大鼠BMP-7重组腺病毒构建及在肾小管上皮细胞中表达

目的:利用AdEasy~(TM) system构建携带大鼠BMP-7基因的重组腺病毒,并鉴定其在原代肾小管上皮细胞中的表达.方法:将BMP-7全长序列分为46个片段分段合成,再采用PCR方法将合成的46个寡核苷酸片段拼接成BMP-7基因,并克隆到测序载体中测序鉴定.经Not I和Hind Ⅲ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-BMP-7.EcoRI酶切重组pShuttle-BMP-7,与pAdEasy~(TM)DNA电穿孔共转化BJ5183感受态菌, 抽提重组AdBMP-7质粒,PacI酶切线性化重组质粒AdBMP-7后,转染Ad293细胞进行病毒包装和扩增,腺病毒荧光检测试剂盒测定其滴度.用AdBMP-7感染大鼠肾小管上皮细胞,以ELISA法和Western-blot法检测BMP(-7)在大鼠肾小管上皮细胞中的表达.RT-PCR方法检测α-SMA mRNA的表达水平,以鉴定重组腺病毒AdBMP-7的生物学活性.结果:构建了BMP-7基因的重组腺病毒载体,并制备出高滴度重组腺病毒保存液.该重组腺病毒在体外能有效转染大鼠肾小管上皮细胞并高表达BMP-7蛋白,所表达的BMP-7蛋白能有效逆转由TGF-β1诱导的α-SMA表达增高.结论:成功地构建了携带大鼠BMP-7基因的重组腺病毒,并初步证实其具有一定的抗肾纤维化功能.     

Estimating the date of origin of an HIV-1 circulating recombinant form

HIV is capable of frequent genetic exchange through recombination. Despite the pandemic spread of HIV-1 recombinants, their times of origin are not well understood. We investigate the epidemic history of a HIV-1 circulating recombinant form (CRF) by estimating the time of the recombination event that lead to the emergence of CRF33_01B, a recently described recombinant descended from CRF01_AE and subtype B. The gag, pol and env genes were analyzed using a combined coalescent and relaxed molecular clock model, implemented in a Bayesian Markov chain Monte Carlo framework. Using linked genealogical trees we calculated the time interval between the common ancestor of CRF33_01B and the ancestors it shares with closely related parental lineages. The recombination event that generated CRF33_01B (t_rec) occurred sometime between 1991 and 1993, suggesting that recombination is common in the early evolutionary history of HIV-1. The proof-of-concept approach provides a new tool for the investigation of HIV molecular epidemiology and evolution.     

Genomic organization and evolution of immunoglobulin kappa gene enhancers and kappa deleting element in mammals

We have studied the genomic structure and evolutionary pattern of immunoglobulin kappa deleting element (KDE) and three kappa enhancers (KE5′, KE3′P, and KE3′D) in eleven mammalian genomic sequences. Our results show that the relative positions and the genomic organization of the KDE and the kappa enhancers are conserved in all mammals studied and have not been affected by the local rearrangements in the immunoglobulin kappa (IGK) light chain locus over a long evolutionary time (120 million years of mammalian evolution). Our observations suggest that the sequence motifs in these regulatory elements have been conserved by purifying selection to achieve proper regulation of the expression of the IGK light chain genes. The conservation of the three enhancers in all mammals indicates that these species may use similar mechanisms to regulate IGK gene expression. However, some activities of the IGK enhancers might have evolved in the eutherian lineage. The presence of the three IGK enhancers, KDE, and other recombining elements (REs) in all mammals (including platypus) suggest that these genomic elements were in place before the mammalian radiation.     

The Complete Nucleotide Sequence of a Pakistani Isolate of Watermelon Mosaic Virus Provides Further Insights into the Taxonomic Status in the Bean Common Mosaic Virus Subgroup

Watermelon mosaic virus (WMV) is a potyvirus with a worldwide distribution, but is mostly found in temperate and Mediterranean regions. The complete nucleotide (nt) sequence of a Pakistani isolate of WMV (WMV-Pk) was determined and compared with French isolate (WMV-Fr) and other closely related potyviruses. WMV-Pk showed overall identities of 94.4% (nt) and 96% (amino acid; aa) with the WMV-Fr. However, variability was observed in the 5′ UTR and P1 region. Although sequence identities over most of the genome were well above 90% at both the nt and aa levels, reaching 99.6% (aa) in the CP and 100% (aa) in the 6K1 and 6K2, thereby suggesting that WMV-Pk and WMV-Fr are identical strains, but the sequence identities in the P1 region were only 80.6% (aa) and 82.8% (nt), while that in the 5′ UTR was 82%. These differences may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in two different agro-ecological zones. The sequence of WMV-Pk is very close to that of Soybean mosaic virus (SMV) over most of the genome, except for the N-terminal region, which is subject to recombination between SMV and Peanut stripe virus (PSV)/Bean common mosaic virus (BCMV), as revealed by Simplot and phylogenetic analyses of N- and C-terminal P1, HC-Pro, and 5′ UTR regions of the genome.     

Risk of HIV-1 transmission by breastfeeding among mothers infected with recombinant and non-recombinant HIV-1 genotypes

BACKGROUND: Viral genotype and intersubtype recombination may influence the rate and/or timing of mother-to-child HIV-1 transmission. METHODS: We determined the HIV-1 subtype of the C2-C5 env and 5'LTR regions from milk and blood samples of 61 Tanzanian mothers who transmitted the virus through breastfeeding and their HIV-1 positive non-transmitting controls. Cases and controls were matched on infant's age at sample collection. All mothers resided in Dar es Salaam, Tanzania. RESULTS: Most infections among cases were due to recombinant viruses (41.0%), followed by HIV-1 subtype A (26.2%), subtype D (19.7%), and subtype C (13.1%). In multivariate analysis including maternal CD4+ cell counts, HIV disease stage, and proviral load in breast milk, the odds of breast milk transmission were 7.2 times higher if the mother carried an intersubtype recombinant genome in comparison to a subtype C virus (p=0.02). Viruses with recombinant LTRs were 4.9 times more likely to be transmitted through breastfeeding than viruses with non-recombinant LTRs of subtype A, C or D combined (p=0.01). CONCLUSIONS: This suggested that intersubtype recombinant genomes, and especially recombination within the LTR, might render HIV-1 more fit for transmission via breast milk in comparison with non-recombinant subtypes A, C, and D.     

Independent acquisition of site-specific recombination factors by asn tRNA gene-targeting genomic islands

Two genomic islands, namely the high-pathogenicity island (HPI) and Ecoc54N target the same asn tRNA genes to integrate into the bacterial chromosome. The HPI encodes the siderophore yersiniabactin in the highly pathogenic Yersinia group (Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica 1B) whilst the Ecoc54N island possibly encodes a polyketide synthase with an unknown function in the uropathogenic Escherichia coli CFT073 strain. HPI encodes the recombinase that promotes site-specific recombination (both integrative and excisive) with its corresponding attachment targets. A recombinase orthologue is also present in Ecoc54N. In addition, the HPI(Yps) of the Y. pestis/Y. pseudotuberculosis evolutionary lineage encodes the excisionase (recombination directionality factor, Xis(HPI)) that facilitates excision of the island. However, no sequence resembling the excisionase gene could be found in Ecoc54N. The rate of the HPI(Yps) excision estimated by real-time PCR was 10(-6) in Y. pseudotuberculosis. The presence of the excisionase increased the efficiency of the excisive recombination only eight fold. However, the introduction of the xis(HPI) in E. coli CFT073 did not influence the excision of Ecoc54N. The Xis(HPI) is encoded by the variable AT-rich part of the HPI(Yps) and substantially differs from its cognate recombinase in A+T content and codon usage. Also the Xis(HPI)-protected region, defined in the HPI attachment site, has suffered several nucleotide substitutions in Ecoc54N that could influence interaction with the excisionase. We propose that the pathogenicity islands (PAIs) targeting asn tRNA genes (PAIs(asn tRNA)) might have acquired recombinase and excisionase (HPI) genes independently and sequentially.     

Chromosome pairing and meiotic recombination in Neurospora crassa spo11 mutants

Some organisms, such as mammals, green plants and fungi, require double-strand breaks in DNA (DSBs) for synapsis of homologous chromosomes at pachynema. Drosophila melanogaster and Caenorhabditis elegans are exceptions, achieving synapsis independently of DSB. SPO11 is responsible for generating DSBs and perhaps for the initiation of recombination in all organisms. Although it was previously suggested that Neurospora may not require DSBs for synapsis, we report here that mutation of Neurospora spo11 disrupts meiosis, abolishing synapsis of homologous chromosomes during pachynema and resulting in ascospores that are frequently aneuploid and rarely viable. Alignment of homologues is partially restored after exposure of spo11 perithecia to ionising radiation. Crossing over in a spo11 mutant is reduced in two regions of the Neurospora genome as expected, but is unaffected in a third.     

Repair of lost 5′ terminal sequences in tombusviruses: Rapid recovery of promoter- and enhancer-like sequences in recombinant RNAs

Maintenance of genome integrity is of major importance for plus-stranded RNA viruses that are vulnerable to degradation by host ribonucleases or to replicase errors. We demonstrate that short truncations at the 5′ end of a model Tomato bushy stunt virus (TBSV) RNA could be repaired during replication in yeast and plant cells. Although the truncations led to the loss of important cis-regulatory elements, the genome repair mechanisms led to the recovery of promoter and enhancer-like sequences in 92% of TBSV progeny. Using in vitro approaches, we demonstrate that the repaired TBSV RNAs are replication-competent. We propose three different mechanisms for genome repair: initiation of RNA synthesis from internal sequences and addition of nonviral nucleotides by the tombusvirus replicase; and via RNA recombination. The ability to repair cis-sequences makes the tombusvirus genome more flexible, which could be beneficial to increase the virus fitness and adaptation to new hosts.     

Constructive episodic simulation of the future and the past: Distinct subsystems of a core brain network mediate imagining and remembering

Recent neuroimaging studies demonstrate that remembering the past and imagining the future rely on the same core brain network. However, findings of common core network activity during remembering and imagining events and increased activity during future event simulation could reflect the recasting of past events as future events. We experimentally recombined event details from participants’ own past experiences, thus preventing the recasting of past events as imagined events. Moreover, we instructed participants to imagine both future and past events in order to disambiguate whether future-event-specific activity found in previous studies is related specifically to prospection or a general demand of imagining episodic events. Using spatiotemporal partial-least-squares (PLS), a conjunction contrast confirmed that even when subjects are required to recombine details into imagined events (and prevented from recasting events), significant neural overlap between remembering and imagining events is evident throughout the core network. However, the PLS analysis identified two subsystems within the core network. One extensive subsystem was preferentially associated with imagining both future and past events. This finding suggests that regions previously associated with future events, such as anterior hippocampus, medial prefrontal cortex and inferior frontal gyrus, support processes general to imagining events rather than specific to prospection. This PLS analysis also identified a subsystem, including hippocampus, parahippocampal gyrus and extensive regions of posterior visual cortex that was preferentially engaged when remembering past events rich in contextual and visuospatial detail.     

Olaparib plus bevacizumab as maintenance therapy in patients with newly diagnosed,advanced ovarian cancer: Japan subset from the PAOLA-1/ENGOT-ov25 trial

ObjectiveThe addition of maintenance olaparib to bevacizumab demonstrated a significant progression-free survival (PFS) benefit in patients with newly diagnosed, advanced ovarian cancer in the PAOLA-1/ENGOT-ov25 trial ({"type":"clinical-trial","attrs":{"text":"NCT02477644","term_id":"NCT02477644"}}NCT02477644). We evaluated maintenance olaparib plus bevacizumab in the Japan subset of PAOLA-1.MethodsPAOLA-1 was a randomized, double-blind, phase III trial. Patients received maintenance olaparib tablets 300 mg twice daily or placebo twice daily for up to 24 months, plus bevacizumab 15 mg/kg every 3 weeks for up to 15 months in total. This prespecified subgroup analysis evaluated investigator-assessed PFS (primary endpoint).ResultsOf 24 randomized Japanese patients, 15 were assigned to olaparib and 9 to placebo. After a median follow-up for PFS of 27.7 months for olaparib plus bevacizumab and 24.0 months for placebo plus bevacizumab, median PFS was 27.4 versus 19.4 months, respectively (hazard ratio [HR]=0.34; 95% confidence interval [CI]=0.11–1.00). In patients with tumors positive for homologous recombination deficiency, the HR for PFS was 0.57 (95% CI=0.16–2.09). Adverse events in the Japan subset were generally consistent with those of the PAOLA-1 overall population and with the established safety and tolerability profiles of olaparib and bevacizumab.ConclusionResults in the Japan subset of PAOLA-1 support the overall conclusion of the PAOLA-1 trial demonstrating that the addition of maintenance olaparib to bevacizumab provides a PFS benefit in patients with newly diagnosed, advanced ovarian cancer.Trial RegistrationClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02477644","term_id":"NCT02477644"}}NCT02477644