Extracellular progeny virion production of 293T-BAC36, 293T-BACΔ6 and 293T-BACΔ6 complemented with ORF6.

<p>(<b>A</b>) Infectivities of supernatants from 293T-BAC36, 293T-BACΔ6 and 293T-BACΔ6 cells complemented with ORF6 in the absence or presence of TPA/NaB. 293T cells were innoculated with supernatants from uninduced 293T-BAC36 (1) and 293T-BACΔ6 (3), induced 293T-BAC36 (2), 293T-BACΔ6 (4) and 293T-BACΔ6 complemented with ORF6 (5). GFP expression was examined under a fluorescence microscope at 2 days postinfection. (<b>B</b>) Quantitation of viral genomic DNA in supernatants from 293T-BAC36, 293T-BACΔ6 and 293T-BACΔ6 complemented with ORF6 in the absence or presence of TPA/NaB. At 4 days post-induction, viruses in the supernatants were harvested and concentrated 100-fold. Viral stocks were treated with DNase I for 1 h at 37°C, and viral DNAs were extracted. Viral DNAs were analyzed by a real-time PCR assay using primers to LANA. Copy numbers were normalized and are expressed as copy number per milliliter of supernatant.</p

<b>Table1.</b> Primers used for PCR

<p><b>Table1.</b> Primers used for PCR</p

Lytic DNA replication of 293T-BAC36 and 293T-BACΔ6 cells.

<p>293T cells harboring BAC36, BACΔ6 and BACΔ6 infected with lentiviruses expressing ORF6 were collected at different time points postinduction (0, 48 and 72 h), and total intracellular DNA were extracted. Intracellular viral DNAs were measured by a real-time PCR with primers directed to the ORF73 gene. The viral genome copies were normalized to 20,000 copies of GAPDH.</p

Ori-Lyt dependent lytic replication of 293T-BAC36 and 293T-BACΔ6 cells.

<p>293T-BAC36, 293T-BACΔ6 cells were transfected with ori-Lyt plasmids (pOri-A) plus others as indicated (PCR3.1-ORF50 and FLAG-ORF6). KSHV lytic replication was induced by the expression of ORF50. Total DNAs were isolated from the transfected cells. Replicated DNAs were distinguished from input DNAs by <i>Dp</i>nI digestion and detected by southern blotting with Dig-labeled pOri-A probe.</p

Generation of stable 293T cells carrying BAC36 and BACΔ6 genomes.

<p>(<b>A</b>) Transfection of 293T cells with BAC36 and BACΔ6 DNAs. Cells were transfected with BAC36 (1 and 3) and BACΔ6 (2 and 4) with lipofectamine 2000. GFP expression levels were monitored by fluorescent microscopy 2 days post-transfection (1 and 2). Then, the transfected cells were split and selected with hygromycin. Hygromycin-resistant clones of 293T-BAC36 and 293T-BACΔ6 cells were established (3 and 4). (<b>B</b>) RT-PCR confirmation of the absence of ORF6 in 293T-BACΔ6 cells. Total RNA from uninduced or TPA/NaB induced 293T-BAC36 cells and 293T-BACΔ6 cells were used to prepare cDNAs. The RT-PCR generated products were analyzed on a 1.5% agarose gel. Shown are the KSHV ORFs 6, 7, 73, 50, 59 and 65, respectively. β-actin was used as the normalization control for input RNA. Absence of contaminating DNA in the samples was tested by reverse transcriptase-negative control reactions (RT control). (<b>C</b>) Western blot confirmation of the absence of ORF6 in 293T-BACΔ6 cells. The 293T-BAC36 cells and 293T-BACΔ6 cells were induced with TPA/NaB for 2 days. Whole cell lysates were immunoblotted with antibodies against ORF6, LANA, RTA, ORF45 and ORF59. The same blots were probed with anti-β-actin antibody to ensure equal loading of all samples. (<b>D</b>) Real-time PCR analysis of the mRNA levels of ORF4 and ORF7 from 293T-BACΔ6 cells induced with TPA/NaB. cDNAs were prepared as described previously and analyzed by real-time PCR using primers specific for ORF4 and ORF7 transcripts. β-actin was used as an internal standard. The data are shown as the fold increase compared to the untreated 293T-BAC36 cells.</p

Construction and analysis of ORF6-null KSHV genome.

<p>(<b>A</b>) Schematic diagrams of the structures of ORF6 and its neighboring ORFs in the wild-type and mutant BACs. (<b>B</b>) Confirmation of KSHV ORF6 replacement with Kan/SacB cassette by PCR amplification. The BAC36 (lane 1 and 3) and BACΔ6 (lane 2 and 4) were amplified with flanking PCR primers A and B and internal PCR primers C and D. (<b>C</b>) Confirmation of BAC36 and BACΔ6 genome with <i>Bg</i>lII digestion and Southern blot analysis. BAC36 (lane 1) and BACΔ6 (lane 2) DNAs were digested with <i>Bg</i>lII. The digested DNAs were electrophoresed, blotted, and hybridized with Dig-labeled ORF6 or Kan/SacB DNA probes, respectively.</p

Table_1_The effects of positive leadership on quality of work and life of family doctors: The moderated role of culture.XLS

BackgroundQuality of work and life (QWL) of family doctors is highly valued in improving access and equity of healthcare; however, the current low level of QWL in many countries and regions needs to be improved urgently.MethodsThis study explored the effect of positive leadership on the QWL of family doctors, as well as the moderating role of culture, via analysis of data from 473 valid questionnaires of family doctors in China as a sample using SEM, hierarchical linear regression, and a simple slope test.ResultsThe empirical results show that positive leadership promoted the QWL of family doctors by improving their achievement motivation and coordinating supportive resources. In addition, our hierarchical linear regression analysis found that the interactive items of positive leadership and culture had a positive effect on achievement motivation (β(a)  = 0.192), QWL (β(b)  = 0.215) and supportive resources (β(c)  = 0.195). Meanwhile, culture had a moderated mediating effect on the relationship between positive leadership and QWL via the achievement motivation of family doctors and supportive resources.ConclusionThese findings suggest that the interaction among multiple factors, including environmental factors, individual physiological features and culture, may influence the impact of positive leadership on the QWL of family doctors. The possible reasons of these findings and theoretical and practical implications are discussed in this study.</p

Additional file 1 of MRI-based tumor shrinkage patterns after early neoadjuvant therapy in breast cancer: correlation with molecular subtypes and pathological response after therapy

Additional file 1. Table S1. Participants characteristics in the primary analysis cohort. Table S2. Participants characteristics in the subgroup analysis cohorts. Table S3. Inter-reader agreement for tumor shrinkage patterns in each cohort. Table S4. Inconsistent shrinkage pattern distribution between two readers. Table S5. MRI-based tumor shrinkage patterns association with pNR in HR+/HER2− subtype. Table S6. Univariate and multivariate analysis of factors associated with pNR in HR+/HER2− subtype. Table S7. The diagnostic efficacy of factors in each molecular subtype. Table S8. MRI-based tumor shrinkage patterns association with pCR according to different molecular subtypes in the subgroup analysis cohorts. Table S9. Univariate and multivariate analysis of factors associated with pCR according to different molecular subtypes in the subgroup analysis cohorts. Figure S1. Receiver operating characteristic (ROC) curves of the change in tumor size (continuous variable) at 1st-timepoint and 2nd-timepoint for pathologic complete response (pCR) prediction in the breast

Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain

<div><p>Nicotinic acetylcholine receptors containing α4 subunits (α4β2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 μg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.</p></div

Nicotine CPP is reduced in mice with vMB α4 deletion.

<p>a) CPP schematic. Prior to a 5-day, biased CPP procedure to establish nicotine CPP, mice were mildly food-restricted and handled. Drug-free pre-test and post-test days flanked 3 consecutive conditioning days that consisted of morning and afternoon saline (SAL) and nicotine (NIC) conditioning sessions. b) Nicotine CPP in C57Bl/6 WT mice. Groups of WT mice were conditioned with saline (n = 12), 0.25 mg/kg NIC (n = 12), or 0.5 mg/kg NIC (n = 12) to validate our CPP assay and identify a dose to be used subsequently in α4-flox mice. Mean (± SEM) place preference score is shown for the three drug doses. <i>p</i> values are for Dunnett’s multiple comparisons test. c) Nicotine CPP in α4-flox mice. AAV-GFP or AAV-Cre-GFP vectors were infused into vMB of α4-flox mice (GFP(+), n = 10; Cre(+), n = 10), and mice were subsequently conditioned with 0.25 mg/kg NIC. Mean (± SEM) place preference score is shown for both groups. <i>p</i> value is for unpaired t-test. d) Mean (± SEM) locomotor activity during the pre-test and post-test is shown for α4-flox mice conditioned with NIC. e) Mean (± SEM) locomotor activity during conditioning sessions 1, 2, and 3 is shown for α4-flox mice conditioned with NIC.</p