Cloning of flanking sequences of double-copy gene is a challenge in molecular biology. We developed a method to solve this problem by combining an optimized inverse PCR (iPCR) with TAIL-PCR. First, ...Southern blotting analysis was used to determine a proper restriction enzyme that could obtain proper-length restriction fragments that contained the target gene. Then optimized iPCR was performed to amplify the restriction fragments that contained the separated copies of the gene. Based on the obtained sequences, TAIL-PCR was performed to amplify further flanking regions of the gene. With this method, we obtained all of the EcoR I restriction fragments (2.2-5.1 kb) and Hind III restriction fragments (8.5-11.7 kb) of mitochondrial atpA gene in cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton. The results showed that this method was an efficient approach to clone flanking sequences of double-copy gene.
The effects of magnesium (MgSO
4) on sodium currents (Na
+ currents) in freshly dissociated rat hippocampal neurons were studied using the whole-cell patch clamp techniques. MgSO
4 caused a ...concentration-dependent and voltage-dependent reversible decrease of Na
+ currents. The half-blocking concentration (IC
50) of MgSO
4 on Na
+ currents was 4.05 mM. But the action was frequency-independent. In addition, 4 mM MgSO
4 shifted the steady state activation curve of Na
+ currents toward positive potential (control
V
h=−55.83±6.79 mV, MgSO
4
V
h=−34.15±6.18 mV,
n=8,
P≤0.01 without changing the slope factor). However, the steady state inactivation curve was not affected. These results suggested that blockade of MgSO
4 on Na
+ currents might be an interpretation for its neuroprotection against damages induced by ischemia and oxygen deprivation.
Floral MADS-box genes encode transcription factors that play critical roles in the development and evolution of the flower. Proteins of floral MADS-box genes regulate the expression of their ...downstream genes by forming various homodimers/heterodimers and quaternary complexes. Interactions among proteins of floral MADS-box genes have been documented in several model species, yet the information accumulated so far is still not sufficient to draw a general picture of the evolution of the interactions. We have characterized 28 putative floral MADS-box genes from three representative basal eudicots (i.e., Euptelea pleiospermum, Akebia trifoliata, and Pachysandra terminalis) and investigated the protein-protein interactions (PPIs) among the proteins encoded by these genes using yeast two-hybrid assays. We found that, although the PPIs in basal eudicots are largely consistent with those in core eudicots and monocots, there are lineage-specific features that have not been observed elsewhere. We also reconstructed the evolutionary histories of the PPIs among members of seven MADS-box gene lineages (i.e., AP1, AP3, PI, AG, STK, AGL2, and AGL9) in angiosperms. We revealed that the PPIs were extremely conserved in nine (or 32.1%) of the 28 possible combinations, whereas considerable variations existed in seven (25.0%) of them; in the remaining 12 (or 42.9%) combinations, however, no interaction was observed. Notably, most of the PPIs required for the formation of quaternary complexes, as suggested by the "quartet model," were highly conserved. This suggested that the evolutionarily conservative PPIs may have played critical roles in the establishment of the basic structure (or architecture) of the flower and experienced coevolution to maintain their functions. The evolutionarily variable PPIs, however, seem to have played subsidiary roles in flower development and have contributed to the variation in floral traits.
Autophagy plays an important role in the growth and survival of hepatocellular carcinoma (HCC) cells through several target proteins or signaling pathways. Glypican-3 (GPC3) is a new reliable HCC ...marker, which is involved in tumor growth in HCC, primarily mediated by wnt/β-catenin signaling.
The present study aimed to identify the role of autophagy in the proliferation of HepG2 cells through GPC3/wnt/β-catenin signaling.
Results demonstrated that induction of autophagy by nutrition starvation and rapamycin treatment led to the downregulation of GPC3 expression in HepG2 cells, accompanied by the decreased expression of wnt downstream target genes (β-catenin, c-myc and cyclin D1). On the other hand, inhibition of autophagy by 3-methyl adenine (3-MA) could rescue rapamycin-directed downregulation of GPC3 and wnt/β-catenin target genes and augment the proliferation of HepG2 cells. Furthermore, interference of GPC3 by siRNA suppressed wnt/β-catenin signaling and attenuated 3-MA stimulation of HepG2 cell proliferation. More interestingly, the mRNA of GPC3 remained unchanged when the protein levels of GPC3 were decreased by autophagy activation, suggesting that induction of autophagy may accelerate the degradation of GPC3.
These results suggest that autophagy suppresses proliferation of HepG2 cells partially by inhibition of GPC3/wnt/β-catenin signaling.
Although liquid biopsy has been reported in detecting actionable mutations, and is becoming standard of care in non-small cell lung cancer (NSCLC) under certain circumstances, the relationship ...between plasma mutant allele fractions (MAFs) and treatment responsiveness remains unclear.
This study enrolled 134 patients with advanced NSCLC. Tumor tissue specimens were collected after surgery, and peripheral blood samples were collected at baseline (before surgery) and during treatments. Amplification-refractory mutation system (ARMS) was performed for EGFR mutation screening in all tumor tissues. Circulating single-molecule amplification and resequencing technology (cSMART) was performed in all cfDNA samples using a panel of nine genes including EGFR. EGFR status were compared between tumor tissue and matched cfDNA samples. Moreover, the association between MAFs of EGFR mutations in cfDNA and the responsiveness to tyrosine-kinase inhibitor (TKI) treatment was analyzed.
EGFR mutations were detected in 56 tumor samples (56/134, ∼41.8%) and 67 matched baseline plasma cfDNA samples (67/134, 50.0%), the concordance rate between tumor and cfDNA samples is 82.8%. Specifically, EGFR exon19 non-frameshift deletions were detected in 24 tumor tissue and 23 matched cfDNA samples (concordance rate 97.8%), EGFR p.L858R mutations were detected in 24 tumor tissue and 22 matched cfDNA samples (concordance rate 97.0%). The overall response rate (ORR) and disease control rate (DCR) for EGFR mutation positive patients (TKI sensitive mutations only) were 47.6% and 73.8%, respectively. More importantly, for TKI-treated patients with original plasma EGFR MAFs higher than 0.1% and lower than 0.1%, the ORRs were 58.6% and 23.1% (17/29 versus 3/13 patients, P=0.047), respectively.
In this study we confirmed that liquid biopsy based screening is reliable for actionable somatic mutation detection. The plasma mutant allele fraction of EGFR driver mutation tend to associate with the responsiveness to TKI targeted treatment. Larger cohorts are needed to clarify the impact of plasma MAFs in predicting treatment responsiveness.
The authors.
Has not received any funding.
All authors have declared no conflicts of interest.
Abstract Background Accurate quantitation of hepatitis B viral load is a critical aspect in screening and monitoring HBV infection. Objectives We used loop-mediated isothermal amplification (LAMP) to ...develop a real-time fluorogenic (RtF-LAMP) protocol to quantitate HBV DNA. Quantitative analysis was obtained by measuring time-to-positive (TTP), a biomarker similar to cycle threshold (Ct) in real-time PCR. Study design Sensitivity, specificity, reproducibility, and dynamic range for RtF-LAMP were evaluated using molecular and biological standards. Four hundred and two patient samples were then used to compare the performance of RtF-LAMP to a commercial real-time PCR assay (DaAn Gene Co, China). Results The lower detection limit (LDL) of RtF-LAMP by Probit analysis at the 95% detection level was 210 copies/ml, and the dynamic range was 8 orders of magnitude. The conversion factor for results obtained with the RtF-LAMP assay was 1 IU/ml equals to 4.4 copies/ml. Coefficients of variation (CV) reflected low intra-assay and inter-assay variability (4.24–12.11%). In a large number of serum samples, there was excellent an correlation between RtF-LAMP and real-time PCR ( R2 = 0.96). There was a good agreement between the two tests except at the detection cutoff of the real-time PCR assay. Conclusion Our RtF-LAMP protocol appears to be precise, accurate and rapid. It could be a valuable tool for the detection of HBV in large clinical and epidemiological studies.
We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A
1 ...receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague–Dawley rats. Forskolin (0.1, 0.5 and 1.0
pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal
N
6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A
1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice–ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A
1-selective agonist,
N
6-cyclohexyladenosine (24
ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal
N
6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal
N
6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal
N
6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500
ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (G
i, G
o) and possibly of the adenosine A
1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal
N
6-cyclohexyladenosine.
The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A
1 receptor level.
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