic conditions in a humidified atmosphere (95% air/5% CO2) at 37°C for the indicated time . Normoxic control cells were incubated at 37°C under 95% air/5% CO2. In the Rapamycin experiments, the cells were pretreated for 1 hour with the autophagy inducer Rapamycin (100 nM) and then subjected to hypoxia and reoxygenation. In the ZnPP experiments, the cells were pretreated for 6 hours with the HO-1 inhibitor ZnPP (10 μM) and then subjected to hypoxia and reoxygenation.The H9c2 cardiomyocyte cell line (rat embryonic cardiomyoblasts) was maintained in our laboratory and obtained from the American Type Culture Collection . The H9c2 cells were maintained in high-glucose DMEM supplemented with 10% v/v fetal calf serum at 37°C in a humidified atmosphere containing 5% COA lentiviral vector was constructed, and a lentiviral shuttle plasmid and its secondary packaging of the original vector plasmid were prepared; the three plasmid vectors were subjected to endotoxin-free extraction to obtain materials of high purity. Next, the plasmid vectors were co-transfected into 293T cells. The medium was replaced with complete medium at 6 hours after transfection. The supernatant, which was rich in lentivirus particles, was collected after 48 and 72 hours of culture and filtered using a 0.45 μm filter (Millipore), and the virus was then concentrated by ultracentrifugation. Next, a high-titer lentivirus concentrate was obtained after concentration. A vector map of pHBLV-CMVIE-IRES-puro is shown in The pHBLV-CMVIE-IRES-puro vector contains a CMVIE promoter to initiate the expression of target genes and contains a puromycin resistance gene as a marker. The HO-1primer designs as ATGGAGCGTCCGCAACCCGACAGCATGCCCCAGGATTTGTCAGAGGCCCTGAAGGAGGCCACCAAGGAGGTGCACACCCAGGCAGAGAATGCTGAGTTCATGAGGAACTTTCAGAAGGGCCAGGTGACCCGAGACGGCTTCAAGCTGGTGATGGCCTCCCTGTACCACATCTATGTGGCCCTGGAGGAGGAGATTGAGCGCAACAAGGAGAGCCCAGTCTTCGCCCCTGTCTACTTCCCAGAAGAGCTGCACCGCAAGGCTGCCCTGGAGCAGGACCTGGCCTTCTGGTACGGGCCCCGCTGGCAGGAGGTCATCCCCTACACACCAGCCATGCAGCGCTATGTGAAGCGGCTCCACGAGGTGGGGCGCACAGAGCCCGAGCTGCTGGTGGCCCACGCCTACACCCGCTACCTGGGTGACCTGTCTGGGGGCCAGGTGCTCAAAAAGATTGCCCAGAAAGCCCTGGACCTGCCCAGCTCTGGCGAGGGCCTGGCCTTCTTCACCTTCCCCAACATTGCCAGTGCCACCAAGTTCAAGCAGCTCTACCGCTCCCGCATGAACTCCCTGGAGATGACTCCCGCAGTCAGGCAGAGGGTGATAGAAGAGGCCAAGACTGCGTTCCTGCTCAACATCCAGCTCTTTGAGGAGTTGCAGGAGCTGCTGACCCATGACACCAAGGACCAGAGCCCCTCACGGGCACCAGGGCTTCGCCAGCGGGCCAGCAACAAAGTGCAAGATTCTGCCCCCGTGGAGACTCCCAGAGGGAAGCCCCCACTCAACACCCGCTCCCAGGCTCCGCTTCTCCGATGGGTCCTTACACTCAGCTTTCTGGTGGCGACAGTTGCTGTAGGGCTTTATGCCATGTGAAfter H9c2 and Lv-HO1-H9c2 cells were cultured in 96-well plates and received the described treatments, 10 μl of CCK-8 solution was added to each well (1/10 dilution); the plates were then incubated for a further 3 h. The absorbance was measured at 450 nm using a microplate reader . The mean optical density (OD) of five wells in the indicated groups was used to calculate the percent cell viability according to the following formula: Percent cell viability = OD treatment group/OD control group×100%. The experiments were repeated 3 times.®Premix Ex Taq™ Perfect Real-Time Kit. To measure mouse gene expression, the following SYBR Green real-time PCR primers were used:Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) and purified using the RNeasy Total RNA Isolation Kit (Invitrogen). Real-time quantitative PCR was performed using the ABI 7900 Real-Time PCR System (Applied Biosystems) and the SYBR5′- TGCACATCCGTGCAGAGAAT -3′HO-1: Forward 5′-CTGGGTTCTGCTTGTTTCGC -3′; product length = 147    Reverse 5′—CACCATCTTCCAGGAGCGAG -3′GAPDH: Forward 5′—AAATGAGCCCCAGCCTTCTC -3′; product length = 114    Reverse Proteins were isolated from the cells that had been subjected to hypoxia and reoxygenation using standard protocols, and the protein concentrations in the lysates were determined using the Bradford Protein Assay Kit . Equal quantities of proteins (30–50 lg/lane) were subjected to 8%–12% SDS-PAGE, depending on the target proteins, electrotransferred onto nitrocellulose membranes, and incubated with primary antibodies against HO-1 , p62 , LC-3-II , GAPDH , and antiRabbit . After incubating the membranes with the corresponding secondary antibodies, protein bands were detected using a chemiluminescence imaging analysis system (Fujifilm), and quantitation was performed using Quantity One 4.4.0 software (Bio-Rad).4 cells/mL) was added. Cells were transfected with Ad-mCherry-LC3-GFP adenovirus (MOI = 50); 36 hours later, the plates were pre-incubated for 24 hours in an incubator . Next, 4% PFA  was used to fix the cells for more than 10 minutes; the cells were then washed in PBS (5 minutes x 3 times). Then, Antifade mounting medium was added, and the cells were imaged under a fluorescent microscope. RFP-GFP-LC3 double-labeled adenovirus was used for testing. GFP degrades in the acidic environment used. When red and green fluorescent images are merged, the yellow spots that appear indicate autophagosomes. Red spots indicate autophagic lysosomes. In normal phagosome-lysosome fusions, there will be more red fluorescence than yellow fluorescence. In impeded autophagy, where phagosome-lysosome fusion does not work, yellow fluorescence will be dominant.To 6-well plates , 2 mL of Lv-HO1-H9c2 cell suspension  and reactive nitrogen species (RNS) rapid oxidation. After binding nucleic acid oxidation products, the probe can produce large amounts of fluorescence by flow cytometry, thus, the changes of mitochondrial reactive oxygen species content may be reflected.Using flow cytometry to detect the 5,5',6,6'-tetrachloro-1,1',3,3'-. tetraethylbenzi-midazolylcarbocyanine iodide (JC-1) staining. After incubation with the JC-1 staining solution at 37°C in the cell incubator for 10 min, cells were washed with JC-1 staining buffer two times and then analyzed using a fluorescence microplate reader. In the normal mitochondria, JC-1 aggregate to form a polymer in the mitochondrial matrix, the polymer sends a strong red fluorescence ; While in the unhealthy mitochondrial, due to the decline or loss of the mitochondrial membrane potential, JC-1 monomers just can be present in the cytoplasm, resulting in a green fluorescence . Therefore, using flow cytometry to observe the color changes reflects very directly the early change of mitochondrial membrane potential.5 cells were harvested and placed in EP tubes. The cells were rinsed with 0.1 mmol/L PBS (pH = 7.4) and collected brief centrifugation with trypsin EDTA-free. After that, the cells were resuspended in 500 μL of binding buffer, mixed with 5 μL of Annexin V-FITC, and then kept in darkness for 10 min at room temperature. Then, 5 μL of propidium iodide (PI) was added and the samples were kept in darkness for 10 min at room temperature. Approximately 300 μL of binding buffer was added, and the samples were analyzed using flow cytometry within 1 h. The blank control was the cells of the control group without Annexin V-FITC and PI. The Annexin V-FITC single-labeled control consisted of cells labeled with Annexin V-FITC. The PI single-labeled control consisted of cells labeled with PI.After treatment, 1~5×10The data are presented as means ± SEM. Statistical analysis was performed using the Mann-Whitney test for two-group comparisons. Significant differences between multiple treatments were calculated using one-way analysis of variance followed by the Bonferroni post hoc test when appropriate. Western blot densities were analyzed using the Kruskal-Wallis test followed by Dunn's post hoc test. Probabilities of 0.05 or less were considered statistically significant.S1 Fig(A) Cell viability; (B) HO1 QPCR; (C)WB analysis.(RAR)Click here for additional data file.S2 Fig(A) Cell viability (CCK-8); (B) WB analysis; (C) Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (F) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S3 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.S4 Fig(A) Cell viability (CCK-8); (B) HO1 QPCR; (C) WB analysis; (D)Confocol-RFP-GFP-LC3;  Flow cytometry detection of changes in JC-1 and MitoSox; (G) apoptosis rate (AnnexinV-PI).(RAR)Click here for additional data file.


Monoclonal antibodies (mAbs), because of their unique specificity, are irreplaceable tools for scientific research. Precise mapping of the antigenic determinants allows the development of epitope tagging approaches to be used with recombinant proteins for several purposes. Here we describe a new family of tags derived from the epitope recognized by a single highly specific mAb (anti-roTag mAb), which was obtained from a pool of mAbs reacting with the rotavirus nonstructural protein 5 (NSP5). The variable regions of the anti-roTag mAb were identified and their binding capacity verified upon expression as a single-chain/miniAb. The minimal epitope, termed roTag, was identified as a 10 amino acid sequence (SISSSIFKNE). The affinity of the anti-roTag/roTag interaction was found to be comparable to that of the anti-SV5/SV5 tag interaction. roTag was successfully used for detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation. In biological sciences development of new specific monoclonal antibodies (mAbs) is a pressing requirement for several aspects in the field: from basic research on protein function, to medical diagnosis, prophylaxis and therapy of several pathogenic conditions As tags are often short (6–15 amino acids in length), they are generally presumed to have no effect on the biological functions of the tagged proteins. However, if located in inappropriate positions, they might interfere with protein structure, function and interactions. In addition, not all mAb are suitable for every immunodetection method, as in the case of mAb specific for non-linear epitopes. For those reasons, it is useful to develop mAbs and epitope tags of different sequence characteristics  or that can be fused in different positions of the target protein to increase the chances of success in tagging applications.Here we describe and characterize a new 10 amino acids long epitope tag (roTag) derived from the sequence of the rotavirus (RV) non-structural protein 5 (NSP5). NSP5 has an essential role during the RV replication cycle, as it is essentially required for the assembly of viroplasms, the sites of viral genome replication and initial assembly of progeny virus ++-purified His-tagged NSP5 protein of the RV porcine OSU strain A panel of anti-NSP5 mAbs were generated from BALB/c mice immunized with a NiTo establish the epitope on NSP5 we took advantage of a series of NSP5 deletion mutants To further define the epitope, a series of progressive N-terminal deletion mutants, NSP5-ΔN3, NSP5-ΔN8, NSP5-ΔN13, NSP5-ΔN18, NSP5-ΔN23 and NSP5-ΔN28, were assayed with a polyclonal anti-NSP5 serum or with 1F2 by WB. As shown in PSISSSIFKNE)  suggesting involvement of S12 and S14. In fact, a tag initiating in S12 (tag 12–24) resulted in a pattern of bands equally recognized by anti-SV5 and 1F2, both in the intracellular and in the secreted material  and because of the relevant impact of P11 on O-glycosylation, we defined S12 as the N-terminal border of the epitope and termed roTag the peptide 12–21 and P-roTag the one starting in P11 (11–21). The C-terminal border of the anti-roTag/1F2 epitope was confirmed to be E21, as peptides 9–18, 9–19 and 9–20 were not detected by anti-roTag . All theper se sufficient to cause retention in the ER and to completely prevent O-glycosylation, according to three different criteria: unchanged mobility, lack of secretion and detection by mAb anti-roTag.O-glycosylation was confirmed for roTagO, P-roTag and OG-tag by treatment of supernatants containing the reporter protein (with either of the three tags) with a glycosidase cocktail. As shown in L-linker-VH) fused to the hinge-CH2-CH3 domains of the γH chain of murine IgG2b. The resulting recombinant antibodies  containing the scFv  the 10 amino acids long roTag (peptide 12–21) that can be efficiently used with different proteins, in N-terminal, middle and C-terminal positions and in different cellular compartments  , approved by the ICGEB Trieste Ethics Committee for Animal Experimentation. Animals were anesthetized (isoflurane) before venipuncture and sacrifice (asphyxiation in carbon dioxide). All efforts were made to minimize suffering.++-purified His-tagged NSP5 protein Balb/c mice have been immunized with NiNSP5 deletion mutants were previously described 5′-GATATTGTGATGACCCAGTCTCCA-3′, CK-2 3′: 5′-TGGATACAGTTGGTGCAGC-3′, VHS-2 5′: 5′-TGTGCACTCYSAGGTSMARCT-3′, CHγ 3′: 5′-GGCCAGTGGATAGAC-3′. From pUC18 VL and VH regions were amplified with primers 1F2-VL-for: 5′TATGGTGCACTCTGATGTTGTGATGACCCAGACTCCA-3′, 1F2-VL-rev: 5′-TATAACTAGTGCTGCCTTTCAGCTCCAGCTTGGT-3′, VH-for 5′: 5′-TCTCTCGAGCAAAGGTCAGGTCCAACTGCAGCAGTC-3′, 1F2-VH rev: 5′-AGTTCCGGAGGAGACGGTGACTGA-3′ and cloned ApaLI/BspEI into pUT vector downstream of a leader peptide H2 e CH3 of IgG2b, which were previously amplified from murine splenocytes and inserted in pcDNA3 by BspEI/XbaI (primers MHG2B-1 5′: 5′-CCCTCCGGACCCATTTCAACAATCA-3′ and MHG2B-2 3′: 5′-TCTTCTAGAGCTCATTTACCCGGAGA-3′).After cDNA synthesis VL and VH were amplified and cloned in pUC18 using primers VKB-2 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAATCGFor mapping the anti-roTag epitope the following synthetic oligonucleotides encoding the different epitope tags were cloned as BamHI/EcoRI, downstream of SV5 in a plasmid containing scFv-SV5-BAP previously described 5′-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATCTTCTTAAG-3′, 9-24-2 3′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTGAGGGAAGG-3′, 9-24G-1 5′: 5′-AATTCTTAAGAAGAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3′, 9-21-1 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3′, 9-21-2 3′: 5′-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3′, 12-24-1 5′: 5′-GATCCATTTCTTCTAGTATCTTTAAAAATGAATCGTCTTCTTAAG-3′, 12-24-2 3′: 5′-AATTCTTAAGAAGACGATTCATTTTTAAAGATACTAGAAGAAATG-3′, 9-16-1 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTTAAG-3′, 9-16-2 3′: 5′-AATTCTTAACTAGAAGAAATTGAGGGAAGG-3′, 10-21-1 5′: 5′-GATCCGGTGGCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3′, 10-21-2 3′: 5′-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGGCCACCG-3′, 11-21-1 5′: 5′-GATCCGGTGGCCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3′, 11-21-2 3′: 5′-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGGCCACCG-3′, 12-21-1 5′: 5′-GATCCATTTCTTCTAGTATCTTTAAAAATGAAGGTTAAG-3′ 12-21-2 3′: 5′-AATTCTTAACCTTCATTTTTAAAGATACTAGAAGAAATG-3′, 9-20-1 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATTAAG-3′, 9-20-2 3′: 5′-AATTCTTAATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG-3′, 9-19-1 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAATAAG-3′, 9-19-2 3′: 5′-AATTCTTATTTAAAGATACTAGAAGAAATTGAGGGAAGG-3′, 9-18-1 5′: 5′-GATCCCTTCCCTCAATTTCTTCTAGTATCTTTTAAG-3′, 9-18-2 3′: 5′-AATTCTTAAAAGATACTAGAAGAAATTGAGGGAAGG-3′, roTagG KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTAAAAATGAAGGTAAGGATGAGCTTTAAG, roTagG KDEL-2: AATTCTTAAAGCTCATCCTTACCTTCATTTTTAAAGATACTAGAAGAAATTGAGGGAAGG, OG-Tag KDEL-1: GATCCCTTCCCTCAATTTCTTCTAGTATCTTTGGAAAGGATGAGCTGTAAG, OG-Tag KDEL-2: AATTCTTACAGCTCATCCTTTCCAAAGATACTAGAAGAAATTGAGGGAAGG.TTCTTCTTAAG-3′, 9-24G-2 3′: 5 cells/well) by standard calcium phosphate technique Sp2/0 myeloma cells (ATCC CRL-1581) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS); hybridoma clones were grown in the same medium supplemented with 2% Hybridoma Media Supplement (Sigma-Aldrich) and 1 mM sodium pyruvate. MA104 and HEK 293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum (FCS). Cells were co-transfected in 6-well plates  supplemented with protease inhibitors cocktail (Sigma-Aldrich). In the experiments shown in Immunofluorescence experiments were performed after cells were fixed in 3.7% paraformaldehyde in PBS for 10 min at room temperature. Coverslips were washed in PBS and blocked with 1% bovine serum albumin (BSA) in PBS for 30 min and incubated with mouse anti-NSP5 serum (1:500) in PBS–1% BSA, supernatants of anti-roTag hybridoma for 1 h at room temperature. After three washes in PBS, the slides were stained for 45 min with rhodamine isothiocyanate-conjugated secondary antibody (Sigma), washed, and mounted with ProLong mounting medium (Molecular Probes). Samples were analyzed by confocal microscopy (Zeiss LSM510).3-Na2CO3 50 mM, pH 9.5 (100 µl/well). After reaction with HRP-labeled goat anti-mouse gamma Fc (Jackson Immunoresearch) plates were developed with tetramethylbenzidine (TMB) reagent (Sigma) and blocked with H2SO4. O.D. at 450 nm was read on a BioRad microplate reader 550. ELISA comparison of relative affinity of recombinant anti-SV5 and anti-roTag was performed by capturing the double-tagged (SV5-roTag) reporter ELISA to determine relative antibody concentrations was performed by capturing supernatants containing Abs on polystyrene microplates (Nunc Maxisorp C96) coated with 0.1 µg/ml of goat anti-mouse gamma in buffer NaHCOFigure S1(A) Full gel, of non-infected and RV-infected MA104 cell extracts reacted with mAb 1F2. (B) Full gel of lanes shown in (TIF)Click here for additional data file.